CHAPTER I

4.9 Time-dependent effects of 1-, 4-, and 8-week restraint stress induction on the hypothalamic and hippocampal GR protein levels.

Numbers of animals are noted in parentheses. *P < 0.05 and ***P < 0.001 compared to non-stressed control group. 76 4.10 Time-dependent effects of the 1-, 4-, and 8-week restraint stress induction on the NET protein levels. Numbers of animals are noted in parentheses. *P < 0.05 compared to non-stressed control group. 77 4.11 Time-dependent effects of the 1-, 4-, and 8-week restraint stress induction on the SERT protein levels. Numbers of animals are noted in parentheses. *P < 0.05 compared to non-stressed control group. 78 4.12 Time-dependent effects of the 1-, 4-, and 8-week restraint stress induction on the 5-HT 2C R protein levels. Numbers of animals are noted in parentheses. *P < 0.05, and **P < 0.01 compared to nonstressed control group. 79 4.13 The effects of the 4-week voluntary wheel running on (A) starting body weight, (B) final body weight, (C) body weight gain, and (D) daily food intake. Numbers of animals are noted in parentheses. **P < 0.01, ***P < 0.001 exercise group compared to non-stressed sedentary control. # P < 0.05 sedentary stressed group compared to non-stressed sedentary control group. † P < 0.05 combined stressed and exercised group compared to sedentary stressed group.

LIST OF TABLES

Table Page

2.1 Functional anatomy and physiology of brain regions in stress responses.

2.2 Summary of localization of selected target proteins in the present study.

3.1 Descriptions of animal groups in the experiment 1.

3.2 Descriptions of animal groups in the experiment 2.

3.3 Descriptions of animal groups in the experiment 3.

3.4 Summarizes the parameters and interpretations of behavioral evaluations.

3.5 Primary antibodies used in Western blotting analysis.

3.6 The MT1-/MT2-LacZ genotyping and primer designs. Changes in bio-behavioral parameters of time dependent effects of the 1-, 4-, and 8-week restraint stress induction in male rats.

Changes in bio-behavioral parameters of effects of the 4-week voluntary wheel running under non-stressed condition and/or after the 4-week restraint stress induction in male rats.

Changes in bio-behavioral parameters of preventive effects of the 4week agomelatine, venlafaxine, and voluntary wheel running in stressed male rats.

Localization of MT1 and MT2 melatonin receptors in transgenic MT1-/MT2-LacZ knock-in mouse brains.

v

LIST OF FIGURES

2.1 The hypothalamic-pituitary-adrenal (HPA) axis in stress responses

showing positive feedbacks (+) and negative feedbacks ().

Elevated glucocorticoids (GCs) are induced by the release of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) from the hypothalamic paraventricular nucleus (PVN) and anterior pituitary, respectively. Negative feedback inhibition by GCs acts on the pituitary gland and the hypothalamus.

The release of GCs is also regulated by circadian signals () from the suprachiasmatic nucleus (SCN) in the hypothalamus. 6 2.2 Neuroanatomical organization of stress responses consist of three levels, i.e., the cortico-limbic system, the hypothalamus-brain stem system, and brain-to-adrenal gland system. ACTH, adrenocorticotropic hormone; ACH, acetylcholine; BNST, bed nucleus of the stria terminalis; CRH, corticotropin releasing hormone; EPI, epinephrine; GC, glucocorticoids; NE, norepinephrine; PFC, prefrontal cortex; and subcortical structures, amygdala [START_REF] Gunnar | The neurobiology of stress and development[END_REF]. Prolactin (Modified from [START_REF] Dubocovich | Functional MT1 and MT2 melatonin receptors in mammals[END_REF]. 17 pH, posterior hypothalamus; A, amygdala; VTA, ventral tegmentum area; and H, hippocampus [START_REF] Heffner | A rapid method for the regional dissection of the rat brain[END_REF]. 54 3.9 Coronal brain sections at the levels of periaqueductal grey and dorsal raphe nuclei (A) and locus coeruleus (B) [START_REF] Paxinos | The rat brain in stereotaxic coordinates[END_REF]. LIST OF FIGURES (cont.) Numbers of animals are noted in parentheses. † † P < 0.01, † † † P < 0.001 compared to vehicle (Veh)-treated group. aaa P < 0.001 compared to percent sucrose preference after completing 4-week pre-treatment period. Numbers of animals are noted in parentheses. † P < 0.05, † † P < 0.01, and † † † P < 0.001 compared to vehicle (Veh)-treated group. becoming a global burden of disability and comorbidity [START_REF] Kessler | The global burden of mental disorders: an update from the WHO World Mental Health (WMH) surveys[END_REF]. Severe and chronic stress exposures are believed to cause anxiety, depression, and memory impairment later in life [START_REF] Sareen | Combat and peacekeeping operations in relation to prevalence of mental disorders and perceived need for mental health care: findings from a large representative sample of military personnel[END_REF][START_REF] Li | Prenatal infection affects the neuronal architecture and cognitive function in adult mice[END_REF]Ikeda et al., 2017), and therefore, preventive intervention for these stress complications is worth exploring. In many situations, such as extreme military combat or war, infectious disease outbreak, and natural disaster, preventive strategies become more important in reducing incidence of stress-related psychiatric problems in veterans or victims.

The underlying mechanisms of mood disorders in stressed individuals are associated with deregulation of the hypothalamic-pituitary adrenal (HPA) axis and an imbalance of the monoamines serotonin (5-HT) and norepinephrine (NE) [START_REF] Charney | Monoamine dysfunction and the pathophysiology and treatment of depression[END_REF][START_REF] Bowman | Chronic stress effects on memory: sex differences in performance and monoaminergic activity[END_REF][START_REF] Belmaker | Major depressive disorder[END_REF]. Chronic stress increases the glucocorticoid stress hormones, that glucocorticoids can pass the blood-brain barrier and induce deleterious changes in the neuronal function in brain regions regulating memory formation and behavior. Sustained high levels of glucocorticoids decrease glucocorticoid receptor (GR), brain-derived neurotrophic factor (BDNF), and neurogenesis in the hippocampus leading to anxiety, depression and memory impairment [START_REF] Mcewen | Physiology and neurobiology of stress and adaptation: central role of the brain[END_REF][START_REF] Schmidt | The role of neurotrophic factors in adult hippocampal neurogenesis, antidepressant treatments and animal models of depressivelike behavior[END_REF].

Besides the aberrant HPA axis, stress could disturb the circadian rhythms and the pineal-adrenal secretory response [START_REF] Konakchieva | Chronic melatonin treatment and the hypothalamo-pituitary-adrenal axis in the rat: attenuation of the secretory response to stress and effects on hypothalamic neuropeptide content and release[END_REF][START_REF] Mcclung | Circadian rhythms and mood regulation: insights from pre-clinical models[END_REF].

Melatonin (MT) plays a crucial role in the synchronization of circadian rhythmicity.

Actions of MT are mediated by membrane MT receptor subtypes, MT1 and MT2.

Despite the finding that MT levels were decreased in depressed patients (Claustrat et al., 1984), it is still important to know where MT1 and MT2 are localized because they are targets for the development of novel antidepressants and MT derivatives in the HPA axis and hippocampus. So far, the cellular localization of MT1 and MT2 has not been visualized clearly despite the use of 125 I-iodomelatonin binding, in situ hybridization, and some developed antibody for immunocytochemistry (Liu et al., 1997;Fugieda et al., 1999;[START_REF] Poirel | MT1 melatonin receptor mRNA expression exhibits a circadian variation in the rat suprachiasmatic nuclei[END_REF]. MT1 has been identified in the suprachiasmatic nucleus (SCN), but site of MT2 remain elusive. Therefore, a genetic modified rodent model has been generated for mapping and phenotyping MT receptor expression.

The use of animal models in the stress-related studies provides useful insights into the behavioral and physiological mechanisms involved in the stress response. Restraint stress is a simple but effective method that produces both physical and behavioral changes in mouse and rats. However, the variations of the procedures such as the intensity and duration of restraint stress have so far failed to produce a consensus in the results for stress-induced anxiety, depression, and memory impairment [START_REF] Buynitsky | Restraint stress in biobehavioral research: Recent developments[END_REF]. Although chronic restraint stress could produce anxiety-and depression-like behavior in male rats [START_REF] Lapmanee | Anxiolytic-like actions of reboxetine, venlafaxine and endurance swimming in stressed male rats[END_REF][START_REF] Lapmanee | Beneficial effects of fluoxetine, reboxetine, venlafaxine, and voluntary running exercise in stressed male rats with anxiety-and depression-like behaviors[END_REF], but whether total durations of stress induced learning and memory impairment is still unclear. In addition, the mechanisms triggering the restraint stress-related behaviors are still not known

The involvement of the dysregulation of 5-HT and NE system in stressrelated mood disorders has been primarily supported by the enhancing effects of antidepressants on the binding of 5-HT to the serotonin transporter (SERT) and NE to the noradrenaline transporter (NET), thus increasing storage and release of these neurotransmitters into the synaptic cleft. A recent study showed that a serotoninnorepinephrine-dopamine reuptake inhibitor (SNDRI), venlafaxine, showed a greater potency in the treatment of anxiety-like behavior in stressed male rats than a selective serotonin reuptake inhibitor (SSRI), fluoxetine or a norepinephrine reuptake inhibitor (NRI), reboxetine [START_REF] Lapmanee | Anxiolytic-like actions of reboxetine, venlafaxine and endurance swimming in stressed male rats[END_REF]. However, it is not known whether venlafaxine can prevent stress-induced anxiety, depression, and memory impairments.

Interestingly, due to the anxiolytic and antidepressant effects [START_REF] Papp | Effect of agomelatine in the chronic mild stress model of depression in the rat[END_REF][START_REF] Millan | Anxiolytic properties of agomelatine, an antidepressant with melatoninergic and serotonergic properties: role of 5-HT2C receptor blockade[END_REF][START_REF] Taylor | Antidepressant efficacy of agomelatine: meta-analysis of published and unpublished studies[END_REF] of the melatoninergic antidepressant agomelatine, the a melatonin MT1 and MT2 receptor agonist and 5-HT 2C receptor (5-HT 2C R) antagonist could be a candidate drug for the prevention of stress-induced behavioral change. Unlike venlafaxine, agomelatine is without side effects, such as nausea, diarrhea, or sexual dysfunctions, which can be attributed to excessive stimulation of some peripheral and central 5-HT receptors (Kennedy and Emsley, 2007). Furthermore, there are no discontinuation symptoms after cessation of agomelatine treatment compared to paroxetine [START_REF] Montgomery | Absence of discontinuation symptoms with agomelatine and occurrence of discontinuation symptoms with paroxetine: a randomized, double-blind, placebo-controlled discontinuation study[END_REF]. However, it is unclear whether agomelatine acts directly on brain region responsible for stress responses. Since localization of MT receptors in the rodent central nervous system is not completely understood, and there is limitation in the use of immunolocalization for MT1 and MT2 [START_REF] Wu | Alterations of melatonin receptors MT1 and MT2 in the hypothalamic suprachiasmatic nucleus during depression[END_REF][START_REF] Lacoste | Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain[END_REF], the present study attempted to use transgenic mice with β-galactosidase (LacZ) reporter gene in the study of MT receptor distribution in in brain regions associated with stress responses.

Since the pharmacological intervention in mood disorders are timeconsuming and might cause side effects, it is better to search for a novel medication in parallel with alternative interventions. Therefore, non-pharmacological intervention such as physical exercise has recently become popular, and could be a low cost-stressrelieving therapy with no side effects for stressed individuals. Exercise has been reported to be an alternative treatment for a variety of mood disorders. Compared to forced exercise, e.g., treadmill running and swimming, voluntary exercise such as voluntary wheel running in rodents should be a motivational exercise with less associated stress. Moreover, voluntary exercise was found to have both antidepressant and anxiolytic effects in rodents and human studies [START_REF] Burghardt | The effects of chronic treadmill and wheel running on behavior in rats[END_REF][START_REF] Ströhle | Physical activity, exercise, depression and anxiety disorders[END_REF][START_REF] Lapmanee | Beneficial effects of fluoxetine, reboxetine, venlafaxine, and voluntary running exercise in stressed male rats with anxiety-and depression-like behaviors[END_REF]. Voluntary exercise is a potent stimulator of the HPA axis [START_REF] Stranahan | Central mechanisms of HPA axis regulation by voluntary exercise[END_REF]. Voluntary wheel running increases hippocampal BDNF and neurogenesis (Cotman and Berchtold, 2002) and also enhances brain function by altering monoaminergic neurotransmission [START_REF] Dishman | Brain monoamines, exercise, and behavioral stress: animal models[END_REF]. However, the modulating effects of voluntary wheel running exercise on the chronic stress response are not well understood, and its preventive effect on stress-induced mood and memory deficits remains unclear.

Therefore, the objectives of thesis research were:

1. To examine the time-dependent effects of the 1-, 4-and 8-week stress induction on anxiety-, depression-, and memory impairment-like behaviors and changes in the expression of selected target proteins in brain regions associated with stress responses.

Neuroanatomy of stress

Various brain regions involved in stress responses can be described at three levels (Figure 2.2), i.e., the cortico-limbic system, the hypothalamus-brain stem system, and brain-to-adrenal gland system (as reviewed by [START_REF] Gunnar | The neurobiology of stress and development[END_REF]. At the level of the cortico-limbic system, the anterior cingulate (ACC) and orbital frontal cortex (OFC) are connected to the amygdala, which in turn connects with the hippocampus and bed nucleus of the stria terminalis (BNST). The BNST i.e., the cortico-limbic system, the hypothalamus-brain stem system, and brain-toadrenal gland system. ACTH, adrenocorticotropic hormone; ACH, acetylcholine;

BNST, bed nucleus of the stria terminalis; CRH, corticotropin releasing hormone; EPI, epinephrine; GC, glucocorticoids; NE, norepinephrine; PFC, prefrontal cortex; and subcortical structures, amygdala [START_REF] Gunnar | The neurobiology of stress and development[END_REF].

The PFC, hippocampus, and amygdala are the key regions responsible for GC feedback in the regulation of the HPA axis [START_REF] Sairanen | Chronic antidepressant treatment selectively increases expression of plasticity-related proteins in the hippocampus and medial prefrontal cortex of the rat[END_REF]. The hippocampus and PFC are involved with depression, while subcortical regions, i.e., nucleus accumbens (NA), amygdala, and hypothalamus are involved with reward, fear, and motivation. The brain regions responsible for stress responses are summarized in Table 2.1 [START_REF] Drevets | Neuroimaging and neuropathological studies of depression: implications for the cognitive-emotional features of mood disorders[END_REF][START_REF] Liotti | The role of functional neuroimaging in the neuropsychology of depression[END_REF].

Brain region Physiological roles in stress responses Amygdala

Triggers the fear response, aggressive behaviors, memory, and acts as an emotion center.

Hippocampus

Forms conscious memories of emotional events.

Hypothalamus

Controls body homeostasis, detects and responds to stress through the HPA axis via PVN, and generates defensive behaviors.

Thalamus

Integrates and relays sensory inputs, modulates motivation and emotion, and regulates motor programs.

Prefrontal cortex (PFC)

Supports learning and memory processes, and expresses subjective feelings (e.g., nausea and anxiety).

Caudate putamen

Provides a feedback loop to the cortex, and voluntary motor movement.

Globus pallidus

Consists of inhibitory GABAergic projection neurons, controls coordinate/fine movement, and processes learning and memory. Nucleus accumbens (NA) Receives stress-responsive DA projection neurons from the VTA, mediates by interfacing limbic, cognitive and motor circuitry and processes rewarding and reinforcing, exploratory, and spontaneous activity.

Septal nuclei

Plays a role in reward and reinforcement along with the NA and promotes active stress coping behavior involved in a HPA-inhibitory mechanism.

Raphe nuclei

Produces serotonin (5-HT) in dorsal raphe nuclei, promotes attention, vigilance, alertness, arousal and attention.

Locus coeruleus (LC)

Produces NE, modulates emotion, attention, pain, autonomic, respiratory, and cardiovascular functions.

Ventra tegmental area (VTA)

Produces DA, processes for reinforcement, reward, memory, and attention.

Periaqueductal gray (PAG)

Generates defensive responses and strong feeling of fear in panic attacks and promotes fear and defensive behaviors, arousal, hypoalgesia

Substantial nigra (SN)

Contains nigrostriatal dopaminergic neurons and regulates movement and coordination. Its dysfunction is implicated in Parkinson's disease.

Neurochemistry of stress

Corticotropin-releasing hormone (CRH)

CRH is an amino acid neuropeptide, which plays a major role in the adaptation to stress [START_REF] Vale | Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and betaendorphin[END_REF]. Release of CRH depends on circulating GC and ACTH, which are associated with circadian patterns of the HPA axis. CRH is required for the activation of anterior pituitary ACTH gene expression. CRH is present throughout the central nervous system, adrenal and sympathetic ganglion of autonomic nervous system and some fetal organs such as lung and gastrointestinal tract.

Regarding localization of CRH receptors, CRH 1 and CRH 2 are expressed in the brain. About 50% of CRH neurons colocalize with AVP in the the dorsomedial parvocellular part of the PVN [START_REF] Smith | The role of the hypothalamic-pituitary-adrenal axis in neuroendocrine responses to stress[END_REF]. CRH neurons directly synapse with glutamatergic and GABAergic neurons in the PVN and the cerebral cortex, i.e., the layers 2-3 of the rat brain [START_REF] Yan | Co-localization of corticotropinreleasing hormone with glutamate decarboxylase and calcium-binding proteins in infant rat neocortical interneurons[END_REF][START_REF] Kubota | Selective coexpression of multiple chemical markers defines discrete populations of neocortical GABAergic neurons[END_REF]. In addition, CRH neurons and CRH receptors are expressed in the hippocampus, stria terminalis, solitary nucleus of hypothalamus, amygdala, locus coeruleus, cerebellum and spinal cord [START_REF] Aguilera | The molecular physiology of CRH neurons[END_REF]. CRH is not only responsible for metabolic, endocrine and autonomic responses in the perceived acute stress, but CRH also leads to psychological problems in the perceived chronic stress [START_REF] Korosi | The central corticotropin releasing factor system during development and adulthood[END_REF].

Therefore, CRH can mediate behavioral responses such as anxiety, arousal, depression, and learning and memory [START_REF] Binder | The CRF system, stress, depression and anxiety-insights from human genetic studies[END_REF].

Glucocorticoid (GC)

GCs are essential for life. Normally, the release of GC is driven by a circadian rhythm originating in the SCN of the hypothalamus. Signal inputs from SCN result CRH release, which activates the HPA axis and releases GCs (Figure 2.1). In addition, sympathetic nerve fibers directly connect the SCN to the HPA axis; therefore GC release exhibits a circadian pattern. GC promotes energy mobilization, i.e., increase in blood glucose and free-fatty acids in the fight to flight response, and also disturbs the functions of the immune, digestive, and reproductive systems in long-term stress exposure. GRs and MRs are nuclear receptors with transcription factor activity, which is activated by the binding of GCs to the GR and MR. GR is highly expressed throughout the whole brain, whereas MR is expressed in specific restricted brain regions such as the amygdala, the hippocampus, the LC, the PFC, the solitary nucleus and the PVN (de Kloet et al., 2005;[START_REF] Mcewen | Physiology and neurobiology of stress and adaptation: central role of the brain[END_REF]. GC mediates the negative feedback to inhibit the secretion of CRH and ACTH.

Hyperactivity or dysregulation of the HPA axis leads to hypercortisolism contributing to metabolic, cardiovascular, and mental diseases. It has been well established that chronic stress is a risk factor for mood disorders (e.g.

anxiety disorders and depression). Particularly, GCs cause impaired learning and memory formation, changes in the distribution pattern of GR and MR, atrophy of hippocampal cells, reduced hippocampal neurogenesis, reduced synaptic plasticity and impaired learning ability, resulting in memory problems in stressed individuals [START_REF] Sapolsky | The neuroendocrinology of stress and aging: the glucocorticoid cascade hypothesis[END_REF].

GABAergic neurotransmission

GABA plays a role in the modulation of emotional behaviors.

GABAergic neurons synthesize GABAs to inhibit neuronal excitability. GABA is synthesized by glutamic acid decarboxylase (GAD), which exists as two isoforms based on the protein size of glutamic acid decarboxylase (GAD), i.e., 67 kDa (GAD1)

and 65 kDa (GAD2). In the mouse brain, 80-90% of GAD activity is produced by GAD1 [START_REF] Asada | Cleft palate and decreased brain gammaaminobutyric acid in mice lacking the 67-kDa isoform of glutamic acid decarboxylase[END_REF], Condie et al. 1997). Changes in GABA, GABA receptor, and GAD expression can occur in stress exposure. Stress can inhibit the GABAergic plasticity, which contributes to the development of stress-related disorders (reviewed by [START_REF] Maguire | Stress-induced plasticity of GABAergic inhibition[END_REF].

During stress, increased levels of GCs lead to increased GABA levels in the hippocampus of rats (de [START_REF] De Groote | Exposure to novelty and forced swimming evoke stressordependent changes in extracellular GABA in the rat hippocampus[END_REF]. Alteration of GAD expression causes marked inhibitory effects on postsynaptic neurons in the hippocampus [START_REF] Stone | Effects of preand postnatal corticosterone exposure on the rat hippocampal GABA system[END_REF][START_REF] Maggio | Differential corticosteroid modulation of inhibitory synaptic currents in the dorsal and ventral hippocampus[END_REF]. Both GAD1 and GAD2 expressions are increased in the bed nucleus of the stria terminalis, preoptic area of hypothalamus, PVN and CA3 of the hippocampus after chronic intermittent stress [START_REF] Bowers | Region-specific regulation of glutamic acid decarboxylase (GAD) mRNA expression in central stress circuits[END_REF]. Moreover, chronic immobilization stress increases the expression of GABA A receptors in the PFC as indicated by greater binding of 3 Hflunitrazepam [START_REF] Braestrup | Benzodiazepine receptors in the brain as affected by different experimental stresses: the changes are small and not undirectional[END_REF]. In contrast, the reduction of GAD was reported in the striatum and olfactory bulb in chronic cold stress [START_REF] Acosta | Area-dependent changes in GABAergic function after acute and chronic cold stress[END_REF] leading to reduced levels of GABA. These results indicate that the GABAergic inhibitory control over glutamatergic cells is reduced [START_REF] Kalkman | GAD(67): the link between the GABA-deficit hypothesis and the dopaminergic-and glutamatergic theories of psychosis[END_REF]. Therefore, the reduction of GABAergic inhibitory control in these areas could be implicated in the pathophysiology of fear, anxiety, and depression.

Monoaminergic neurotransmission

Monoamines, organic compounds with one amino group, are associated with mood stabilization. Imbalance of monoaminergic neurotransmitters contributes to the impairments of structural plasticity and changes in the size of of specific limbic areas. The members of monoamine neurotransmitters include catecholamines (dopamine, DA and NE) and 5-hydroxytryptamine (serotonin; 5-HT).

Therefore, dysregulation of monoaminergic neurotransmission has been implicated in mood disorders such as such as depression, anxiety, and schizophrenia [START_REF] Kandel | Principles of neural science[END_REF][START_REF] Siegla | Price DL Basic neurochemistry moleclar, cellular, and medical aspects[END_REF] 3.4.1 Catecholamines

The dopaminergic system plays a role in emotional reward, motivation, desire, addiction, pleasure, learning and motor fine tuning. The central component of the dopaminergic system is the ventral tegmental area (VTA).

DA is synthesized in the nigrostriatal neurons that are involved in body movement and mood. In rats, dopaminergic neurons are present in the midbrain, i.e., the substantia nigra (SN), VTA and related nuclei. DA and the limbic system are essential in forming behavior, sensory and environmental/emotional-memory connections. The VTA forms a connection with a key pleasure center known as NA. DA release into the NA is responsible for rewarding and addictive behaviors. In addition, the NE system plays a role in wakefulness, alertness, energy, arousal and motivation. The LC, a nucleus located in the pons of the brainstem, is the central norepinephrine system. The LC produces NE and sends projections to areas of the hindbrain, forebrain, and spinal cord stressed rats. Therefore, elevated TH levels could be an indicator of the stressed status in the NE system [START_REF] Melia | Coordinate regulation of the cyclic AMP system with firing rate and expression of tyrosine hydroxylase in the ratlocus coeruleus: effects of chronic stress and drug treatments[END_REF]. Moreover, high NE may facilitate long-term potential (LTP) induction in the hippocampus by decreasing the threshold to LTP generation, facilitating memory formation [START_REF] Hu | Emotion enhances learning via norepinephrine regulation of AMPA-receptor trafficking[END_REF]. NETs are located on the presynaptic membrane of NE nerve terminals. NET expression has not been found in serotonergic and dopaminergic neurons, but the NET is located on the presynaptic part of an NE terminal [START_REF] Barker | Norepinephrine and serotonin transporters: molecular targets of antidepressant drugs[END_REF]. Moreover, alterations of NET expression could contribute to the pathophysiology of depressive symptoms [START_REF] Klimek | Reduced levels of norepinephrine transporters in the locus coeruleus in major depression[END_REF][START_REF] Haenisch | Knockout of the norepinephrine transporter and pharmacologically diverse antidepressants prevent behavioral and brain neurotrophin alterations in two chronic stress models of depression[END_REF][START_REF] Haller | Behavioral responses to social stress in noradrenaline transporter knockout mice: effects on social behavior and depression[END_REF]. Although the role of the NET in NE signaling is well known, its regulation in response to treatment with NET inhibiting antidepressants remains uninvestigated. Moreover, reports of effects of stress on NET expression are inconsistent. Either membrane trafficking or degradation of NET proteins may contribute to the loss of NET ligand binding and protein levels. Altered expression of the NET gene may contribute to the loss of NET proteins in response to antidepressant treatment [START_REF] Zhu | Effects of desipramine treatment on norepinephrine transporter gene expression in the cultured SK-N-BE(2)M17 cells and rat brain tissue[END_REF][START_REF] Chen | Chronic social defeat up-regulates expression of norepinephrine transporter in rat brains[END_REF].

3.4.2 Serotonin (5-hydroxytryptamine, 5-HT)

The 5-HT system plays a role in mood, emotion, social disposition, sleep cycle, wakefulness, dreaming, appetite, spinal pain modulation and the modulation of the endogenous opioidergic system. The raphe nuclei are located in the brainstem, and are the primary source of 5-HT in the brain. 5-HT is a hydrophilic substance and that can pass the blood-brain barrier. Ascending serotonergic projections innervating the cerebral cortex and other regions of the forebrain arise from the dorsal raphe (DR) and the median raphe. The median raphe projects to the hippocampus, the septum and the hypothalamus, whereas the striatum is innervated predominantly by the DR. Raphe neurons send collateral axons to brain areas that are functional related, i.e., the amygdala and hippocampus or the SN and caudate putamen. The serotonergic pathways project to areas of the cerebral cortex, and the spinal cord. When activated, the ascending projections of the 5-HT neurons in the DR facilitate neuronal activity in the amygdala and suppress neurons commanding flight in the periaqueductal grey (PAG). Neurons in the median raphe nucleus could be stimulated by uncontrollable stress. In order to prevent the occurrence of depression, 5-HT would promote a disconnecting mechanism that results in tolerance to stress that takes place in the hippocampus. Abnormalities of 5-HT function in the brain can lead to psychiatric disorders such as schizophrenia, anxiety, panic and depression [START_REF] Graeff | Role of 5-HT in stress, anxiety, and depression[END_REF].

5-HT is synthesized from the amino acid tryptophan by the rate limiting enzymes tryptophan hydroxylase (TPH) and 5hydroxytryptophan (5-HTP) decarboxylase. TPH is similar to TH in term of catalytic mechanism and the amino acid sequences. 5-HT is taken up from the cytoplasm into the storage vesicle by active transport. The 5-HT receptors are divided into 5-HT 1 , 5-HT 2 , and 5-HT 3 receptors. The 5-HT 1 and 5-HT 2 receptor families are G-protein coupled receptor, while the 5-HT 3 receptor is a ligand-gated sodium and potassium ion channel [START_REF] Lf | Serotonin: a review[END_REF].

Among the 5-HT receptor families, the 5-HT 2C receptor (5-HT 2C R) is a prominent serotonin receptor that is distributed throughout the central nervous system. 5-HT 2C Rs are widely distributed in the choroid plexus, frontal cortex, hippocampus, hypothalamus, ventral tegmental area and amygdala [START_REF] Xu | 5-HT2CRs expressed by proopiomelanocortin neurons regulate energy homeostasis[END_REF][START_REF] Greenwood | 5-HT2C receptors in the basolateral amygdala and dorsal striatum are a novel target for the anxiolytic and antidepressant effects of exercise[END_REF]. Activation of 5-HT 2C R induces a decrease in food intake and increases anxiety [START_REF] Dryden | Differential effects of the 5-HT 1B/2C receptor agonist mCPP and the 5-HT1A agonist flesinoxan on hypothalamic neuropeptide Y in the rat: evidence that NPY may mediate serotonin's effects on food intake[END_REF][START_REF] Gatch | Discriminative stimulus effects of m-chlorophenylpiperazine as a model of the role of serotonin receptors in anxiety[END_REF] because 5-HT 2C R is expressed on CRF neurons that regulate ACTH and corticosterone secretion (Heisler et al., 2007a). Furthermore, 5-HT 2C R knockout mice display reduced anxiety when exposed to novel environments and show decreased neuronal activities, specifically in the amygdala (Heisler et al., 2007b). In addition, altered RNA editing of the 5-HT 2C R was observed in the PFC of suicide victims with major depressive disorder as well as in stressed rats [START_REF] Niswender | RNA editing of the human serotonin 5-References / 186 HT2C receptor: alterations in suicide and implications for serotonergic pharmacotherapy[END_REF][START_REF] Iwamoto | Altered RNA editing of serotonin 2C receptor in a rat model of depression[END_REF]. Therefore, the activation of this receptor could be involved in anxiety and fear responses and could be a target for anxiolytic drugs, i.e., agomelatine.

Serotonin reuptake transporter (SERT)

In order to regulate the availability of 5-HT in the synaptic cleft, both binding of 5-HT to its autoreceptor and the activity of the SERT located on the presynaptic membrane are required. Stimulation of the 5-HT autoreceptor decreases further release of 5-HT, while the SERT removes 5-HT from the synaptic cleft back into the presynaptic neuron. Changes in SERT expression and function are correlated with the pathophysiology of depression and anxiety disorders.

Effects of stress on SERT expression in the raphe nuclei have been studied but the results are still controversial. For example, single immobilization reduced SERT mRNA in the raphe pontis [START_REF] Vollmayr | Acute stress decreases serotonin transporter mRNA in the raphe pontis but not in other raphe nuclei of the rat[END_REF]. Single social defeat reduced the SERT densities in hippocampus, but did not change those in the midbrain where the DR nuclei are located [START_REF] Berton | Behavioral, neuroendocrine and serotonergic consequences of single social defeat and repeated fluoxetine pretreatment in the Lewis rat strain[END_REF]. In contrast, chronic restraint plus cold stress resulted in an increase in SERT mRNA levels in the DR of both male and female rats [START_REF] Pare | Gender differences in acute and chronic stress in Wistar Kyoto (WKY) rats[END_REF]. These studies with inconsistent results clearly emphasize the need for further studies on the effects of chronic stress on the SERT in serotoninergic neurons. SERT is the primary target of the clinically successful class of antidepressant and anxiolytic drugs known as the selective serotonin reuptake inhibitors (SSRIs). Although the action of the SSRIs is generally understood to involve an increase in 5-HT concentrations in the synapse, resulting in increased postsynaptic receptor binding, this has not been clearly established [START_REF] Schloss | The serotonin transporter: a primary target for antidepressant drugs[END_REF].

The complex neural pathways of the serotonergic system allow this transmitter substance to broadly affect behavior and mood. Changes in the serotonergic system have been shown to be strongly correlated with mental disorders. At the cellular level, abnormalities may include abnormal regulation of 5-HT synthesis, release and/or reuptake or abnormal responsiveness to the 5-HT signal.

In addition, alteration of this neurotransmission involves the presynaptic autoreceptors

(5-HT 1A ), the SERT site, and the postsynaptic 5-HT 2C R, in several brain regions, that are believed to be potentially important in mood and memory disorders.

Melatonin

Melatonin (MT) is an important regulator of circadian rhythms and seasonal physiology. MT was first isolated and identified by Lerner and co-workers (1958). MT synthesis in the pineal gland is modulated by the light/dark information that is detected by the retina. The relative contributions of the intrinsically photosensitive retinal ganglion cells (ipRGCs) and the classical photoreceptors in the control of MT synthesis are subject to discussion. IpRGCs alone are sufficient in photoreceptor deficient animals, but that does not mean that in animals with functional retinal photoreceptors only the ipRGC control melatonin production. MT production in the pineal gland and locally in the retina follows a circadian rhythm, with the highest levels produced during the dark phase. In mammals, the light/dark information is sent through the retino-hypothalamic tract and the SCN to the pineal gland, where MT is synthesized and secreted by pinealocytes.

Glutamate released from the retinohypothalamic tract stimulates the SCN GABAergic neurons, which in turn inhibit the stimulatory action of the PVN on MT secretion by the pineal gland through its sympathetic innervation at the intermediolateral nucleus of the spinal cord and the superior cervical ganglion. MT binds to the MT1 and/or MT2 receptors on the target cells (reviewed by [START_REF] Liu | MT1 and MT2 Melatonin Receptors: A Therapeutic Perspective[END_REF].

Stress-induced activation of the HPA axis can modulate the MT rhythm, and thus the MT production and secretion pattern [START_REF] Mcclung | Circadian rhythms and mood regulation: insights from pre-clinical models[END_REF]. There is a reduction in MT secretion in the dark phase after stress exposure, which probably results from a reduction in tryptophan precursor for MT synthesis. However, nocturnal illumination did not further suppress MT production in stressed animals [START_REF] Persengiev | Circadian patterns of melatonin, corticosterone, and progesterone in male rats subjected to chronic stress: effect of constant illumination[END_REF]. Corticosterone apparently alters 5-HT and MT synthesis by modulating the mRNA levels of TH (Clark and Russo, 1997).

Although corticosterone reportedly inhibited nuclear factor-B translocation, thereby enhancing the NE-induced synthesis of MT in the pineal gland [START_REF] Ferreira | Corticosterone modulates noradrenaline-induced melatonin synthesis through inhibition of nuclear factor kappa B[END_REF], chronic stress indirectly impaired sympathetic inputs to the pineal gland, leading to disruption of MT rhythm (Dagnino-Subiabre et al., 2006). It is therefore possible that derangement of the melatonergic system in stressed rats may stem from inappropriate MT production and its irregular rhythm. Also, MT levels are decreased in depressed patients (Claustrat et al., 1984) receptors (Vanecek et al., 1987). Although the tissue distribution of MT1 can be detected by receptor binding, there is no specific mapping for MT2 and this technique does not allow the study of cellular receptor localization. In situ hybridization has proven not sensitive enough for the mapping and phenotyping of MT receptor expressing cells [START_REF] Poirel | MT1 melatonin receptor mRNA expression exhibits a circadian variation in the rat suprachiasmatic nuclei[END_REF].

In addition, MT receptor protein localization using the immunocytochemistry in rodents is controversial because reliable antibodies with high sensitivity are not available [START_REF] Fujieda | Expression of mt1 melatonin receptor in rat retina: evidence for multiple cell targets for melatonin[END_REF]. Therefore, a genetically modified rodent model has been generated for mapping and phenotyping MT receptor expression. Tissues characterized functional MT1 and/or MT2 melatonin receptors, e.g., retina, SCN, pars tuberalis, cerebral, arteries, kidney, pancreas, adrenal cortex, testes and immune cells [START_REF] Dubocovich | Functional MT1 and MT2 melatonin receptors in mammals[END_REF]. However, MT receptor localization is not clearly and controversial finding.

Brain-derived neurotrophic factor (BDNF)

Besides affecting neurotransmitter levels underlying the regulation of neurogenesis such as GABA, 5-HT, DA, and NE, cannabinoids, opioids and nitric oxide [START_REF] Balu | Adult hippocampal neurogenesis: regulation, functional implications, and contribution to disease pathology[END_REF], stress also decreases the expression of growth factors, such as BDNF, insulin-like growth factor-1 (IGF-1), nerve growth factor (NGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) that also influence neurogenesis [START_REF] Schmidt | The role of neurotrophic factors in adult hippocampal neurogenesis, antidepressant treatments and animal models of depressivelike behavior[END_REF].

Sarawut Lapmanee

Literature Review / 18

BDNF is a member of the nerve growth factor family and is expressed in the hippocampus and the cortex where it modulates neuronal plasticity, inhibits cell death cascades and increases the cell survival proteins that are responsible for the proliferation and maintenance of central nervous system neurons [START_REF] Duman | Role of neurotrophic factors in the etiology and treatment of mood disorders[END_REF][START_REF] Murakami | Chronic stress, as well as acute stress, reduces BDNF mRNA expression in the rat hippocampus but less robustly[END_REF]. BDNF mRNA was transcribed and regulated by 8 various promoters containing untranslated exons (I to VIII). Especially, promoter IV is mediated by calcium and regulatory components, leading to protein translation [START_REF] Zheng | NMDA-mediated and self-induced bdnf exon IV transcriptions are differentially regulated in cultured cortical neurons[END_REF]. The function of BDNF is mediated by its binding to specific receptors, i.e., tropomycin receptor kinase B (TrkB) receptors and pan75 neurotrophin receptor (p75NTR). TrkB signaling promotes neurogenesis while p75NTR activation mediates proteolytic mechanisms in survival and apoptosis inducing mechanisms [START_REF] Lee | The roles of BDNF in the pathophysiology of major depression and in antidepressant treatment[END_REF]. BDNF is involved in the pathophysiology of mood disorders and plays an important role in the mechanism of action of antidepressant drugs [START_REF] Duman | A neurotrophic model for stress-related mood disorders[END_REF]. Clinical studies have found a decreased BDNF level in the blood of depressed patients [START_REF] Aydemir | The effect of chronic antidepressant treatment of serum brain-derived neurotrophic factor levels in depressed patients: a preliminary study[END_REF].

Taken together, these proteins involved in neuronal remodeling of the noradrenergic and serotoninergic systems and which were among the primary targets of the effects of stress, pharmacological treatment, and voluntary wheel running were brain-derived neurotrophic factor (BDNF), glucocorticoid receptor (GR), norepinephrine transporter (NET), serotonin transporter (SERT), and 5-HT type 2C receptor (5-HT 2C R). [START_REF] Kawamoto | Immunohistochemical localization of brain-derived neurotrophic factor in adult rat brain[END_REF][START_REF] Yan | Expression of brain-derived neurotrophic factor protein in the adult rat central nervous system[END_REF][START_REF] Zhou | Distribution and localization of pro-brain-derived neurotrophic factor-like immunoreactivity in the peripheral and central nervous system of the adult rat[END_REF][START_REF] Avwenagha | Patterns of expression of brain-derived neurotrophic factor and tyrosine kinase B mRNAs and distribution and ultrastructural localization of their proteins in the visual pathway of the adult rat[END_REF] [START_REF] Charney | The neurobiological basis of anxiety disorders[END_REF].

Dysregulation of 5-HT release and reuptake or decreased responsiveness to the 5-HT signal lead to anxiety and mood disorders [START_REF] Graeff | Role of 5-HT in stress, anxiety, and depression[END_REF]. It was reported that stress caused a reduction of 5-HT and/or 5-hydroxyindoleacetic acid (5-HIAA)metabolite of 5-HT in the brain of rats exposed to electrical foot shock (Malyzko et al., 1994). Maternal deprivation and uncontrollable stress induced the reductions in the 5-HT 1A autoreceptor and 5-HT 2C R in the hippocampus and the basolateral amygdala of rats (Christianson et al., 2010, Maniam and[START_REF] Maniam | Voluntary exercise and palatable high-fat diet both improve behavioural profile and stress responses in male rats exposed to early life stress: role of hippocampus[END_REF].

Apart from the serotoninergic system, the central noradrenergic system has been implicated in both anxiety and regulation of the HPA axis [START_REF] Sullivan | The noradrenergic system in pathological anxiety: a focus on panic with relevance to generalized anxiety and phobias[END_REF]. After being activated by stress, this system modulates the activity of forebrain regions involved in behavioral and neuroendocrine responses to stress, such as the BNST that receives dense noradrenergic innervations. Tanaka and co-workers (2000) demonstrated that immobilization stress led to a marked increase in NE release in several brain regions. In addition, stressed rats exhibited increased anxiety in the elevated plus-maze (EPM) and a high dendritic arborization in the basolateral amygdala neurons [START_REF] Mitra | Stress duration modulates the spatiotemporal patterns of spine formation in the basolateral amygdala[END_REF]. They also displayed aggressive behaviors and an increase learned fear in elevated T-maze (ETM) [START_REF] Wood | Chronic immobilization stress alters aspects of emotionality and associative learning in the rat[END_REF][START_REF] Lapmanee | Anxiolytic-like actions of reboxetine, venlafaxine and endurance swimming in stressed male rats[END_REF][START_REF] Herman | Neural control of chronic stress adaptation[END_REF].

Stress induced-depression

Depression is a common feeling that disturbs daily life if present for long periods. The risk factors for depression include personal or family history, physical illness and stress exposure. Besides the genetic background, dysfunction of HPA axis can also cause depression. Maladaptive responses to stress and GC hypersecretion can induce extreme emotional imbalance, mood dysfunction and cognitive impairments in depressed patients. PFC and hippocampus mediate memory impairments and feelings of worthlessness, helplessness, guilt and suicidal thoughts. The volume of these brain structures was reduced in severe depression [START_REF] Cerqueira | The stressed prefrontal cortex. Left? Right![END_REF][START_REF] Kempton | Structural neuroimaging studies in major depressive disorder: metaanalysis and comparison with bipolar disorder[END_REF]. In addition, the striatum and amygdala control the emotional memory and could be the cause of anhedonia. The CRH expressing neurons and the amount of CRH-mRNA in the PVN are increased and disrupted CRH signaling by CRH1 receptors promotes stress-related depressive conditions [START_REF] Raadsheer | Corticotropin-releasing hormone mRNA levels in the paraventricular nucleus of patients with Alzheimer's diseaseand depression[END_REF].

Concerning the monoamine deficiency hypothesis, many studies suggested that there is a decrease in 5-HT, as indicated by its metabolite (5-HIAA) in the cerebrospinal fluid of depressed suicide victims [START_REF] Fisher | Contributors to depression in Latino and European-American patients with type 2 diabetes[END_REF]. Moreover, SERT mRNA levels were upregulated in PFC, hippocampus, amygdala, and PGA of ovariectomized rats subjected to chronic mild stress [START_REF] Charoenphandhu | Upregulated mRNA levels of SERT, NET, MAOB, and BDNF in various brain regions of ovariectomized rats exposed to chronic aversive stimuli[END_REF].

Alteration of the noradrenergic system in depression is associated with a synaptic deficiency of NE that is consistent with up-regulation of TH and down-regulation of the NET observed in postmortem studies [START_REF] Klimek | Reduced levels of norepinephrine transporters in the locus coeruleus in major depression[END_REF]. Therefore, monoamine modulators, such as monoamine oxidase inhibitor (MAOI), SSRIs, norepinephrine reuptake inhibitors (NRIs) and selective norepinephrine reuptake inhibitors (SNRIs), are widely used to alleviate the symptoms by elevating the levels of monoamines in the synaptic cleft. This suggests that availability of 5-HT and NE plays an important role in the pathogenesis of depression.

Stress induced-memory impairment

Memory is ability to recall or recognize that can be divided into declarative and non-declarative memory. Declarative memory processes consciously recollect events and facts, whereas non-declarative memory processes implicitly through performance and skill without consciousness [START_REF] Squire | Declarative and nondeclarative memory: multiple brain systems supporting learning and memory[END_REF]. The hippocampus consolidates information while the amygdala plays a role in emotional declarative memory. The basal ganglia and cerebellum are associated with nondeclarative memory, such as procedural or habit learning and motor skill learning [START_REF] Foerde | Feedback timing modulates brain systems for learning in humans[END_REF]. Declarative memory is associated with the storage of consciously learned facts, and includes both general knowledge (e.g., figures, word meanings, object uses, scientific facts) and episodic knowledge in the specific events.

The hippocampus, amygdala, entorhinal cortex, and areas of the cerebral cortex participate in declarative (explicit) memory. Non-declarative memory also includes new skills through repeated trials and learning [START_REF] Searleman | Memory from a broader perspective[END_REF].

Learning refers to the encoding of information. For example, learning new words is defined as one type of non-declarative memory. The formation and storage of longterm memory are derived from transient changes in neuronal efficiency followed by long-lasting functional and morphological modifications in the hippocampus, related cortical regions and different nuclei of the amygdala [START_REF] Izquierdo | Different molecular cascades in different sites of the brain control memory consolidation[END_REF]. The cellular and molecular cascade of memory involves glutamate release from presynaptic neurons and activation of the postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and the N-Methyl-Daspartic acid (NMDA)

receptors. These events are associated with increased protein levels of BDNF and activation of TrkB which is coupled to mitogen-activated protein kinases (MAPK/ERK/Ark), which in turn regulate the phosphorylation of the CREB [START_REF] Yamada | Brain-derived neurotrophic factor/TrkB signaling in memory processes[END_REF]Nabeshima, 2003, Izquierdo et al., 2008).

A study on physiological stress responses demonstrated that GCs potentially act on the hippocampus to exert their effect on memory (Sadni, 1996).

High levels of GCs from stress exposure affect LTP processes, facilitate long-term depression (LTD) and also induce cognitive impairment [START_REF] Sapolsky | The neuroendocrinology of stress and aging: the glucocorticoid cascade hypothesis[END_REF].

Studies in rats with hippocampal damage showed impairment in learning the location of the hidden platform in the radial arm water maze, whereas chronic restraint stress caused object-recognition memory defects. Similarly, the infusion of NMDA receptor antagonists, which block hippocampal LTP, impaired object-recognition memory in rats [START_REF] Beck | Food deprivation modulates chronic stress effects on object recognition in male rats: Role of monoamines and amino acids[END_REF]Dimond et al., 1999). Memory impairment may also occur in the onset and progression of Alzheimer's disease [START_REF] Huang | Elevated basal cortisol level predicts lower hippocampal volume and cognitive decline in Alzheimer's disease[END_REF].

Pharmacological treatment of mood and memory disorders

Current treatments involve pharmacological approaches and psychological interventions. However, pharmacological treatments are largely alleviative and may potentially offer superior benefits in the management of patients with mood and memory disorders [START_REF] Garner | Research in anxiety disorders: from the bench to the bedside[END_REF][START_REF] Li | Review of pharmacological treatment in mood disorders and future directions for drug development[END_REF].

GABA receptor agonist

Benzodiazepines (BDZs) are the most widely used anxiolytic drugs. The target for BDZs actions is GABA neurotransmission, an inhibitory neurotransmitter in central nervous system). BDZs, such as flurazepam, temazepam, triazolam, alprazolam, clonazepam and diazepam are usually considered as a second line treatment because of their side effects, risk of dependence and withdrawal problems.

BDZs are frequently used for the treatment of anxiety associated with panic disorder and generalized anxiety disorder. Since anxiety co-exists with depression, combinations of BDZs and antidepressant drugs are more effective than antidepressants alone. The side effects of BDZs are related to their sedating action, i.e., cognitive impairment, impaired coordination of muscle movement, and muscle relaxation.

Antidepressants

MAOIs and tricyclic antidepressants (TCAs) were the effective first-line antidepressants in clinical use during the early period of psychotropic drug discovery [START_REF] Youdim | Monoamine oxidase: isoforms and inhibitors in Parkinson's disease and depressive illness[END_REF]. Recently SSRIs, SNRIs, and other antidepressants, such as mirtazapine and nefazodone, have improved side-effect profiles but are no more effective than MAOIs and TCAs. All of these drugs share similar mechanisms of enhancing or modulating 5-HT and NE neurotransmission in order to alleviate the symptoms.

Monoamine oxidase inhibitors (MAOI)

These drugs inhibit the MAO, which destroys excessive monoamine within the axon terminals. MAOs are a group of enzymes that metabolize and subsequently inactivate monoamine and indolamine neurotransmitters, i.e., DA, NE, and 5-HT [START_REF] Amsterdam | Monoamine oxidase inhibitors revisited[END_REF]. The precursor amine, similar to catecholamines, stimulates the sympathetic nervous system. Thus a depressed patient who is treated with MAOI may develop a serious sympathetic reaction after eating food containing the precursor amine. MAOIs, such as phenelzine, isocarboxazid, and tranylcypromine show effectiveness in anxiety disorders, e.g., panic disorder and social anxiety disorder in cases in which no success was found with BDZs. Phenelzine prevents degradation and accumulation of neurotransmitters. Hypotension, dizziness, drowsiness, insomnia, nausea, weight gain, edema, muscle soreness, paresthesias, sexual dysfunction and abnormal sensation such as tingling, tickling, pricking, numbness have been reported as the side effects of MAOIs [START_REF] Evans | Early and late side-effects of phenelzine[END_REF][START_REF] Fiedorowicz | The role of monoamine oxidase inhibitors in current psychiatric practice[END_REF].

Tricyclic antidepressants (TCA)

TCAs are antidepressant drugs that increase the availability of 5-HT and NE in the post-synaptic cleft by inhibiting reuptake of these neurotransmitters. TCAs mediate nonselective interaction between 5-HT and NE neurotransmitter systems, anticholinergic action, direct alpha-adrenergic blockade and membrane-stabilizing effects on the myocardium resulting from over-dosage [START_REF] Biggs | Tricyclic antidepressant overdose: incidence of symptoms[END_REF]. TCAs such as imipramine also have beneficial effects in depression, panic attack, generalized anxiety disorder and obsessive-compulsive disorder.

Selective antidepressants

Selective reuptake inhibitor-antidepressants promote availability of monoamines in the synaptic cleft by inhibiting the specific reuptake transporters. SSRIs and SNRIs cause adverse effects resulting from drug-induced spillover of 5-HT and NE. SNRIs block central  2 -AR, hetero-, 5-HT 2 and 5-HT 3 receptors. NE/DA reuptake inhibitors (NDRIs) are effective in bipolar depression.

However, these antidepressant drugs are effective only after several weeks of continued administration. But SSRIs can cause gastrointestinal discomfort, weakness and sleep disturbances [START_REF] Isbister | Relative toxicity of selective serotonin reuptake inhibitors (SSRIs) in overdose[END_REF].

2.3.2 NRIs such as atomoxetine, daledalin, edivoxetine, mazindol, and viloxazine have a positive effect on concentration and motivation. Reboxetine, in particular, is efficacious in the acute treatment of manic disorder and panic disorder [START_REF] Eyding | Reboxetine for acute treatment of major depression: systematic review and meta-analysis of published and unpublished placebo and selective serotonin reuptake inhibitor controlled trials[END_REF].

2.3.3 SNRIs selectively inhibit the reuptake of both 5-HT and NE. SNRIs are used in the treatment of depressions, and anxiety disorders (i.e., obsessive-compulsive disorder), neuropathic pain, menopausal-related symptoms, and are also used as an appetite suppressant for body weight control [START_REF] Spina | Clinically relevant pharmacokinetic drug interactions with second-generation antidepressants: an update[END_REF]. SNRIs in general use are venlafaxine, sibutramine, duloxetine, desvenlafaxine, milnacipran, and levomilnacipran.

Interestingly, venlafaxine inhibits 5-HT reuptake at a low dose. And at high dose, it inhibits both, 5-HT and NE reuptake. Venlafaxine was the first to be introduced in this class and has been estimated to be more effective than the SSRIs [START_REF] Bauer | A pooled analysis of two randomised, placebo-controlled studies of extended release quetiapine fumarate adjunctive to antidepressant therapy in patients with major depressive disorder[END_REF]. Venlafaxine and duloxetine are effective in panic disorder, generalized anxiety disorder, and social phobia. Their adverse effects include nausea, dizziness, insomnia, sedation and constipation. After oral administration, this drug is absorbed from the gut and undergoes hepatic first-pass metabolism. The peak plasma concentration is obtained within 2 h. The oral bioavailability of venlafaxine is 45%, and its biotransformation pathway is mediated by cytochrome P450 (CYP) 2D6.

The active metabolite is o-desmethylvenlafaxine. The elimination half-life of venlafaxine is at least 4 h. Both venlafaxine and its metabolites have little affinity for muscarinic, cholinergic, H1-histaminergic or -adrenergic receptors (AR), and have no MAOA or B inhibitory activity. Venlafaxine is available in both immediate-release and extended release formulations, and its metabolites are effective in depression, panic disorder, generalized anxiety disorder, and social anxiety disorder. Its adverse effects include nausea, dizziness, insomnia, sedation and constipation [START_REF] Dell'osso | Serotonin norepinephrine reuptake inhibitors (SNRIs) in anxiety disorders: a comprehensive review of their clinical efficacy[END_REF]. Therefore, venlafaxine has a greater potency in the treatment of anxiety-like behavior in stressed male rats than fluoxetine or reboxetine [START_REF] Lapmanee | Anxiolytic-like actions of reboxetine, venlafaxine and endurance swimming in stressed male rats[END_REF].

However, further investigation is required to demonstrate whether venlafaxine could protect and directly modulate neurotransmission in the stress circuit of male rats.

Melatonergic antidepressants

Agomelatine (N-[2-(7-methoxy-1-naphthyl)ethyl] acetamide)

is the first melatonergic antidepressant in an alternative new pharmacological approach for depression with no side effects. Even though the role of the different melatonin (MT) receptors in the treatment of depression is still unknown, it is suggested that the antidepressant action of agomelatine is caused by its agonistic action at MT receptors and antagonistic action at 5-HT 2C Rs. It shows a high affinity for the cloned human MT1 and MT2 subtypes (Ki = 6.15×10-11 mol/L and 2.68×10-10 mol/L, respectively) and displaces iodomelatonin from its binding sites in the central SCN and peripheral organs, the biological clock [START_REF] Bonnefond | Melatonin and the circadian clock in mink: effects of daily injections of melatonin on circadian rhythm of locomotor activity and autoradiographic localization of melatonin binding sites[END_REF]. This drug is more than 80% absorbed after oral administration. The peak plasma concentration is obtained within 1-2 hours. It is metabolized by the CYP1A2, CYP2C9, and CYP2C19 isoenzymes. The elimination is rapid as the mean plasma half-life is 1-2 hours and clearance is about 1,100 mL/min with excretion of 80% in urine. Previous studies reported that agomelatine had antidepressant-like effects in the learned helplessness test, chronic mild stress and in a transgenic mouse model of depression, as well as anxiety. Agomelatine-treated rats showed reduced duration of immobility in the forced swimming test (FST) and tail suspension [START_REF] Bourin | Antidepressant-like activity of S20098 (agomelatine) in the forced swimming test in rodents: involvement of melatonin and serotonin receptors[END_REF], Païzanis et al., 2006). Agomelatine also increased the capacity of rats to wait for a delayed but larger reward in ETM and EPM [START_REF] Loiseau | Antidepressant-like effects of agomelatine, melatonin and the NK1 receptor antagonist GR205171 in impulsive-related behaviour in rats[END_REF][START_REF] Morley-Fletcher | Chronic agomelatine treatment corrects behavioral, cellular, and biochemical abnormalities induced by prenatal stress in rats[END_REF]. Moreover, treatment with agomelatine reversed stress affected dentate gyrus neurogenesis and proliferating progenitor cells deficits [START_REF] Dagytè | Agomelatine reverses the decrease in hippocampal cell survival induced by chronic mild stress[END_REF][START_REF] Morley-Fletcher | Chronic agomelatine treatment corrects behavioral, cellular, and biochemical abnormalities induced by prenatal stress in rats[END_REF]. Taken together, these data show that agomelatine has beneficial effects on hippocampal neurogenesis in stress. However, it is still not clearly known whether agomelatine can prevent stress-induced changes in behavior and monoamine secretion.

Anti-manic and mood-stabilizing agents

Lithium is an anti-manic drug which freely penetrates into neurons and interacts with multiple intracellular molecules, e.g., inositol phosphatases and glycogen synthase kinase-3 associated with the neurotrophic effects [START_REF] Berridge | Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands[END_REF][START_REF] Klein | A molecular mechanism for the effect of lithium on development[END_REF].

Other drugs

Buspirone, the action of which appears to be mediated by the serotonin receptor 5HT 1A , is particularly useful in the treatment of generalized anxiety disorders and elderly patients. Buspirone is also used to augment antidepressants in the treatment of depression [START_REF] Howland | Buspirone: Back to the Future[END_REF]. Its side effects include dizziness, nausea, headache, but not sedation or loss of coordination.

Beneficial effects of exercise on stress-induced mood and memory disorders

It is generally accepted that physical activity and exercise have positive effects on mental health reducing the incidence and severity of psychiatric disorders such as anxiety and depression. These interventions could relieve symptoms and are as effective as meditation or psychotherapy.

Physical exercise is cost-efficient and also provides protection against harmful consequences of stress in healthy people and patients [START_REF] Salmon | Effects of physical exercise on anxiety, depression, and sensitivity to stress: a unifying theory[END_REF]. Lightto-moderate intensity of aerobic exercise produces antidepressant-and anxiety-like action [START_REF] De Moor | Regular exercise, anxiety, depression and personality: a population-based study[END_REF]. In human studies, aerobic exercise training shows a comparable effect when compared to antidepressant drugs or placebo in middle-age and older adults with major depression disorder [START_REF] Blumenthal | Exercise and pharmacotherapy in the treatment of major depressive disorder[END_REF]. Carta and co-workers (2008) showed that long term-exercise provided better physical quality of life in depressed patients who received antidepressant and/or physical exercise as evaluated by self-report survey questionnaire. In addition, both high and low exercise intensities could reduce anxiety sensitivity as indicated by an anxiety sensitivity index.

On the other hand, exercise may paradoxically induce anxiety in some groups of patients, e.g., inducing acute panic attacks or increasing the anxiety levels in patients with panic disorder [START_REF] Ströhle | Physical activity, exercise, depression and anxiety disorders[END_REF]. The aforementioned human studies indicate the strong effects of aerobic exercise on the emotional states, such as anxiety, depression and mood disorders. Although, the mechanism responsible for the exercise-induced anxiolytic-like effect is not completely understood, the improvement could result from the released endorphins and some monoamines. Leasure and Jones (2008) reported that the increased emotional state (as indicated by anxiety-like behaviors, defecation and increased circulating levels of corticosterone)

were found in forced exercising rats as compared to those subjected to wheel running.

Indeed, most exercise models, such as treadmill and swimming may be considered as forced exercise. Swimming might induce additional stress on animals since the swimming tank represents an inescapable environment [START_REF] Charoenphandhu | Long-term swimming in an inescapable stressful environment attenuates the stimulatory effect of endurance swimming on duodenal calcium absorption in rats[END_REF].

Therefore, voluntary exercise is better for the study of behavioral and neuroendocrine changes.

The effects of voluntary wheel running on brain NE and its metabolites in male rats have been previously investigated. Two major metabolites of NE, 3,4dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG), as indicators of neuronal NE release [START_REF] Scheinin | Plasma 3,4dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG) are insensitive indicators of alpha 2-adrenoceptor mediated regulation of norepinephrine release in healthy human volunteers[END_REF]. For example, during running, there was an increase in NE in the cell bodies of the pons, the LC, the DR and the spinal cord MHPG, whereas DHPG levels were unaltered in spinal cord, pons medulla, hippocampus, and PFC, which was correlated with increased freezing in contextual fear conditioning [START_REF] Dunn | Brain norepinephrine and metabolites after treadmill training and wheel running in rats[END_REF][START_REF] Dishman | Brain monoamines, exercise, and behavioral stress: animal models[END_REF]. The enhancement of -1B adrenoceptor mRNA expression in DR depended on exercise duration, but not the distance ran [START_REF] Greenwood | Wheel running alters serotonin (5-HT) transporter, 5-HT1A, 5-HT1B, and alpha 1b-adrenergic receptor mRNA in the rat raphe nuclei[END_REF]. Moreover, previous studies also reported that wheel running exercise increased 5-HT release and metabolism. The effects of exercise on the 5-HT system appeared to be highly dependent on the receptor subtypes and brain regions investigated, and exercise effects may not be as immediate as in the NE system [START_REF] Garcia | Hippocampal lowfrequency stimulation and chronic mild stress similarly disrupt fear extinction memory in rats[END_REF]. The levels of 5-HIAA were reduced in the amygdala and CA1 only after uncontrollable stress. The ratio of 5-HIAA to 5-HT was also reduced in the PVN [START_REF] Dishman | Activity wheel running reduces escape latency and alters brain monoamine levels after footshock[END_REF]. SERT mRNA in the MR and DRN subregions was reduced but this effect was not correlated with the distance ran.

Furthermore, the increase in 5-HT 1A receptor in the MR and in DR was dependent on the duration of exercise [START_REF] Greenwood | Freewheel running prevents learned helplessness/behavioral depression: role of dorsal raphe serotonergic neurons[END_REF][START_REF] Greenwood | Wheel running alters serotonin (5-HT) transporter, 5-HT1A, 5-HT1B, and alpha 1b-adrenergic receptor mRNA in the rat raphe nuclei[END_REF]. Voluntary exercise reversed maternal deprivation induced reductions in 5-HT 1A mRNA in the hippocampus [START_REF] Maniam | Voluntary exercise and palatable high-fat diet both improve behavioural profile and stress responses in male rats exposed to early life stress: role of hippocampus[END_REF]. On other hand, 5-HT 1B receptor mRNA in the DR was decreased by exercise and this decrease was dependent on exercise length [START_REF] Greenwood | Wheel running alters serotonin (5-HT) transporter, 5-HT1A, 5-HT1B, and alpha 1b-adrenergic receptor mRNA in the rat raphe nuclei[END_REF]. As mentioned before, NE and 5-HT, the two classical central monoamine neurotransmitters, can regulate synaptic plasticity, enhance neuronal survival, promote neurogenesis and improve mental health. Therefore, NE and/or 5-HTcould be important in the exercise-induced protective effects on the brain, and may participate in the up-regulation of BDNF in the response to voluntary exercise.

The effects of exercise on depressive behavior and spatial performance in stressed rats were examined by determining the alterations of corticosterone, hippocampal BDNF mRNA expression and hippocampal GRs. Voluntary wheel running should reverse the adverse effects of variable stressors on both behavior and hippocampal BDNF mRNA level. Although, exercise alone increased corticosterone, exercise did not affect the hippocampal GR mRNA expression, whereas stress showed increased corticosterone and decreased hippocampal GR mRNA. These results suggested that exercise had beneficial effect on depressive behavior that might be associated with corticosterone and BDNF mRNA levels [START_REF] Zheng | Beneficial effects of exercise and its molecular mechanism on depression in rats[END_REF]. In hyperactive rats, there were higher extracellular NE levels and 2-ARs but lower MAOA activity. Wheel running activity reduced the level of depression (as indicated by immobility time in FST) by increasing the number of 2-ARs and elevating the extracellular NE via a reduction of MAOA [START_REF] Morishima | Monoamine oxidase A activity and norepinephrine level in hippocampus determine hyperwheel running in SPORTS rats[END_REF].

The anxiolytic effects of voluntary wheel running associated with adaptation of mental and physiological effects on brain and behavior in laboratory rodents and their implications to humans have been well studied [START_REF] Pietropaolo | The impact of voluntary exercise on mental health in rodents: a neuroplasticity perspective[END_REF]. After being trained with a running wheel for 4 weeks, mice spent more time in the open arm, had a higher percentage of open arm entries in the EPM test and increased open-field locomotion. Furthermore, chronic wheel running exercise resulted in antidepressant-like behavior, which was associated with increased BDNF levels [START_REF] Duman | Voluntary exercise produces antidepressant and anxiolytic behavioral effects in mice[END_REF]. van Praag and co-workers ( 2005) reported that voluntary wheel running enhanced the number of hippocampal DG cells and LTP, thus improving performance of mice in the Morris water maze (MWM). This result suggested a close relationship between neurogenesis and the hippocampal function involved in the behavioral performance. Voluntary exercise also led to a reduction in anxiety-like behaviors in mice, and the experimental outcome was consistent with

those measured with acoustic startle, stress-induced hyperthermia, social interaction and light-enhanced startle [START_REF] Salam | Voluntary exercise in C57 mice is anxiolytic across several measures of anxiety[END_REF].

Therefore, it is possible that voluntary exercise can be an alternative treatment for mood and memory disorders in humans or animals. However, little is known regarding the preventive effect of exercise on behavior and brain neurochemical changes in stressed individuals.

Behavioral testing of depression

Elevated plus-maze (EPM) test

The EPM test has become one of the most popular behavioral tests for investigations on anxiety [START_REF] Hogg | A review of the validity and variability of the elevated plus-maze as an animal model of anxiety[END_REF][START_REF] Belzung | Measuring normal and pathological anxiety-like behaviour in mice: a review[END_REF]. The EPM test is based on the conflict between an innate fear to exposed spaces and the elevation of the maze. This test is typically used for the investigation of anxiolytic drugs. The the open arm activities [START_REF] Pinheiro | Elevated mazes as animal models of anxiety: effects of serotonergic agents[END_REF].

Elevated T-maze (ETM) test

The ETM test is developed to discriminate between conditioned and unconditioned fear in animals. It is derived from the elevated X-or plus-maze by closing the entrance to one of the enclosed arms [START_REF] Graeff | The elevated T maze, a new experimental model of anxiety and memory: effect of diazepam[END_REF][START_REF] Charney | Monoamine dysfunction and the pathophysiology and treatment of depression[END_REF] that TCAs (e.g., imipramine), SSRIs (e.g., sibutramine, fenfluramine, and paroxetine), 5-HT 1A R partial agonist (e.g., buspirone, and isapirone), 5-HT 2A/B/C R antagonist (e.g., SR46349B, SER082, and SB200646A) impaired inhibitory avoidance and one-way escape latencies [START_REF] Pinheiro | Elevated mazes as animal models of anxiety: effects of serotonergic agents[END_REF].

Open field test (OFT)

The OFT test is used to determine locomotion, exploration, and anxiety by exposure to an open area [START_REF] Prut | The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review[END_REF]. The number of crossed lines and the frequency of rearing (i.e. lifting the forepaws from the floor) are used as measures of locomotor activity. A high frequency of these behaviors indicates increased locomotion, while the duration of time spent in the inner zone are a measure of exploration and anxiety. A high frequency or duration of these behaviors indicates high exploratory behavior and low anxiety levels. Behavioral and pharmacological validation of the OFT has reported that BDZs but not SSRIs or 5-HT 1A R partial agonist enhanced time spent in the inner zone [START_REF] Prut | The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review[END_REF]. Rearing and grooming behaviors are a displacement response and are expected to be displayed in a novel environment [START_REF] Thanos | A noninvasive method for detecting the metabolic stress response in rodents: References / 192 Characterization and disruption of the circadian corticosterone rhythm[END_REF].

Experimental models of depression

Preclinical studies in experimental animals have been an important part of the development of antidepressants. Equipments used to assess the behavior of rodents include swimming tanks, drinking tubes and an open field apparatus. Forced swimming test and the sucrose preference test are used to evaluate helplessness, anhedonia, behavioral despair and other neurovegetative changes such as changes in sleep and appetite patterns. The use of animal models for behavioral tests can reveal depression-like behavioral profiles.

Forced swimming test (FST)

The FST is high the predictive validity models of depression that is most commonly used to evaluate antidepressant-like action. The FST described by Porsolt and co-workers (1979), aimed to measure the effects of antidepressant compounds in mice. This procedure involves placing an animal in a water-filled cylinder which it is unable to exit. At the beginning, the animal will try to escape, and then it adopts a posture of immobility defined by a lack of movements except for keeping the body afloat in the water. The test for rats consists of 2 swimming sessions.

The first session lasts 15 minutes and the second is performed 24 h after the first session, with a test period of 5 minutes. The FST is used for the selection of new antidepressant drugs. Various antidepressants reduce immobility time by increasing the swimming and/or climbing time. Antidepressant drugs affecting noradrenergic neurotransmission such as imipramine and reboxetine increase the climbing behavior, whereas antidepressant drugs affecting serotonergic neurotransmission such as fluoxetine, sertraline, paroxetine and citalopram increase swimming time [START_REF] Castagné | Rodent models of depression: forced swim and tail suspension behavioral despair tests in rats and mice[END_REF][START_REF] Lapmanee | Beneficial effects of fluoxetine, reboxetine, venlafaxine, and voluntary running exercise in stressed male rats with anxiety-and depression-like behaviors[END_REF]. The swimming time is measured by the horizontal and vertical movements as animals try to scale the cylinder walls with their paws. The effects of antidepressants on FST behavior are relatively specific, since they do not increase spontaneous motor activity, unlike stimulants such as amphetamine and cocaine [START_REF] Kang | Effects of reboxetine and citalopram pretreatment on changes in cocaine and amphetamine regulated transcript (CART) expression in rat brain induced by the forced swimming test[END_REF]. Besides the effects of antidepressant drugs, the FST can also be used to evaluate the type of depressive behavior; for example, it has been demonstrated that animals subjected to a protocol of maternal deprivation and chronic restrain stress exhibit increased immobility time in the FST [START_REF] Reus | Maternal deprivation induces depressive-like behaviour and alters neurotrophin levels in the rat brain[END_REF][START_REF] Lapmanee | Beneficial effects of fluoxetine, reboxetine, venlafaxine, and voluntary running exercise in stressed male rats with anxiety-and depression-like behaviors[END_REF].

Sucrose preference test

The sucrose preference test is used for the assessment of hedonic sensitivity whereby animals learn to associate an environment with a rewarding experience. Measurement of consumption of sweetened fluids (sucrose or saccharin) is commonly conducted for assessing stress-induced anhedonia in depression models. Rats habituated to sucrose are typically given a choice of drinks between sucrose and water in a two-bottle test. While control rats typically show a preference for drinking weak sucrose solutions, rats exposed to chronic stress such as chronic mild stress lose this preference. The effects of an antidepressant treatment could reverse this symptom in a time dependent manner. However, the factors affecting sucrose consumption or preference include sucrose concentration, temperature, test duration and body weight. Anhedonic measures after chronic mild stress are sensitive to strain effects for both rats and mice [START_REF] Willner | Decreased hedonic responsiveness following chronic mild stress is not secondary to loss of body weight[END_REF][START_REF] Pothion | Strain differences in sucrose preference and in the consequences of unpredictable chronic mild stress[END_REF]. In addition, Strekalova and co-workers (2011) have reported that a four week chronic stress paradigm resulted in a decrease in the preference for sucrose solutions by ≤ 65% in 50-70% of mice and rats without any changes at the beginning of the stress paradigm.

Experimental models of learning and memory

Animal models have been widely used to study the physiology and the cellular and molecular mechanisms of learning and memory and human cognition.

Spatial memory involves processes such as encoding, retaining and retrieving information in the hippocampus, whereas non-spatial memory (implicit) relies on the cerebellum and the amygdala.

Morris water maze (MWM) test

The MWM test is one of the most commonly used methods in assessing hippocampal-dependent spatial memory in rodents [START_REF] Morris | Spatial localization does not require the presence of local cues[END_REF][START_REF] Morris | Developments of a water-maze procedure for studying spatial-learning in the rat[END_REF]. Spatial learning is assessed across repeated trials and reference memory is determined by preference for the platform area when the platform is absent. The test includes a pool of water that is made opaque, and surrounded by a wall with different patterns at various directions. The principle of the test is that rodents can learn to swim towards a hidden escape platform from any starting position. They do this using distal extra-maze cues that are remote from the actual place in the pool to which the animal is heading. Therefore, the room containing the tank should have permanently positioned distinctive objects such as posters placed outside of the pool and/or on the walls. Animals are expected to find the hidden platform under the water using these visual cues. After several trials, the platform is removed, and experimenter further measures the time spent by animals in the corrected quadrant to evaluate their spatial memories [START_REF] Paul | Spatial memory: theoretical basis and comparative review on experimental methods in rodents[END_REF].

Novel objective recognition (NOR) test

The NOR test has a large field of application not only to test the pharmacological effects of a drug, but also to characterize which brain regions are involved in memory and learning, as well as to understand Alzheimer's disease (e.g., [START_REF] Redrobe | Translational Aspects of the Novel Object Recognition Task in Rats Abstinent Following Sub-Chronic Treatment with Phencyclidine (PCP): Effects of Modafinil and Relevance to Cognitive Deficits in Schizophrenia[END_REF]2012;[START_REF] Antunes | The novel object recognition memory: neurobiology, test procedure, and its modifications[END_REF].

CHAPTER III

MATERIALS AND METHODS

Part I: Effects of restraint stress induction, monoamine modulators (i.e., agomelatine and venlafaxine), and voluntary wheel running in male rats.

Animals

Eight-week-old Wistar male rats, weighing 180-220 g, were obtained from the National Animal Center of Thailand, Mahidol University. Rats were individually housed in standard stainless steel shoe box at 25 ± 2 °C in a humidity-controlled room at 55 ± 5% humidity with a 12-h (06:0018:00) light/dark cycle (average illuminance of 200 lux). Rats were acclimatized for 1 week with food (CP Co., Ltd., Thailand) and water available ad libitum before the start of the experiments. Body weight (BW) and food intake were measured daily. All procedures were approved by the Animal Care and Use Committee of the Faculty of Medicine, Thammasat University, Thailand.

Experimental protocols

Experiment 1. To examine the time-dependent effect of the 1-, 4-and 8week stress induction on anxiety-, depression-, and memory impairment-like behaviors and changes in the expression of selected target proteins in the brain regions associated with stress responses.

Rats were subdivided into 2 groups as follows.

1. Non-stress control group (16 rats/group) 2. Stressed group were divided into 3 subgroups -1-week restraint stress induction (16 rats/group) -4-week restraint stress induction (16 rats/group) -8-week restraint stress induction (16 rats/group)

In the 1-and 4-week stressed groups, there was a preceding stress-free period of 7 and 4 weeks (5-min of gentle handling to reduce stress), respectively, in order to commence the behavioral tests at the same age.

At the end of the restraint stress induction, rats were evaluated for behavioral changes. The anxiety-like behavior was evaluated by elevated plus-maze

Experimental group Definition Control

Rats were subjected to regular handling for 8 weeks.

1-week stress

Rats were subjected to regular handling for 7 weeks and exposed to restraint stress for 1 week.

4-week stress

Rats were subjected to regular handling for 4 weeks and exposed to restraint stress for 4 weeks.

8-week stress

Rats were exposed to restraint stress for 8 weeks.

Experiment 2. To determine the effect of the 4 weeks of voluntary wheel running on the hypothalamic-pituitary-adrenal (HPA) axis under non-stress condition or after the 4-week exposure to restraint stress and changes in the expression of selected target proteins in brain regions associated with stress responses.

Rats were subdivided into 2 groups as follows. Table 3.2 Descriptions of animal groups in the experiment 2.

Experimental group Definition Sedentary control

Rats were kept undisturbed and subjected to regular handling for 4 weeks.

Exercise

Rats were subjected to voluntary wheel running for 4 weeks.

Stress control

Rats were exposed to restraint stress for 4 weeks.

Stress + Exercise

Rats were exposed to restraint stress and subjected to voluntary wheel running for 4 weeks.

Experiment 3. To evaluate the effectiveness of agomelatine, venlafaxine, and voluntary wheel running exercise in the prevention of stress-induced anxiety-, depression-, and memory impairment-like behaviors and changes in the expression of selected target proteins in brain regions associated with stress responses.

Rats were subdivided into 2 groups as follows.

1 Table 3.3 Descriptions of animal groups in the experiment 3.

Experimental group Definition Sedentary

Rats were administered a 4-week vehicle treatment before exposure to restraint stress for 4 weeks.

Monoamine modulators

Rats were administered a 4-week monoamine modulator treatment (i.e., agomelatine and venlafaxine) before exposure to restraint stress for 4 weeks.

Voluntary exercise

Rats were administrated a 4-week combined vehicle treatment and voluntary wheel running before exposure to restraint stress for 4 weeks.

Stress Stress

Experimental procedures 1. Restraint stress induction

The stress protocol was followed from the methods of [START_REF] Lapmanee | Beneficial effects of fluoxetine, reboxetine, venlafaxine, and voluntary running exercise in stressed male rats with anxiety-and depression-like behaviors[END_REF]. Briefly, stressed rats were immobilized 2 h/day (8:0010:00 a.m.) in a quiet room, 5 days/week for 1-, 4-, or 8-weeks. Restraint technique involved placing the rat in a transparent plastic cylinder (24 cm long × 6 cm wide) with a 1-cm hole at the end of the cylinder for breathing. Rat was fixed with plastic tape. The restraint procedure was performed after 4-weeks of pharmacological treatments or voluntary wheel running.

Monoamine modulator administration

Drugs were daily administrated orally by gavage between 15:00 p.m. and 16:00 p.m. every day for 4 weeks. The dosage was based on those reported previously [START_REF] Bourin | Antidepressant-like activity of S20098 (agomelatine) in the forced swimming test in rodents: involvement of melatonin and serotonin receptors[END_REF][START_REF] Lapmanee | Anxiolytic-like actions of reboxetine, venlafaxine and endurance swimming in stressed male rats[END_REF]. Drugs used were agomelatine (10 mg/kgBw, Les Laboratoires Servier Industrie, GidyServier, Paris, France) and venlafaxine (10 mg/kgBW, venlafaxine hydrochloride; Pfizer Ireland pharmaceuticals, Newbridge, Co. Kildare, Ireland). Sterile saline (5 mL/kg/day) was used as vehicle.

Voluntary wheel running

Mild to moderate intensity aerobic voluntary exercise provides beneficial effects on both physical and psychological health. The voluntary exercise protocol was adapted from the methods of [START_REF] Droste | Voluntary exercise impacts on the rat hypothalamic-pituitary-adrenocortical axis mainly at the adrenal level[END_REF][START_REF] Lapmanee | Beneficial effects of fluoxetine, reboxetine, venlafaxine, and voluntary running exercise in stressed male rats with anxiety-and depression-like behaviors[END_REF]. Rats were individually housed in polycarbonate living chamber (50 cm long  40 cm wide  20 cm high) with a running wheel system. The running distance was calculated by the number of turns multiplied by the circumference (144 cm) of the running wheel (model 86060; Lafayette Instrument Company, Lafayette, IN, USA). The number of turns was automatically recorded by an electronic counter. In preparation for wheel running, rats were housed in cages with a running wheel for 1 week to familiarize them with the running wheel. Exercise on the running wheel was voluntary (7 days/week for 4 weeks). Rats not completely familiarized with the running wheel were excluded from the study. As for the sedentary control groups, rats remained in their cages without a wheel for the duration of the experiment.

Behavioral tests

In all experiments, rats were subjected to a weekly sucrose preference test and evaluated for anxiety-, depression-, and memory impairment-like behaviors at the end of the restraint stress induction (8:00-12:00 a.m.). Each rat was brought to the behavioral testing room with average illuminance of 20 lux, and left undisturbed in a quiet environment for at least 5 min. Behavioral profiles were recorded by an infrared video camera (model HDR-XR200E; Sony, Tokyo, Japan). The behavioral test apparatus and the surrounding area were cleaned after each test with a wet towel and 70% ethanol to eliminate odorants, feces and urine. Each rat was subjected to one test at a time without the presence of other rats in the room. The EPM, ETM, OFT, NOR, MWM, and FST tests were performed, respectively as described in the following section. All measured parameters and interpretation are noted in Table 3.4. [START_REF] Hogg | A review of the validity and variability of the elevated plus-maze as an animal model of anxiety[END_REF][START_REF] Rodgers | Animal models of anxiety: an ethological perspective[END_REF][START_REF] Pandaranandaka | Anxiolytic property of estrogen related to the changes of the monoamine levels in various brain regions of ovariectomized rats[END_REF].

Elevated T-maze test (ETM)

The ETM was made of black painted wood and consisted of three arms of equal dimensions (50 cm long × 10 cm wide) and was elevated 50 cm above the floor. One arm, enclosed by walls (40 cm high) was perpendicular to the two opposed open arms. These three arms were connected by a square (10 cm long ×10 cm wide). To prevent rats falling, the open arms were surrounded by a 1 cm high Plexiglas rim (Figure 3.5). The ETM test consisted of three inhibitory avoidance trials (i.e., baseline, avoidance 1, and 2) and one escape trial held at 30 s intervals. In the first three inhibitory avoidance trials, the rat was gently placed at the terminal end of the enclosed arm facing the center of the maze. The same measurement was repeated in two subsequent trials. One escape trial was performed by placing rat at the end of the right open arm facing the center of the maze and recording the latent time the rats took to exit this arm with all four paws. The ETM was fully cleaned with a wet towel after each test. Increased inhibitory avoidance latency represented learned or conditioned fear, whereas one-way escape latency represented innate or unconditioned fear [START_REF] Graeff | The elevated T-maze as an experimental model of anxiety[END_REF][START_REF] Pandaranandaka | Differential effects of exogenous and endogenous estrogen on anxiety as measured by elevated T-maze in relation to the serotonergic system[END_REF].

Open-field test (OFT)

The OFT was used to evaluate both locomotor activity and anxiety-like behavior. The apparatus was made of black painted wood (76 cm long × 57 cm wide × 35 cm high) with a 48-square grid floor (6 × 8 squares, 9.5 cm per side).

The arena was divided into two zones, i.e., inner and outer zones (24 peripheral squares) as shown in Figure 3 Each rat was subjected to OFT after EPM and ETM tests. The rat was gently placed in one of the four corner squares facing the wall and allowed 5 min to explore the apparatus. The time spent in each zone, the number of lines crossed in the 1st 30-s and 5-min test, and numbers of rearing, grooming, and fecal pellets were recorded by an infrared video camera. The OFT was fully cleaned with a wet Sarawut Lapmanee Materials and Methods / 48 towel between tests. An increase in time spent in the inner zone or a decrease in time spent in the outer zone indicated anxiolysis or increased exploration. A change in the number of lines crossed represented change in locomotor activity [START_REF] Mccarthy | Infusions of diazepam and allopregnanolone into the midbrain central gray facilitate open-field behavior and sexual receptivity in female rats[END_REF][START_REF] Treit | Thigmotaxis as a test for anxiolytic activity in rats[END_REF][START_REF] Pandaranandaka | Differential effects of exogenous and endogenous estrogen on anxiety as measured by elevated T-maze in relation to the serotonergic system[END_REF].

Novel object recognition test (NOR)

NOR was performed in a black rectangular plastic box (63 cm long × 63 cm wide × 45 cm high) in 360 lux room light. The procedure was modified from the method of [START_REF] Redrobe | Translational Aspects of the Novel Object Recognition Task in Rats Abstinent Following Sub-Chronic Treatment with Phencyclidine (PCP): Effects of Modafinil and Relevance to Cognitive Deficits in Schizophrenia[END_REF]2012. A video camera was installed on a movable trolley above the box to record behaviors. The objects to be discriminated were made of glass or ceramic. On the day before the NOR test, each rat was allowed to habituate the empty box (2 sessions, 10 min/session). During the test, each rat was gently placed in the box and exposed for 3 min to an acquisition session with identical objects (two ceramic pepper bottles; 3 cm long × 3 cm wide × 7 cm high) placed, approximately 10 cm apart at the center of the box. Rats were then transferred to their home cages for a 1 h inter-trial interval. Meanwhile, the box and objects were cleaned.

One object in the box was replaced with a novel object (glass paperweight; 5 cm long × 5 cm wide × 12 cm high). The same rat was then returned to the box and allowed to explore the new object for 3 min. Object-exploring behaviors included sniffing, licking, or touching each object. Rats that did not reach a total exploration time of 15 s, after exploring both objects for a minimum of 2 s, were excluded from the data analysis. The discrimination ratio was calculated from the following equation: A decrease in discrimination ratio indicated cognitive and memory impairment in stressed rats [START_REF] Redrobe | Translational Aspects of the Novel Object Recognition Task in Rats Abstinent Following Sub-Chronic Treatment with Phencyclidine (PCP): Effects of Modafinil and Relevance to Cognitive Deficits in Schizophrenia[END_REF][START_REF] Redrobe | Negative modulation of GABAA α5 receptors by RO4938581 attenuates discrete sub-chronic and early postnatal phencyclidine (PCP)-induced cognitive deficits in rats[END_REF][START_REF] Antunes | The novel object recognition memory: neurobiology, test procedure, and its modifications[END_REF]).

Morris water maze (MWM)

Learning and memory were evaluated by using the MWM, which was modified from the methods of [START_REF] Morris | Spatial localization does not require the presence of local cues[END_REF] Prior to learning trials, a rat was individually placed on the platform for 10 s to familiarize it to the task. When the rat moved away from the platform before the end of 10 s, it was placed back onto the platform. This orientation procedure was repeated 3 times, and the learning trials were performed immediately thereafter. Each rat was placed in different location (North, South, East, or West) near the edge of the pool with its front paws first touching the wall. Then, it was allowed to find the hidden platform, and time spent for locating the platform (escape latency) was recorded. The hidden platform was placed at a fixed location, i.e., in the Southeast quadrant. When the rat was unable to locate the platform within 60 s, it was placed on the platform and an escape latency of 60 s was recorded. Finally, the rat was given 10 s to remain on the platform to familiarize itself with the surrounding visual cues. Rats performed 8 trials on day 1 and 2, and 4 trials on day 3 (total 20 trials). There was a 5min inter-trial interval, during which each rat was kept warm in a cage under a heating lamp. Spatial memory was evaluated by a probe test 1 h after the last learning trial on day 3. The rat was individually placed in the North quadrant in the absence of a platform. Each rat was allowed to swim for 60 s, and time spent in each quadrant was recorded by a video camera. Percentage time spent in the correct quadrant was calculated as follows.

(

( ) )
 100

An increase in escape latency indicated poor spatial learning, whereas an increase in correct quadrant time (i.e., time to reach the correct quadrant) or a decrease in percentage time spent in the correct quadrant indicated spatial memory impairment. To eliminate confounding factors (e.g., urine, fecal pellets, stress odors, and pheromones), the pool was drained and refilled with fresh tap water before the training of each group of rats. Finally, all learning trials and memory probes were recorded by an experimenter unaware of the grouping of the animals.

Forced swimming test (FST)

All rats were individually forced to swim in a cylindrical container (25 cm diameter and 45 cm high) filled with 25 C tap water up to 35 cm. In the first session, rats were placed in water for a 15-min assessment. Then, they were removed from water, dried and cleaned with towel before returning to their cages.

Twenty-four hours later, the second session was performed in which rats were put back into the swimming tank for 5 min. Duration of immobility behavior (floating in water with only movement necessary to keep the head above water), swimming behavior (active movement of the forepaws with goal-directed horizontal actions, such as crossing between quadrants of the cylinder and turning), climbing (upward goaldirected movements of the forepaws along the side of the cylindrical container), and number of fecal pellets were recorded. An increase in immobility duration or decreases in swimming duration and climbing duration indicated depression-like behaviors in rats [START_REF] Porsolt | Immobility induced by forced swimming in rats: effects of agents which modify central catecholamine and serotonin activity[END_REF].

Sucrose preference test

The sucrose preference test was modified from the methods of [START_REF] Charoenphandhu | Upregulated mRNA levels of SERT, NET, MAOB, and BDNF in various brain regions of ovariectomized rats exposed to chronic aversive stimuli[END_REF] and performed once weekly throughout the experimental periods. To confirm that sucrose neophobia was not a confounding factor in this behavioral test [START_REF] Strekalova | Measuring behavior in mice with chronic stress depression paradigm[END_REF], rats were given free access to the sucrose preference test prior to the start of the experiment to make sure that baseline sucrose intake did not differ among groups. For the determination of sucrose preference, rats were deprived of water and food for 4 h (08:00-12:00 h). Then rats were presented for 1 h with two bottles, one containing water and the other containing 2% sucrose with the density of 1.0098 g/cm 3 (Ajax Finechem, NSW, Australia). The volumes of liquid were measured by weighing pre-weighed bottles before and after testing. To prevent possible effects of side-preference in drinking behavior, the position of the bottle in the cage was switched weekly. The amount of water and sucrose intake was measured. The relative sucrose intake per body weight (BW) (g/kgBW) was the absolute sucrose intake per gram of rat body weight, whereas sucrose preference was calculated according to the following ratio:

( ) ( ) ( )  100
Decreased sucrose preference represents anhedonia-like behavior in rodents [START_REF] Willner | Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a tricyclic antidepressant[END_REF][START_REF] Papp | An animal model of anhedonia: attenuation of sucrose consumption and place preference conditioning by chronic unpredictable mild stress[END_REF].

Measurement of body, organ weights, and food intake

Body weights (BW), food intake and water intake were measured daily.

Pre-weighed food or water was provided and 24 h later the amount of remaining food was recorded. The daily mean weight gain (DWG) and the daily mean food intake (DFI) were calculated, as follow: DWG = final BW -initial BW/28 (4weeks) or 56 (8 weeks) DFI = summation of food intake/28 (4weeks) or 56 (8 weeks)

At the end of the experiment, the heart and adrenal glands were removed, washed with ice-cold normal saline solution, and blotted dry with filter paper. The wet weights of the heart (experiment 2) and adrenal glands (experiment 1-3) were recorded. Thereafter, the tissues were dried in an incubator at 80 °C for 3 days to obtain dry weights. The heart and adrenal weights were used to indicate the effectiveness of exercise protocol and stress induction, respectively. The heart-to-body weight ratio (% Heart/BW) and percentage of adrenal gland (% AG/BW) were calculated, as follow:

% AG/BW = adrenal gland (g) /BW (g) × 100 % Heart/BW = heart (g) /BW (g) × 100

Euthanasia

At the end of the experiment, rats were humanely euthanized with an overdose of inhaled isoflurane (Minrad Inc., NY, USA).

Analyses of serum and urinary corticosterone levels

Serum supernatant was collected from blood samples after refrigeration for 24 h at 4°C and centrifugation (1,500×g for 10 min at room temperature). Urine samples were filtered and diluted 1:5 with water then stored -80°C until corticosterone analysis. Corticosterone levels (ng/mL) were indicators of stress response. Serum and urinary corticosterone levels were determined by a competitive enzyme immunoassay (EIA) commercial kit (catalog no. AC-14F1; Immunodiagnostic Systems Ltd, Tyne and Wear, UK), according to the manufacturer's instruction, with a detection limit of 0.23 ng/mL with inter-and intra-assay coefficients of variation of <8 and <4 %, respectively [START_REF] Lertsinthai | Voluntary wheel running mitigates the stress-induced bone loss in ovariectomized rats[END_REF].

Brain dissection

Brain was rapidly removed after decapitation, frozen in liquid nitrogen, and stored at -80 C. On the analysis day, the brain regions associated with stress responses (i.e., frontal cortex, septum, amygdala, hippocampus, periaqueductal gray, dorsal raphe, locus coeruleus, and ventral tegmental area) were microscopically dissected according to the methods of [START_REF] Heffner | A rapid method for the regional dissection of the rat brain[END_REF] and the atlas of Paxinos and

Watson, 2005 (Figure 3.83.9). The isolation procedure was done on an ice-cold plate.

Razor blades were carefully inserted through the cutting channels slicing the brain at right angles to the sagittal axis. The initial razor blade sliced through the coronal plane of the brain at the level of the anterior commissure. The position of the initial razor blade served as a reference point other brain sections. A total of eight razor blades were inserted anteriorly or posteriorly to the first blade and thus divided the brains into 8 sections. The razor blades were removed from the block leaving coronal brain slices adhere to their surfaces. Brain sections were placed on a glass plate on ice. Brain regions were then bilaterally dissected from these slices. Besides the frontal cortex, hippocampus, and amygdala, two independent samples from relatively tiny brain areas (i.e., septum, periaqueductal gray, dorsal raphe, locus coeruleus, and ventral tegmental) were pooled together before protein extraction [START_REF] Charoenphandhu | Upregulated mRNA levels of SERT, NET, MAOB, and BDNF in various brain regions of ovariectomized rats exposed to chronic aversive stimuli[END_REF].

Western blot analysis

The brain regions under study were separately lysed and homogenized with a handheld pestle and mortar in the radioimmunoprecipitation assay (RIPA) buffer containing 150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris, pH 8.0 supplemented with protease inhibitors and a phosphatase inhibitor cocktail (sodium pyrophosphate and sodium orthovanadate) (Sigma, St. Louis, MO, USA). Therefore, membrane-bound proteins should thus be in the supernatant. Brain tissues were then sonicated at power 6 for 25-s pulses (60-s pause between pulses) at 4°C and centrifuged (13,000 rpm for 15 min) at SDS-polyacrylamide gel electrophoresis using vertical minigel system (Bio-Rad Laboratories, Inc., Hercules, USA). Each sample was separated in triplicate.

After electrophoresis, proteins were electrophoretically transferred to nitrocellulose membranes (Amersham Biosciences, Freiburg, Germany) in Trisglycine transfer buffer. Blotted membranes were then blocked with 5% non-fat powdered milk (Sigma) in Tris buffered saline with Tween 20 (TBST) for 2 h at room temperature. For identification of proteins, membranes were incubated overnight at 4 C with the primary antibodies diluted in 5% bovine serum albumin (BSA) (Sigma).

The primary antibodies were summarized in Table 3.5. Following the primary antibody incubation, membranes were washed for 10 min with TBST, 3 times and incubated with anti-mouse or rabbit secondary antibodies horseradish peroxidase (HRP) conjugates (1:2,000; Santa Cruz Biotechnology, CA, USA) for 2 h at room temperature. Membranes were washed 3 times for 10 min with TBST, then the immunoreactive protein bands were visualized using Luminata Classico Western HRP substrate (Millipore Corporation, Billerica, MA, USA). Membranes were exposed to film (Hyperfilm-ECL; Amersham Biosciences) long enough to visualize the chemiluminescent bands. Blots were reprobed with mouse monoclonal anti-β-actin (1:2,000; Santa Cruz) and normalized to verify equivalent protein loading. Comparisons were made with known molecular weight standards using prestained protein ladder (Thermo Scientific, Walthem, MA, USA). Blots were scanned and band density was quantified using ImageJ (Rasband, 19972016). The area of the curve in each gel band were quantified and normalized relative to the β-actin levels of control or vehicle-treated groups.

Statistical analysis

Animals

Adult male and female wild-type and transgenic mice with knocked-in β-

Experimental protocols

Experiment 1. To validate and map MT1/MT2-LacZ expressing neurons in transgenic knock-in mice.

Euthanasia

Mice were euthanized during the light phase (9.0012.00 a.m.) by CO 2 inhalation. Heparin (500 IU, Héparine Choay; Sanofi, Gentilly, France) was injected into the heart before transcardial perfusion first with PBS, and then with fixative solution (4% phosphate-buffered formaldehyde for LacZ histochemistry or periodatelysine-paraformaldehyde (PLP) fixative for immunocytochemistry and in situ hybridization).

Preparation of brain tissues

For LacZ histochemistry, the brains were removed from the skulls and post-fixed with 4% paraformaldehyde in 100 mM phosphate buffer, pH 7.4, cryoprotected in 30% sucrose solution at 4 C for at least 24 hours. After sinking in the 30% sucrose cryoprotectant, the brains were frozen in isopentane, chilled to 78°C

with mixed dry ice and 95% ethanol. The brains which were cut on a cryo-microtome (Microm HM 560, Thermo Scientific) into 40-m-thick slices. Free floating sections were collected in PBS with 0.1% sodium azide (PBS-azide) and processed for detection of LacZ activity by X-gal staining.

For immunohistochemistry and in situ hybridization, we used PLP fixative (4% formaldehyde, 10 mM NaIO 4 in 75 mM phosphate-lysine buffer, pH 7.4). After transcardial perfusion, the brains were removed from the skulls and post-fixed in PLP fixative for 10 hours. Then the brains were rinsed in 50% ethanol and dehydrated with ethanol (70% for 2 times 1 hour, 95% for 2 times 1 hour), ethoxyethanol for 2 times 1 hour and finally n-butanol overnight. Thereafter, brains were embedded in PEG as described by [START_REF] Klosen | PEG embedding for immunocytochemistry: application to the analysis of immunoreactivity loss during histological processing[END_REF]. PEG-embedded brains were cut by microtome (Leica, Germany) into 8-μm-thick coronal sections and mounted onto slides.

LacZ histochemistry

The expression of the LacZ reporter can be detected by enzyme histochemical staining using the chromogenic substrate, 5-bromo-4-chloro-3-indolylβ-d-galactoside (X-gal). The free-floating sections were washed for 3 times 15 min each in LacZ wash buffer (PBS, 2 mM MgCl 2 , 0.01% sodium deoxycholate, 0.02% nonidet-P40) at room temperature. The sections were stained overnight at 37 °C with an X-gal development solution (K 4 Fe(CN) 6 , K 3 Fe(CN) 6 , and X-gal in LacZ wash buffer). On the next day, the sections were washed for 3 times 5 min each in LacZ wash buffer and the floating sections were mounted on gelatinized slides from 0.5X PBS and 0.5% polyvinylacohol. Slides were dehydrated by 5 min incubations in 70%, 95%, and 100% (twice) ethanol and cleared in toluene (twice). Coverslips were mounted using Eukitt (Chem Lab) and allowed to dry. Blue LacZ expressing cells were examined on a Leica DMRB microscope (Leica Microsystems, Rueil-Malmaison, France) equipped with an Olympus DP50 digital camera (Olympus France, Rungis, France). Plasmid DNA was digested by restricted enzymes (e.g., BamH I, EcoR I, and Hind III)

Riboprobe synthesis for in situ hybridization

and its examined sequence by AGOWA Sequencing Service, Germany, in order to confirm the orientation and identity of the plasmid DNA fragments.

Preparation of probes

The transcribed messenger RNA (mRNAs) coded for LacZ, neuropeptides (i.e., AVP, CRH, VIP, and GRP), or enzymes GAD1/2 in neuronal cells that express the corresponding genes were detected. Brain sections were incubated with labeled cDNA probes that form hybrids with the cellular mRNA, and the location of labeled cells was then determined by hapten labeled probes with alkaline phosphatase detection. Neuropeptides AVP and VIP probes were the gifts from Assoc.

Prof. Paul Klosen, Team Melatonin and Seasonal Rhythms, Department of Neurobiology of Rhythms, Institute of Cellular and Integrative Neurosciences, University of Strasbourg, France. In the probe synthesis, mouse CRH, GRP, GAD1, GAD2, and LacZ cDNA primers (Sigma-Aldrich, France) underwent PCR amplification using the following oligonucleotides (Table 3.7). The plasmids were fully linearized with suitable restriction enzymes overnight and the restriction mixtures were phenol-chloroform extracted and ethanol-precipitated. Probe labeling and hybridization were performed using digoxigenin (Roche Biochemicals, Germany). DNA templated RNA transcription was carried out in a reaction mixture containing templates and RNA polymerase e.g., SP6

(2,00010,000 units), T7 (5,00025,000 units), or T3 (5,000 units). These promoters recognized the 5′ end of the reverse primer (Anti-sense RNA). The size of the probes was confirmed by formaldehyde-(N-2-morpholino) propane sulfonic acid agarose gel electrophoresis and Northern blotting, respectively. Non-hydrolyzed (full-length) probes were used. Specificity controls for single in situ hybridizations were performed using sense riboprobes transcribed from the same vectors.

Non-radioactive in situ hybridization (ISH)

ISH was performed as described by [START_REF] Klosen | The mt1 melatonin receptor and RORbeta receptor are co-localized in specific TSH-immunoreactive cells in the pars tuberalis of the rat pituitary[END_REF]. PEG brain sections were postfixed in 4% phosphate-buffered formaldehyde for 10 min at room temperature, washed in PBS, and then digested with 0.5 μg/ml proteinase K (Roche;

Meylan, France) in PBS for 30 min at 37 C. Proteinase K digestion was stopped incubating in 2% cold phosphate-buffered formaldehyde for 5 min. After the sections were washed for 3 times 5 min with PBS and 0.2% dimethylpyrocarbonate (DMPC), the sections were acetylated twice for 10 min each in 100 mM triethanolamine with 0.25% acetic anhydride. After they were rinsed for 5 min in PBS and 0.2% DMPC twice, the sections were rinsed in 5X saline sodium citrate (SSC), 0.05% Tween-20, and 0.02% DMPC before hybridization.

Both sense and antisense probes were denatured for 5 min at 90 C. The sections were then incubated with 200 ng/ml (1:100 dilution) labeled probes in hybridization buffer (50% formamide, 5X SSC, 5X Denhardt's solution, and 500 μg/ml salmon sperm DNA) and placed in the incubator at 62 C for 40 h. After hybridization, the sections were washed in 5X SSC for 10 min at room temperature.

Stringency rinses with 0.1X SSC were performed for 6 times 10 min at 72C followed by A-Dig buffer rinses for 5 min, twice. After 1 h incubation in blocking buffer, hapten-labeled bound probes in the sections were detected with alkaline phosphataselabeled sheep anti-digoxigenin antibodies (Roche). Alkaline phosphatase activity was visualized by bromo-chloro-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT).

Immunocytochemistry

For antigen retrieval, the slide-mounted brain sections were incubated in Tris-Citrate buffer, pH 8.0 for 1 h at 95C, followed by a tap water rinse. After blocking with 3% dry skimmed milk and 0.02% NaN 3 in Tris-buffered saline, 0.05%

Tween 20 (TBST) buffer for 2 h, brain sections were incubated with chicken anti-LacZ (1:5,000; ab9361, Abcam, UK) in antibody diluting buffer (TBS, 0.05% Tween 20, 0.02% NaN 3 , and 1% fetal calf serum) overnight at room temperature. After 3 times 10 min TBST rinses, the brain sections were incubated with the secondary antibody, biotinylated donkey anti-chicken (1:2,000; Jackson ImmunoResearch, PA, USA) in antibody dilution buffer. After a further 3 times 10 min TBST rinses, the slides were finally incubated with a streptavidin-peroxidase conjugate (Pierce) in diluting buffer (TBS, 0.05% Tween 20, and 0.2% cold water fish skin gelatin) for 1 h.

Peroxidase activity was detected using 0.5 mg/ml diaminobenzidine (DAB, Sigma) in 50 mM Tris buffer (pH 7.60) with 10 mM imidazole, and 0.003% H 2 O 2 for 15 min.

The slides were dehydrated and cover-slipped with Eukitt (Chem Lab, Zedelgem, Belgium). The peroxidase-immunostained sections were photographed with a Leica DMRB microscope (Leica Microsystems, Rueil-Malmaison, France) equipped with an Olympus DP50 digital camera (Olympus France, Rungis, France).

Combined ISH with ICC

The brain sections were first hybridized. After visualization of phosphatase activity with NBT/BCIP, the slides were washed with TBST for 3 times for 5 min and then selected for double labeling. These slides were rinsed in 95% ethanol for 5 min to elute background deposits and to change the color of the in situ labeling from dark purple to blue color. Brain sections were then immunolabeled using the standard protocol described above, but with fluorescein, green fluorescent tyramide substrate in PBS-Imidazole buffer instead of DAB. The absence of interactions between the ISH and the ICC was verified by omission of the primary antibody in the ICC detection.

Further tests included ICC on sections that were mock-hybridized to check for antigen inactivation or decloaking by the hybridization procedure. Slides were visualized under bright field or green fluorescence filters. Images were processed using Adobe Photoshop 6 (Adobe Systems, Mountain View, CA, USA). In the stressed group, the 1-and 4-week restraint stress induction could produce high anxiety rather than the 8-week stress induction as evaluated by EPM, ETM, and OFT tests.

Depression-like behavior

There were depression-like symptoms in the 4-week stress group, i.e. decreased swimming duration and increased immobility duration without change in climbing duration in the FST (Figure 4.6A-C). Stressed rats showed high emotionality and then presented anhedonia-like behavior during restraint stress induction. As compared to percent preference in the baseline period, the 1-week stressed rats had increased sucrose preference. Similarly, the 4-or 8-week stressed rats had increased sucrose preference on the 2 nd of the 4-week stress and the 3 rd of the 8week stress. In addition, 8-week stress had decreased sucrose preference at weeks 5, 6, and 8 when compared to non-stressed control rats (Figure 4.6D).

Learning and memory impairment-like behavior

In the MWM test, the 4-week stressed rats spent more time finding the platform in escape latency on day 2 of the test, and on day 3, only 1-week stressed rats showed lower time spent in escape latency. Meanwhile latencies of the 4and 8-week stressed rats were not different from non-stressed control rats (Figure

Restraint stress induced changes in target protein expression in

brain regions associated with stress responses.

Levels of BDNF

The 1-and 8-week restraint stress induction decreased BDNF protein levels in periaqueductal gray and ventral tegmentum area (Figure 4.8F and G).

Levels of GR in the hypothalamus and hippocampus

Hypothalamic GR protein was increased in the 1-week restraint stress induction, whereas hippocampal GR protein was decreased in the 4week stress induction (Figure 4.9). These results suggest stress responses of GR proteins, resulting from hyperactivity of the HPA axis. GR could act back on the hippocampus to exert GR-mediated negative feedback inhibition on the HPA axis

Levels of NET

The 4-week restraint stress induction increased NET proteins in the locus coeruleus when compared to non-stressed controls (Figure 4.10D).

Levels of SERT

The 1-and 8-week restraint induction decreased SERT protein in the ventral tegmental area and hippocampus, respectively (Figure 4.11B and F).

Levels of 5-HT 2C R

The 1-, 4-, and 8-week restraint stress induction increased 5-HT 2C R protein levels in the amygdala and locus coeruleus ( Experiment 2. To determine the effect of 4 weeks of voluntary wheel running on the hypothalamic-pituitary-adrenal (HPA) axis under non-stressed conditions, or after a 4-week exposure to restraint stress, and changes in the expression of selected target proteins in brain regions associated with stress responses.

The 4-week voluntary wheel running induced changes in body

weight, heart weight, adrenal gland weight, food intake, corticosterone levels, and running distance.

There was no difference in starting body weight (Figure 4.13A). The sedentary stressed rats decreased body weight when compared to the non-stressed sedentary control rats. Both non-stressed exercised rats and the combined stressed and exercised rats had decreased body weight when compared to the non-stressed sedentary rats and stressed sedentary rats, respectively (Figure 4.13B). While the nonstressed sedentary rats had gained weight, non-stressed exercised and sedentary stressed rats had less weight gain. The combined stressed and exercised rats also displayed less weight gain when compared to sedentary stressed rats (Figure 4.13C).

Sedentary stressed rats decreased daily food intake when compared to non-stressed control rats (Figure 4.13D). Therefore, the 4-week voluntary wheel running exercise could alter the energy balance including body weight and food intake.

In the exercise group, there was a similar running distance between nonstressed and stressed groups (

The 4-week voluntary wheel running induced the changes in behavioral responses

All groups of rats had increased sucrose preference as compared to the baseline period. There were no significant differences between groups as analyzed by one-way ANOVA. When compared to non-stress sedentary control by using unpaired t-test, stressed rats exhibited reduced sucrose preference in the 4 th week of stress induction. Interestingly, the combined stress and voluntary exercise could prevent stress-induced decrease in sucrose preference in stressed male rats (Figure 4.16).

The 4-week wheel running induced changes in target protein

expression in the brain regions associated with stress responses.

Levels of BDNF

Decreased hippocampal and septal BDNF protein expression in male rats was observed following the 4-week restraint stress induction (Figure 4.17B and H). The protein levels of BDNF in the nucleus accumbens were decreased in the exercise group. In the combined stressed and exercised rats BDNF protein was increased in the hippocampus and septum but decreased in the nucleus accumbens Expression of proteins related to the HPA axis in the stressed male rats showed decreased GR protein levels in the hippocampus but not in the

Levels of 5-HT 2C R

The 5-HT 2C R protein levels in the amygdala of stressed rats were higher than non-stress sedentary control rats ( Numbers of animals are noted in parentheses. † † P < 0.01, † † † P < 0.001 compared to vehicle (Veh)-treated group.

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Results / 100

Veh Ago Vlx Rw (16) ( 16) Numbers of animals are noted in parentheses. † † P < 0.01, † † † P < 0.001 compared to vehicle (Veh)-treated group. aaa P < 0.001 compared to percent sucrose preference after completing 4-week pre-treatment period.

F (3,

Learning and memory impairment-like behaviors

The MWM test showed that the agomelatine-, venlafaxine-, and exercise-treated stressed rats exhibited less escape latencies (on day 1, 2 and 3) and correct quadrant time than vehicle-treated stressed rats (Figure 4.28A-B), but percent correct quadrant was not different among these groups (Figure 4.28C). In the NOR, there was an improvement in the discrimination ratio in agomelatine-and venlafaxine-treated rats when compared to vehicle-treated stressed rats. However, the exercise-treated rats and vehicle-treated rats showed similar discrimination ratios (Figure 4.28D). These results suggest that pre-treatment of agomelatine, venlafaxine, and voluntary wheel running could improve learning and memory in stressed rats.

Levels of BDNF

Venlafaxine and voluntary wheel running resulted in higher levels of hippocampal BDNF as compared to vehicle-treated rats. In contrast, agomelatine or venlafaxine pre-treatment decreased BDNF protein levels in the locus coeruleus and the septum (Figure 4.29B, E, and H).

Levels of GR in hypothalamus and hippocampus

The hypothalamic GR protein expression levels did not change in any of the groups. The hippocampal GR protein levels were increased in venlafaxine-treated stressed rats (Figure 4.30B).

Levels of NET

When compared to vehicle-treated stressed rats, NET protein levels in the locus coeruleus were increased in exercise treated-stressed rats. However, NET protein levels were decreased in the hippocampus and the hippocampus and periaqueductal gray in agomelatine-and venlafaxine-treated rats (Figure 4.31B and E).

Levels of SERT

Pre-treatment of agomelatine and venlafaxine decreased the septal SERT protein levels when compared to vehicle-treated rats (Figure 4.32G).

Levels of 5-HT 2C R

No significant differences in the 5-HT 2C R protein expression were observed among these groups (Figure 4.33). However, there was a tendency towards a downregulation of the 5-HT 2C R in the amygdala of the exercise group. This result might suggest partially anxiolytic effects of voluntary exercise on stress-induced 5-HT 2C R upregulation in stressed rats. Experiment 1. To validate and map MT1/MT2-LacZ expressing neurons in transgenic knock-in mice.

Genotyping of MT1/MT2-LacZ knock-in mice

The knock-in strategy is shown in the Figure 4.34A. The LacZ reporter gene was inserted at the MT1 or MT2 locus in order to investigate the expression pattern of MT1/MT2 gene. For both MT1 and MT2 loci, the LacZ transgene was inserted starting at the original ATG start codon, thus retaining all upstream and downstream regulatory sequences. Only potential regulatory elements contained in the intron of either the MT1 or the MT2 gene have been deleted by this strategy.

Therefore, MT1/MT2-LacZ reporter knock-in mice were considered ideal tools to investigate the sites of expression of MT1/MT2 melatonin receptors in the absence of reliable specific antibodies for the immunocytochemical localization of these receptors.

We first selected heterozygous (MT1-or MT2-LacZ KI+ ) for the initial investigations. These heterozygous animals have one intact MT1 or MT2 allele, thus reducing the risk of loss of the expression of the MT1/2 locus, or ectopic expression of the MT1/2 locus, because of the absence of functional MT1/2 receptors. Heterozygous carriers were also interbred to produce homozygous (MT1-or MT2-LacZ KIKI ) offsprings that were identified by genotyping PCR (Figure 4.34B).

In the genotyping of MT1-LacZ knock-in mice, a product of ∼425 bp indicated the wild-type MT1 allele and a product of ∼196 bp indicated the recombinant MT1-LacZ allele. Similarly, in the genotyping of MT2-LacZ knock-in mice, a product of ∼143 bp indicated the wide-type allele and a product of ∼666 bp indicated the recombinant allele.

LacZ mapping of MT1 and MT2 gene expression in the brain

Detection of LacZ enzymatic activity and LacZ protein expressing neurons were examined by X-gal histochemistry and immunocytochemistry respectively.

Immunocytochemistry detected LacZ protein mostly in lysosomal accumulations as strongly stained dot-like structures, but also in the nucleus and sometimes in the cytoplasm (Figure 4.35A and C). There was also some diffuse background staining in immunocytochemistry that reduced the signal to noise ratio. X-gal histochemistry only stained the lysosomal accumulations of LacZ, and the absence of background resulted in a highly contrasted image (Figure 4.35B and D). Therefore, the staining of LacZ enzymatic activity was selected to map the patterns of MT1 and MT2 expression in these transgenic mice. Immunocytochemical detection of LacZ was used for the fine characterization of the MT1 and MT2 expressing cells by colabeling for phenotypic markers either by immunocytochemistry or by in situ hybridization.

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No LacZ reporter activity was observed in the wild-type control mice. In MT1-LacZ heterozygous and homozygous mice, LacZ enzymatic activity was mainly restricted to the suprachiasmatic nucleus (SCN), the paraventricular nucleus of the thalamus (PVT), the pars tuberalis (PT) of anterior pituitary, and the suprageniculate nucleus of the dorsal thalamus which were consistency with 125 I-iodomelatonin binding in in vitro autoradiography by Liu et al., 1997 (Figure 4.36). Other structures contained only isolated cells expressing the MT1-LacZ transgene (Figure 4.37).

The expression of the MT2-LacZ transgene in heterozygous and homozygous mice was much more wide-spread than that of the MT1-LacZ transgene. Large numbers of MT2-LacZ positive cells were seen in the olfactory bulb, the islands of Calleja, layer 3 of the cortical cortex, the superior colliculus, the amygdale and the arcuate nucleus of the hypothalamus. The MT2-LacZ was also expressed in the SCN, but much less intensely than the MT1-LacZ transgene.

Interestingly in the context of our study, MT2-LacZ knock-in mice had a strong staining in the paraventricular nucleus of the hypothalamus (PVN), the cornu amonis 2 (CA2) of the hippocampus and in a population of small horizontal cells of the molecular layer of the dentate gyrus. In addition, there were low expression levels of both MT1 and MT2 in the cerebellum (Figure 4.37).

The pattern of expression of the MT1-LacZ and MT2-LacZ transgenes was confirmed by immunocytochemistry for the LacZ protein. The results of immunocytochemistry were similar to X-gal histochemistry. These results confirmed that LacZ expression in the mutant mice was faithful to the endogenous MT1 or MT2 protein expression (Figure 4.38-4.39). Experiment 2. To characterize the phenotype of MT1/MT2-LacZ expressing neurons in transgenic knock-in mice by determination of co-expressed neuropeptides and neuronal markers.

Co-localization of MT1-LacZ expressing neurons

Both X-gal histochemical and immunocytochemical staining in the previous experiment showed expression of the MT1-LacZ transgene in the SCN.

Generally, neurons in the SCN subregions are distinguished by their neuropeptide contents. The SCN contains neurons containing AVP, GRP, and VIP in specific subregions. Therefore, these neuropeptides were selected for identifying MT1 expression in the SCN. Combined staining showed that MT1-LacZ expressing neurons co-localized with some, but not all, GRP and VIP neurons in the SCN, but did not colocalize with AVP neurons (Figure 4.40).

Co-localization of MT2-LacZ expressing neurons

MT2-LacZ expression was much more widespread than that of MT1-LacZ.

We did not attempt to identify the phenotype of all MT2-LacZ expressing cells, but concentrated on those structures that are involved in the modulation of stress responses, in this case the PVN and the hippocampus.

The PVN contains CRH neurons, which are an important component of the HPA axis. CRH probes were cloned and applied on brain sections. MT2-LacZ expressing neurons co-localized with CRH in the PVN (Figure 4.41). Furthermore, MT2-LacZ expressing neurons co-localized with CRH in the layer III of cerebral cortex. Therefore, the co-localization of MT1 and CRH suggests that melatonin and agomelatine might directly modulate the HPA axis in the PVN, which may have implications for stress conditions such as depression.

Hippocampal interneurons express both the GAD1 and the GAD2 isoforms which synthesize GABA. Both isoforms are co-localized in 95% of GABA cells in hippocampus. Therefore, GAD 1 and 2 probes were cloned and double staining was performed with in situ hybridization and LacZ immunocytochemistry.

CHAPTER V DISCUSSION

Validation of restraint stress induction

Because restraint stress induces both physical and behavioral changes in animals, and it is often used to model mental illness in human [START_REF] Keim | Plasma corticosterone and brain catecholamines in stress: Effect of psychotropic drugs[END_REF][START_REF] Buynitsky | Restraint stress in biobehavioral research: Recent developments[END_REF]. The sustained circulating level of corticosterone induces a negative feedback that diminishes the release of CRH in the hypothalamus and ACTH from the pituitary gland. Restraint stress induction increases heart rate and blood pressure in rats [START_REF] Irvine | The influence of restraint on blood pressure in the rat[END_REF][START_REF] Mcdougall | Differential cardiovascular responses to stressors in hypertensive and normotensive rats[END_REF]. Stress-induced hyperactivation of HPA axis leads to adrenal hyperplasia and elevated serum and urine corticosterone levels [START_REF] Ulrich-Lai | Chronic stress induces adrenal hyperplasia and hypertrophy in a subregion-specific manner[END_REF].

In the present study, 4-and 8-week chronic restraint stress (2 h/day, 5 days/week) induced an increase in the adrenal gland weight in male rats. However, the absence of increased adrenal gland weight in the 1-week restraint stressed rats suggested that the 1-week duration was too short to produce long-lasting effects.

Elevated corticosterone levels have been shown to cause a catabolic state and muscle wasting via lipolysis and glycogenolysis [START_REF] Hasselgren | Glucocorticoids and muscle catabolism[END_REF][START_REF] Adam | Stress, eating and the reward system[END_REF]. Corticosterone was reported to reduce appetite and body weight by downregulating hypothalamic neuropeptide Y/agouti gene-related protein and amphetamine-regulated transcript peptide mRNA expression levels resulting in a reduction in food intake and body weight gain [START_REF] Liu | Glucocorticoids decrease body weight and food intake and inhibit appetite regulatory peptide expression in the hypothalamus of rats[END_REF]. In addition, the intensity and duration of restraint stress on the body weight in stressed rats were associated with decreased levels of central monoamines and opioid peptides [START_REF] Ely | Effect of restraint stress on feeding behavior of rats[END_REF]. Therefore, a decrease in food intake and an increase in adrenal gland weight could be used as appropriate indicators of physical change in stress induction.

Behavioral response to different durations of restrain induction

It has been shown that acute stress exposure led to anxiety in human and animal studies. Intense fear is present in posttraumatic stress disorders [START_REF] Garner | Research in anxiety disorders: from the bench to the bedside[END_REF]. However, behavioral responses in male rats were found to depend on the intensity of the stressors [START_REF] Mercier | Behavioural changes after an acute stress: stressor and test types influences[END_REF]. Apart from the acute stress reaction, chronic stress-induced increase in corticosterone levels disturbed the balance of monoamine neurotransmitters and neuronal function in several brain regions, thus contributing to the development of mood disorders and memory problems [START_REF] Jezova | Endocrine factors in stress and psychiatric disorders: focus on anxiety and salivary steroids[END_REF]Christoffel et al., 2011).

Herein, the study of different durations of restraint stress induction that stressed rats exhibited anxiety-, depression-, and memory impairment-like behaviors.

Stressed rats had high anxiety as indicated by decreased open arm activity in the EPM test and decreased time spent in the inner zone of open arena with hyperarousal in the first 30s and stress-related behaviors (i.e., rearing and grooming) in the OFT test. The 1-and 4-week stressed rats further showed learned (conditioned) fear as indicated by increased avoidance latency, but not innate fear in one-way escape latency of the ETM test, suggesting that the subchronic and chronic restraint stress could produce a generalized anxiety disorder-like condition in rats without panic which was similar in human [START_REF] Treit | Thigmotaxis as a test for anxiolytic activity in rats[END_REF][START_REF] Wood | Chronic immobilization stress alters aspects of emotionality and associative learning in the rat[END_REF][START_REF] Lapmanee | Anxiolytic-like actions of reboxetine, venlafaxine and endurance swimming in stressed male rats[END_REF][START_REF] Herman | Neural control of chronic stress adaptation[END_REF].

Besides the anxiety-like behavior, the 4-and 8-week stressed rats showed strong emotionality and depression-like behavior as indicated by increased immobility time in the FST and altered sucrose preference. Reduction of sucrose preference in stressed rats indicated a decreased sensitivity to reward and may be homologous to anhedonia [START_REF] Willner | Decreased hedonic responsiveness following chronic mild stress is not secondary to loss of body weight[END_REF]. These results were consistent with previous reports [START_REF] Lapmanee | Anxiolytic-like actions of reboxetine, venlafaxine and endurance swimming in stressed male rats[END_REF][START_REF] Herman | Neural control of chronic stress adaptation[END_REF]. These changes are related to the hopelessness and anhedonic-like symptoms (a decreased ability to experience pleasures) in major depression [START_REF] Strekalova | Stress-induced anhedonia in mice is associated with deficits in forced swimming and exploration[END_REF]Cryan and Holmes, 2005). In addition, chronic stress in rats and mice has been shown to display several physiological changes that are also observed in patients with depression such as abnormal sleep cycle, reduced sexual function, and a disturbed circadian clock [START_REF] Strekalova | Stress-induced anhedonia in mice is associated with deficits in forced swimming and exploration[END_REF] Changes in learning and memory were more complicated to apprehend.

As shown in the MWM test, an increase in escape latency (an indicator of impaired spatial learning) was observed in the 4-week stressed rats (day 2), but the spatial learning was apparently improved in the 1-week stressed rats (day 3). These results

suggest that the transitional period of stress promotes memory formation, but the memory does not last long. This was consistent with a previous finding that acute exposure to mild-to-moderate stress led to better learning in radial arm maze test [START_REF] Luine | Restraint stress reversibly enhances spatial memory performance[END_REF]. These results imply that the first role of stress is to improve the response to a stressful situation. The spatial memory or ability to retrieve previous consolidated information, as indicated by an increase in the time spent to reach the correct quadrant, was found to be impaired in the 1-and 4-week stressed rats.

Furthermore, the NOR test showed that the 4-week stressed rats had poor ability to discriminate a previously explored object from a new object, a sign of cognitive or memory impairment [START_REF] Redrobe | Translational Aspects of the Novel Object Recognition Task in Rats Abstinent Following Sub-Chronic Treatment with Phencyclidine (PCP): Effects of Modafinil and Relevance to Cognitive Deficits in Schizophrenia[END_REF][START_REF] Redrobe | Negative modulation of GABAA α5 receptors by RO4938581 attenuates discrete sub-chronic and early postnatal phencyclidine (PCP)-induced cognitive deficits in rats[END_REF][START_REF] Antunes | The novel object recognition memory: neurobiology, test procedure, and its modifications[END_REF]. Such stress on a short timescale is supposed to be "beneficial" because it improves the responsiveness to stressors, thus increasing the chance of the organism's survival in dangerous situations. But this "reprogramming" of the physiological phenomena by stress does not last long. That is why long term stress becomes detrimental.

However, there was no alteration of motor skills or locomotor activities following chronic stress induction in the present study as suggested by the absence of change in total arm entries in the EPM, total lines crossed in OFT, or climbing duration in FST. Regarding approximately 80% change in the correct quadrant in all studied group, this means that both control and stressed rats spent a similar time in the correct quadrant (i.e., quadrant with a platform). Nevertheless, stressed rats required more time swimming around the correct quadrant before reaching the platform, as indicated by increased in the correct quadrant time. In other words, stressed rats remembered where the correct quadrant was, but required additional time to actually reach the platform.

Interestingly, it was not clear why some behavioral responses observed in the 1-or 4-week stressed groups were absent in the 8-week stressed group. However, it might be explained by habituation to prolonged restraining [START_REF] Stamp | Corticosterone modulates autonomic responses and adaptation of central immediate-early gene expression to repeated restraint stress[END_REF]. The process of habituation involves the reduction of HPA axis activity and deleterious actions of prolonged glucocorticoid (GC) secretion [START_REF] De Kloet | Brain corticosteroid receptor balance in health and disease[END_REF][START_REF] Herman | Neural control of chronic stress adaptation[END_REF]. The relationship between GC levels and behavioral adaptation has been reported as an "inverted-U" shaped curve, wherein an optimal level of GC signaling is required to produce the organismal response [START_REF] Herman | Neural control of chronic stress adaptation[END_REF]. GCs affect behavioral profiles that cause changes in learning task. For example, mice switch from spatial learning to stimulus-response learning by stress or corticosterone administration [START_REF] Schwabe | Corticosteroids operate as a switch between memory systems[END_REF].

Differential target protein expression in the brain regions associated with stress responses

The underlying mechanisms of the restraint stress-nduced led to aberrant behaviors and memory impairment are not completely understood. In the present study, the expressions of several target proteins, i.e., BDNF, GR, NET, SERT, 5-HT 2C R which are related to anxiety, depression, memory and/or stress, were investigated.

BDNF is an important regulator of the formation and plasticity of neuronal connectivity. BDNF is produced as a precursor (proBDNF), which is subsequently cleaved to become mature (m)BDNF. The proBDNF binds to p75NT receptors, whereas mBDNF binds to TrkB receptors. proBDNF-p75NT promotes hippocampal long-term depression (LTD) and apoptosis while mBDNF-TrkB promotes LTP and cell proliferation [START_REF] Chao | Neurotrophins: To cleave or not to cleave[END_REF][START_REF] Ibáñez | Jekyll-Hyde neurotrophins: The story of proNGF[END_REF].

Restraint stress provides contrasting effects on the expression of BDNF mRNA and protein levels. Both intermittent restraint and chronic cold stress upregulated hippocampal BDNF protein levels (Adlard et al., 2004). Downregulation of BDNF expression was observed in depressed patients and a chronic mild stress model of depression in rodents [START_REF] Gonul | Effect of treatment on serum brain-derived neurotrophic factor levels in depressed patients[END_REF][START_REF] Molteni | Chronic Mild Stress Modulates Activity-Dependent Transcription of BDNF in Rat Hippocampal Slices[END_REF]. The present study found a downregulation of BDNF in the periaqueductal gray of the 1-week stressed rats and in the ventral tegmental area (VTA) of the 8-week stressed rats. Four-week restraint stress also downregulated BDNF in the hippocampus and the septum. It was reported that stress-induced inflammatory cytokine production downregulated BDNF in the hippocampus and the prefrontal cortex, but upregulated BDNF in the nucleus accumbens (NA) leading to depression-like behavior in stressed male rats [START_REF] Zhang | Antidepressant effects of TrkB ligands on depression-like behavior and dendritic changes in mice after inflammation[END_REF]. BDNF expression in the VTA-NA pathway plays a crucial role in resilience to social defeat stress [START_REF] Krishnan | Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions[END_REF]. Antidepressant treatment for at least 4 weeks can restore the decreased BDNF levels to normal levels in stressed rats [START_REF] Lee | The roles of BDNF in the pathophysiology of major depression and in antidepressant treatment[END_REF].

Prolonged high GC levels also down-regulate GR expression in the hippocampus and the prefrontal cortex controlling negative feedback [START_REF] Chiba | Chronic restraint stress causes anxiety-and depression-like behaviors, downregulates glucocorticoid receptor expression, and attenuates glutamate release induced by brain-derived neurotrophic factor in the prefrontal cortex[END_REF] leading to an impaired ability to regulate GC homeostasis. GR expression is another indicator of stress response [START_REF] Boyle | Acquired deficit of forebrain glucocorticoid receptor produces depressionlike changes in adrenal axis regulation and behavior[END_REF][START_REF] De Kloet | Stress and the brain: from adaptation to disease[END_REF]. There was the hyperactivity of the HPA axis in the 1-week stressed rats as shown by increased hypothalamic GR protein levels. On the other hand, the 4-week stressed rats had decreased levels of hippocampal GR indicating hyperactivity HPA axis in depression (de [START_REF] De Kloet | Brain corticosteroid receptor balance in health and disease[END_REF][START_REF] Zhu | The different roles of glucocorticoids in the hippocampus and hypothalamus in chronic stress-induced HPA axis hyperactivity[END_REF]. Moreover, the 4-week restraint stress markedly increased the MC2R protein levels suggesting an increased adrenal sensitivity to ACTH [START_REF] Liu | Transcriptional regulation of episodic glucocorticoid secretion[END_REF].

NET is a key protein regulating noradrenergic transmission and a site of action of TCA and NRIs. The 4-week stress induction upregulated NET protein levels in the LC similar to chronic social defeat [START_REF] Chen | Chronic social defeat up-regulates expression of norepinephrine transporter in rat brains[END_REF][START_REF] Fan | Effects of desipramine treatment on stress-induced up-regulation of norepinephrine transporter expression in rat brains[END_REF].

However, the upregulated NET in the locus should reduce noradrenergic transmission in the LC, but the reduction of NET in the PAG should act in this structure much like antidepressants, and thus stimulated noradrenergic transmission. Therefore, NET of the 4-week stressed rats showed adaptive change by downregulating its projections PAG (van Bockstaele and Aston-Jones, 1992). These results indicated that chronic stress activated the LC-noradrenergic system and caused depression-like behaviors.

Serotonergic transmission is critically regulated by serotonin reuptake via the SERT. Dysfunction of the SERT causes depression and anxiety disorders [START_REF] Owens | Role of serotonin in the pathophysiology of depression: focus on the serotonin transporter[END_REF]. The 1-and 8-week restraint stress induction downregulated SERT protein levels in the VTA and the hippocampus. The 4-week restraint stress tended to downregulate SERT in the dorsal raphe. Acute restraint stress and social defeat decreased SERT in the raphe pontis and hippocampus [START_REF] Berton | Behavioral, neuroendocrine and serotonergic consequences of single social defeat and repeated fluoxetine pretreatment in the Lewis rat strain[END_REF][START_REF] Vollmayr | Acute stress decreases serotonin transporter mRNA in the raphe pontis but not in other raphe nuclei of the rat[END_REF]. Where SERT is downregulated, less serotonin is reuptaken and thus longer 5-HT action is observed in the synaptic cleft. In contrast, upregulated SERT protein levels were observed in the chronic social defeat and ovariectomized rats exposed to chronic aversive stimuli [START_REF] Zhang | Chronic social defeat up-regulates expression of the serotonin transporter in rat dorsal raphe nucleus and projection regions in a glucocorticoid-dependent manner[END_REF][START_REF] Charoenphandhu | Upregulated mRNA levels of SERT, NET, MAOB, and BDNF in various brain regions of ovariectomized rats exposed to chronic aversive stimuli[END_REF].

The 5-HT 2C R regulates mood, anxiety, feeding, and reproductive behavior (Heisler et al., 2007). The 1-and 4-week restraint stress upregulated 5-HT 2C R protein expression in the amygdala while the 8-week restraint stress downregulated it in VTA. Changes in 5-HT 2C R RNA site editing were observed in the PFC of suicide patients with major depressive disorder and stressed rats [START_REF] Niswender | RNA editing of the human serotonin 5-References / 186 HT2C receptor: alterations in suicide and implications for serotonergic pharmacotherapy[END_REF][START_REF] Iwamoto | Altered RNA editing of serotonin 2C receptor in a rat model of depression[END_REF]. Activation of the 5-HT 2C R by serotonin inhibits DA and NE release in striatal and PFC rats [START_REF] Alex | Modulation of dopamine release by striatal 5-HT2C receptors[END_REF] leading to depressive-and anxietylike symptoms. In addition, isolation-reared rats showed high anxiety-like behavior and locomotor activities in EPM and OFT due to increased 5-HT 2C R protein levels and responsiveness [START_REF] Fone | Increased 5-HT2C receptor responsiveness occurs on rearing rats in social isolation[END_REF].

Based on the physical and behavioral response to stress, the procedure of stress induction 2h/day, 5day/week for 4 weeks showed the most marked anxiety-, depression-, and memory impairment-like behaviors in male rats. These behavioral changes resulted from increased corticosterone levels, which altered target protein expression in various brain areas of stressed rats, especially BDNF and GR in the hippocampus. Therefore, the 4-week stress induction procedure was used to study the preventive effects of pharmacological and exercise interventions.

Validation of the voluntary exercise protocol

Physical exercise is known to provide several beneficial effects on physical and mental health. Moderate intensity aerobic exercise can prevent development of metabolic syndrome including obesity, diabetes mellitus, dyslipidemia, hypertension and cardiovascular diseases [START_REF] Mcardle | Training for anaerobic and aerobic power[END_REF][START_REF] Booth | Reduced physical activity and risk of chronic disease: the biology behind the consequences[END_REF]. Regular aerobic exercise also reduces body fat accommodation, increases muscle growth and heart weight in both athletes and exercising animals [START_REF] Douglas | Left ventricular hypertrophy in athletes[END_REF][START_REF] Wang | Animal models in the study of exercise-induced cardiac hypertrophy[END_REF][START_REF] Lapmanee | Beneficial effects of fluoxetine, reboxetine, venlafaxine, and voluntary running exercise in stressed male rats with anxiety-and depression-like behaviors[END_REF]. The 4-week voluntary wheel running preserved cardiac performance as determined by an isolated working heart preparation [START_REF] Kuczmarski | Cardiac function is preserved following 4 weeks of voluntary wheel running in a rodent model of chronic kidney disease[END_REF]. Voluntary wheel running alters the energy balance, body weight and composition, food intake and energy expenditure [START_REF] Novak | The use of a running wheel to measure activity in rodents: relationship to energy balance, general activity, and reward[END_REF]. Indeed, the results of voluntary wheel running in rodents are similar to the results of exercise in humans, i.e., increased muscle insulin sensitivity, increased skeletal muscle citrate synthase, and decreased visceral fat [START_REF] Davidson | Training effects in mice after long-term voluntary exercise[END_REF][START_REF] Henriksen | Adaptive responses of GLUT-4 and citrate synthase in fasttwitch muscle of voluntary running rats[END_REF]. Therefore, the physiological responses to voluntary wheel running can be considered a model of aerobic exercise. In this study, 4-week voluntary wheel running exercise resulted in body weight reduction and cardiac hypertrophy in both non-stressed rats and stressed rats. Therefore, the presence of body weight reduction and cardiac hypertrophy were used as indicator of the effective aerobic exercise protocol.

Modulating and protective effects of 4-week voluntary exercise on

HPA axis dysregulation and stress-related behaviors in male rats.

Unlike forced exercise (e.g., treadmill running and swimming), voluntary exercise provides minimal physical and emotional stress [START_REF] Leasure | Forced and voluntary exercise differentially affect brain and behavior[END_REF][START_REF] Charoenphandhu | Long-term swimming in an inescapable stressful environment attenuates the stimulatory effect of endurance swimming on duodenal calcium absorption in rats[END_REF]. Four week voluntary wheel running increased the adrenal gland weight without a change in corticosterone levels in non-stressed male rats. This was consistent with a previous finding in which the 4-week voluntary wheel running had no effect on corticosterone concentrations, either the basal level or the peak level of the circadian rhythm in male Sprague-Dawley rats [START_REF] Fediuc | Effect of voluntary wheel running on circadian corticosterone release and on HPA axis responsiveness to restraint stress in Sprague-Dawley rats[END_REF].

But other studies reported elevated basal corticosterone levels after 3 weeks of wheel running in male Lewis rats [START_REF] Makatsori | Voluntary wheel running modulates glutamate receptor subunit gene expression and stress hormone release in Lewis rats[END_REF]) and C57BL/6 mice [START_REF] Droste | Effects of long-term voluntary exercise on the mouse hypothalamic-pituitaryadrenocortical axis[END_REF]. These finding suggests that duration of voluntary exercise, strain of rats, animal species, and the timing of the circadian cycle can affect hormone levels and are important factors for basal HPA-axis activity.

Moreover, voluntary wheel running increases hippocampal BDNF and neurogenesis, and also enhances brain function by altering monoaminergic neurotransmission [START_REF] Dishman | Brain monoamines, exercise, and behavioral stress: animal models[END_REF]Cotman and Berchtold, 2002). In non-stressed rats, wheel running increased the quantity of 5-HT1A receptor, while decreasing SERT mRNA level in the dorsal raphe, an area responsible for anxiety-like behavior [START_REF] Greenwood | Freewheel running prevents learned helplessness/behavioral depression: role of dorsal raphe serotonergic neurons[END_REF][START_REF] Greenwood | Wheel running alters serotonin (5-HT) transporter, 5-HT1A, 5-HT1B, and alpha 1b-adrenergic receptor mRNA in the rat raphe nuclei[END_REF]. Voluntary wheel running also activated the dentate gyrus granule neurons and increased hippocampal neurogenesis through the upregulation of BDNF mRNA expression [START_REF] Van Praag | Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus[END_REF]. It is already well established that regular exercise provides several physiological and psychological advantages. Voluntary wheel exercise promotes brain health and has both antidepressant and anxiolytic effects in rodents and human [START_REF] Burghardt | The effects of chronic treadmill and wheel running on behavior in rats[END_REF][START_REF] Ströhle | Physical activity, exercise, depression and anxiety disorders[END_REF][START_REF] Greenwood | 5-HT2C receptors in the basolateral amygdala and dorsal striatum are a novel target for the anxiolytic and antidepressant effects of exercise[END_REF].

Anti-stress effects of voluntary exercise possibly involve decreased perception of chronic stress by modulating neurotransmitters, growth factors, cytokines, and HPA axis [START_REF] Sanches | Relationship among stress, depression, cardiovascular and metabolic changes and physical exercise[END_REF]. In the present study, 4-week voluntary wheel running prevented the stress-induced decrease in sucrose preference in experiment 2. Previous reports showed that 4-week voluntary wheel running exerted anxiolytic-and antidepressant-like action as evaluated by the ETM, EPM, FST, and shuttle box escape deficit [START_REF] Zheng | Beneficial effects of exercise and its molecular mechanism on depression in rats[END_REF][START_REF] Duman | Voluntary exercise produces antidepressant and anxiolytic behavioral effects in mice[END_REF][START_REF] Greenwood | Exercise, learned helplessness, and the stress-resistant brain[END_REF][START_REF] Lapmanee | Beneficial effects of fluoxetine, reboxetine, venlafaxine, and voluntary running exercise in stressed male rats with anxiety-and depression-like behaviors[END_REF]. In addition, increased BDNF protein and mRNA was observed after 4 weeks of voluntary wheel running, and thus responses enhanced learning and memory on MWM task (Adlard and Cotman, 2004). functions related to the reward system or the HPA axis activity [START_REF] Gasic | BDNF, relative preference, and reward circuitry responses to emotional communication[END_REF][START_REF] Taliaz | Resilience to chronic stress is mediated by hippocampal brain-derived neurotrophic factor[END_REF]. These results thus suggest that voluntary exercise could reduce the negative effect of chronic stress by modulating sympathetic activity and the HPA axis, which subsequently results in decreased corticosterone secretion [START_REF] Droste | Voluntary exercise impacts on the rat hypothalamic-pituitary-adrenocortical axis mainly at the adrenal level[END_REF][START_REF] Patki | Novel mechanistic insights into treadmill exercise based rescue of social defeatinduced anxiety-like behavior and memory impairment in rats[END_REF].

Therefore, the sympathoadrenomedullary input is likely to play a role in glucocorticoid responses to the 4-week restraint stress. Four-week voluntary wheel running was thus effective in preventing the stress-induced anhedonia by modulating central target proteins in the brain structures and neural pathway of the reward systems.

Effectiveness of monoamine modulators and voluntary exercise in the prevention of stress-induced anxiety-, depression-, and memory impairment in male rats

Both pharmacological treatments (i.e., agomelatine and venlafaxine) and voluntary wheel running successfully prevented anxiety-like behavior, especially the learned fear (i.e., generalized anxiety disorder-like), as well as the impairment of spatial learning and memory in restraint stressed rats. Drugs and exercise had no impact on locomotor activity in the EPM and OFT. However, only pharmacological treatments, but not voluntary wheel running in rats showed a protective action against depression-like behavior and impaired memory in FST and NOR test, even though running concurrently with stress could reduce depression-like behaviors [START_REF] Lapmanee | Beneficial effects of fluoxetine, reboxetine, venlafaxine, and voluntary running exercise in stressed male rats with anxiety-and depression-like behaviors[END_REF]. The finding of agomelatine-induced reduction in depression-like behavior was consistent with the previous reports using other behavioral tests. For example, Païzanis and co-workers (2010) reported that, after agomelatine treatment, immobility was significantly reduced in mice subjected to the tail suspension test.

The corticosterone levels were above basal non-stressed levels in all 4 groups (even worse for the Ago group), thus indeed they all had high corticosterone levels. At the cellular level, stressed rats pre-treated with agomelatine or venlafaxine seemed to be sensitive to sustained high corticosterone levels, which resulted in a downregulation of BDNF through stress in the hippocampus and the septum, and in both locations, this was counteracted and improved by wheel running. The GR was also downregulated but only in the hippocampus. As for SERT and NET, significant changes by stress are solely limited to the hippocampus and dorsal raphe for SERT and to the LC and the PAG for NET. Furthermore, venlafaxine and voluntary wheel running increased the levels of hippocampal BDNF protein in stressed male rats.

Thus, the present findings demonstrate that both venlafaxine and exercise exert a protective effect against anxiety, depression, and memory impairment by restoring hippocampal GR and upregulating BDNF expression in stressed rats. These results suggest that change in corticosterone levels is not associated with the preventive effects of agomelatine treatments. Presumably, the blockade of 5-HT 2C R receptors causes enhanced release of both NE and DA at the fronto-cortical dopaminergic and noradrenergic pathways and promotes hippocampal cell proliferation, which in turn could be the underlying mechanism of the preventive action against stress-induced behavioral abnormality [START_REF] Millan | Mirtazapine enhances frontocortical dopaminergic and corticolimbic adrenergic, but not serotonergic, transmission by blockade of alpha2-adrenergic and serotonin 2C receptors: a comparison with citalopram[END_REF][START_REF] Gatch | Discriminative stimulus effects of m-chlorophenylpiperazine as a model of the role of serotonin receptors in anxiety[END_REF]Dagytė et al., 2010). Although pretreatment did not alter 5-HT 2C R protein levels, there was a tendency towards a downregulation of the 5-HT 2C R in the amygdala of the exercise group. This result might suggest anxiolytic effects of voluntary exercise on stress-induced 5-HT 2C R upregulation in stressed rats [START_REF] Greenwood | 5-HT2C receptors in the basolateral amygdala and dorsal striatum are a novel target for the anxiolytic and antidepressant effects of exercise[END_REF].

Since the beneficial effects of the studied drugs and exercise were observed without changes in serum corticosterone levels, it is plausible that agomelatine, venlafaxine, and exercise might have already induced a long-lasting adaptation in monoaminergic neurotransmission or even neurogenesis in the brain, which lasted throughout the entire stress periods. For instance, agomelatine is known to stimulate cell proliferation within the dentate gyrus of rodents [START_REF] Banasr | Agomelatine, a new antidepressant, induces regional changes in hippocampal neurogenesis[END_REF].

Venlafaxine also enduringly modulated monoaminergic neurotransmission by increasing 5-HT, DA and NE levels in the PFC and striatum [START_REF] Higashino | Effects of acute and chronic administration of venlafaxine and desipramine on extracellular monoamine levels in the mouse prefrontal cortex and striatum[END_REF], and increasing the expression of BDNF protein in rat hippocampus (Cooke et al., 2009). BDNF possibly promoted the function and survival of dopaminergic, GABAergic, noradrenergic, and serotonergic neurons (Connor and Dragunow, 1998).

Improvement of memory by venlafaxine and running exercise could be explained by increased BDNF expression in the hippocampus [START_REF] Li | Dose-related effects of venlafaxine on pCREB and brain-derived neurotrophic factor (BDNF) in the hippocampus of the rat by chronic unpredictable stress[END_REF][START_REF] Liu | Differential effects of treadmill running and wheel running on spatial or aversive learning and memory: roles of amygdalar brain-derived neurotrophic factor and synaptotagmin I[END_REF], which is one of the utmost important brain structures for learning and memory.

Furthermore, voluntary wheel running exhibited SSRI and/or NRI-like actions [START_REF] Engesser-Cesar | Wheel running and fluoxetine antidepressant treatment have differential effects in the hippocampus and the spinal cord[END_REF][START_REF] Lapmanee | Beneficial effects of fluoxetine, reboxetine, venlafaxine, and voluntary running exercise in stressed male rats with anxiety-and depression-like behaviors[END_REF]. During exercise, NE in the cell body and its metabolites were also increased in the pons, medulla oblongata, and spinal cord of rats [START_REF] Dunn | Brain norepinephrine and metabolites after treadmill training and wheel running in rats[END_REF]. The mRNA expression of 5-HT 1B and 1

adrenergic receptors was upregulated in the locus coeruleus and dorsal raphe [START_REF] Greenwood | Wheel running alters serotonin (5-HT) transporter, 5-HT1A, 5-HT1B, and alpha 1b-adrenergic receptor mRNA in the rat raphe nuclei[END_REF], which could in turn alter adrenergic and serotonergic activities, respectively.

As aforementioned, it is possible that agomelatine, venlafaxine and voluntary wheel running can normalize and coordinate the balance of glucocorticoids and neurotransmitter metabolism, and induce long-lasting adaptations in monoaminergic neurotransmission or neurogenesis in the brain (Cotman and Berchtold, 2002;[START_REF] Dagytè | The novel antidepressant agomelatine normalizes hippocampal neuronal activity and promotes neurogenesis in chronically stressed rats[END_REF][START_REF] Huang | Chronic restraint stress promotes learning and memory impairment due to enhanced neuronal endoplasmic reticulum stress in the frontal cortex and hippocampus in male mice[END_REF], throughout the entire stress period.

Finally, the present findings suggest that, in a situation in which future exposure to stress is anticipated such as extreme military combat or war, infectious disease outbreak, and natural disaster, administration of melatonergic modulator (agomelatine), SNDRI (venlafaxine), and voluntary moderate-intensity exercise could be useful in the prevention against the posttraumatic development of mood disorders and memory impairment.

Mapping and characterization of MT1-/MT2-LacZ expressing neurons in transgenic knock-in mouse models

Melatonin (MT) not only plays a role in the mammalian circadian rhythm but also modulates mood, emotion, and memory. It also acts as a neuromodulator by interacting with its central and peripheral MT receptors [START_REF] Dubocovich | Melatonin receptors: role on sleep and circadian rhythm regulation[END_REF][START_REF] Pévet | Melatonin receptors as therapeutic targets in the suprachiasmatic nucleus[END_REF]. Thus, it is important to demonstrate the localization of MT receptor in the central nervous system. In the present study, the mouse brain was examined for the expression of the MT gene at the cellular level, using the LacZ reporter gene to the targeting on MT1 or MT2 receptor gene locus downstream of the endogenous MT receptor regulatory elements.

Previous studies described the distribution of high-affinity binding sites for 125 I-iodomelatonin or localized the mRNA expression of MT receptors [START_REF] Dubocovich | International Union of Basic and Clinical Pharmacology. LXXV. Nomenclature, classification, and pharmacology of G protein-coupled melatonin receptors[END_REF]. These investigations identified numerous structures containing neurons expressing MT receptor genes [START_REF] Masson-Pévet | Are the annual reproductive and body weight rhythms in the male European hamster (Cricetus cricetus) dependent upon a photoperiodically entrained circannual clock?[END_REF].

Unfortunately, there are no reliable specific antibodies for MT1 and MT2 immunocytochemical detection. Therefore, we used X-gal histochemistry and immunohistochemistry to map the distribution of both MT1 and MT2 receptors in these transgenic reporter mice. MT1-LacZ positive cells were restricted to a few structures such as the SCN, the paraventricular nucleus of the thalamus, the posterior limiting nucleus of the thalamus and the pars tuberalis (PT) of the adenohypophysis.

This MT1-LacZ labeling is perfectly superposable to the 125 I-iodomelatonin binding sites, thus validating our approach (Liu et al., 1997). On the other hand, MT2-LacZ positive cells were detected in many structures throughout the brain such as the olfactory bulb, the islands of Calleja, the SCN, the cerebral cortex, the superior colliculus, the amygdala and the arcuate nucleus of the hypothalamus. MT2-LacZ knock-in mice also had a strong staining in the PVN, the CA2 and a population of small horizontal cells in the outer molecular layer of dentate gyrus of the hippocampus.

MT1 and/or MT2 receptors are present in the SCN and PT which have high concentration of MT binding sites [START_REF] Vanĕcek | Hypothalamic melatonin receptor sites revealed by autoradiography[END_REF][START_REF] Morgan | Melatonin receptors on ovine pars tuberalis: characterization and autoradiographicai localization[END_REF][START_REF] Weaver | Localization and characterization of melatonin receptors in rodent brain by in vitro autoradiography[END_REF]. The distinct presence of MT1 and MT2 receptors in different brain areas suggests distinctive functions for each receptor in neurophysiology and neuropathology (Comai and Gobbi, 2014;[START_REF] Lacoste | Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain[END_REF]. MT binding sites are found predominantly in the median eminence, SCN, area postrema, paraventricular thalamic nucleus, hypothalamus, hippocampus, parietal cortex, striatum, PT, medial region of the lateral habenula, arcuate nucleus, and amygdala of the rats and mice [START_REF] Laudon | Melatonin receptors in discrete brain areas of the male rat. Impact of aging on density and on circadian rhythmicity[END_REF][START_REF] Weaver | Localization and characterization of melatonin receptors in rodent brain by in vitro autoradiography[END_REF][START_REF] Masson-Pévet | Are the annual reproductive and body weight rhythms in the male European hamster (Cricetus cricetus) dependent upon a photoperiodically entrained circannual clock?[END_REF][START_REF] Williams | Melatonin receptors in the rat brain and pituitary[END_REF]. MT1 and MT2 receptors are localized in regions known to contribute to anxiety and stress, e.g., the septal area, the habenula, and the dentate gyrus of hippocampus [START_REF] Yadin | The role of the lateral septum in anxiolysis[END_REF][START_REF] Karakaş | The effects of the intraamygdalar melatonin injections on the anxiety like behavior and the spatial memory performance in male Wistar rats[END_REF]. Regarding MT receptor mRNA expression in rodent brain, MT1 receptor mRNA was detected in the rodent dorsal striatum, NA, olfactory tubercle, substantia nigra, and VTA. Also, MT1 and MT2 receptor mRNA expressions have been found in the SCN and vestibular nuclei (Us et al., 2005;[START_REF] Imbesi | Melatonin signaling in mouse cerebellar granule cells with variable native MT1 and MT2 melatonin receptors[END_REF][START_REF] Ahn | Immunohistochemical and biomolecular identification of melatonin 1a and 1b receptors in rat vestibular nuclei[END_REF]. In human studies, MT1 mRNA and protein were detected in the hypothalamus, the cerebellum, the PFC, the NA, the amygdala, and hippocampus [START_REF] Weaver | Melatonin receptors in human hypothalamus and pituitary: implications for circadian and reproductive responses to melatonin[END_REF][START_REF] Mazzucchelli | The melatonin receptor in the human brain: cloning experiments and distribution studies[END_REF][START_REF] Wu | Distribution of MT1 melatonin receptor immunoreactivity in the human hypothalamus and pituitary gland: colocalization of MT1 with References / 194 vasopressin, oxytocin, and corticotropin-releasing hormone[END_REF], and MT2 receptor transcripts were detected in the hippocampus [START_REF] Musshoff | Melatonin receptors in rat hippocampus: molecular and functional investigations[END_REF].

The SCN is the master circadian clock and a target the drug discovery in circadian-dependent pathogeneses. Neurons in the SCN subregions are distinguished by their neuropeptide contents, i.e., AVP, GRP, and VIP, while AVP cells are the output of the SCN central clock, VIP cells are the input and GRP cells the core of the clock [START_REF] Abrahamson | Suprachiasmatic nucleus in the mouse: retinal innervation, intrinsic organization and efferent projections[END_REF][START_REF] Welsh | Suprachiasmatic nucleus: cell autonomy and network properties[END_REF]. MT1-LacZ expressing neurons co-localized with some GRP and VIP neurons in order to synchronize the endogenous circadian rhythms. Therefore, MT can act through MT1 receptors on the input and the inner working of the clock. MT2-LacZ expressing neurons were located more diffusely throughout the SCN and around it.

MT2-LacZ expressing neurons were also located in brain regions associated with stress responses, particularly the PVN and the hippocampus, both of which are important components of the HPA axis [START_REF] Smith | The role of the hypothalamic-pituitary-adrenal axis in neuroendocrine responses to stress[END_REF]. The present study found that MT2-LacZ expressing neurons co-localized with CRH in the PVN, suggesting that melatonin and agomelatine might directly modulate the HPA axis at the PVN. As MT2 receptor signaling is mostly inhibitory, agomelatine action on the CRH neurons should thus reduce the activity of these neurons and reduce the secretion of CRH. Reduced secretion of CRH should result in a decrease in corticosterone levels. We observed however higher levels of corticosterone in the agomelatine-treated animals. Agomelatine being a combined MT1/M2 agonist and 5-HT 2C receptor antagonist, further studies of this complex pharmacological profile might help us to understand this apparently contradictory result.

Furthermore, MT2-LacZ expressing neurons were co-localized with GAD2 in the hippocampus [START_REF] Stone | Localization of cells preferentially expressing GAD(67) with negligible GAD(65) transcripts in the rat hippocampus. A double in situ hybridization study[END_REF]. These results could imply that the actions of MT and agomelatine result in the inhibition of these inhibitory interneurons, and thus an activation of hippocampal activity [START_REF] Chamberland | Inhibitory control of hippocampal inhibitory neurons[END_REF]. However, MT2 receptors also seem to be very selectively placed on pyramidal neurons of the CA2. These pyramidal neurons are known to be glutamatergic, and thus MT or agomelatine action on these neurons should result in the selective inhibition of CA2 mediated functions. Thus MT and agomelatine should activate the hippocampus, but selectively inhibit CA2. How this dual action impacts hippocampal tasks and the hippocampal action on the HPA axis remains to be established.

In addition, MT2-LacZ expressing cells were observed in the dorsal raphe.

This brain region apparently does not contain 5-HT 2C R, but the 5-HT 2C R is involved in negative serotonin feedback to the dorsal raphe through GABAergic neurons around the dorsal raphe, which express 5-HT 2C R [START_REF] Serrats | Expression of serotonin 5-HT2C receptors in GABAergic cells of the anterior raphe nuclei[END_REF]. Thus agomelatine might act directly on dorsal raphe and indirectly through this GABAergic 5-HT 2C feedback. As an agonist on MT2 receptors in the dorsal raphe, it might inhibit these serotonergic neurons. As a 5-HT 2C antagonist, it could block 5-HT negative feedback, which in turn could therefore stimulate the dorsal raphe. Thus this action is probably quite complex because it would both directly inhibit the dorsal raphe and indirectly stimulate the same structure. Therefore, the dorsal raphe is another target of interest for agomelatine.

CHAPTER VI

CONCLUSIONS

The aims of the present study were first, to examine the effects of restraint stress on anxiety-, depression-, and memory impairment-like behaviors at different time points, and second, to determine the preventive therapeutic effects of monoamine modulators (agomelatine and venlafaxine) and voluntary exercise in stressed male rats.

Changes in the physical and behavioral parameters and protein expression levels in the brain regions associated with stress responses were investigated. In addition, possible sites of action for agomelatine administration were identified in melatonin (MT) MT1and MT2-LacZ knocked-in mouse.

The conclusions are as follow with a summary in the Figure 6.1.

Four week restraint stress in male rats resulted in decreases in food intake and body weight gain, adrenal hypertrophy, and increased serum and urine levels of corticosterone. Increased levels of corticosterone could alter central the target proteins, i.e., brain-derived neurotrophic factor (BDNF), glucocorticoid receptor (GR), norepinephrine transporter (NET), and serotonin type 2C receptor (5-HT 2C R) in the amygdala, hippocampus, locus coeruleus, and periaqueductal gray (PAG) leading to manifestation of anxiety-, depression-, and memory impairment-like behaviors.

Four week voluntary aerobic wheel running exercise protocol were found to partly prevent the stress-induced increase in serum corticosterone levels and anhedonia-like behavior, most probably by deregulating adrenal adrenocorticotropic hormone (ACTH) receptors, and altering target protein expression levels (i.e., BDNF and NET) in the brain structures specially involved in stress response, i.e., hippocampus, septum nuclei, locus coeruleus, and periaqueductal gray. Therefore, voluntary exercise could reduce the negative effect of chronic stress by modulating sympathetic activity in the central and peripheral hypothalamic-pituitary-adrenal (HPA) axis.

Sarawut Lapmanee

Conclusions / 140

The study also revealed that agomelatine, venlafaxine as well as voluntary wheel running prevented the restraint stress-induced anxiety/depression-like behaviors and memory impairment in male rats. Upregulation of hippocampal BDNF protein levels might be a possible mechanism underlying the protective effects of venlafaxine and voluntary exercise in stressed male rats.

Regarding the applications of the present findings for clinical use, fente synaptique [START_REF] Charney | Monoamine dysfunction and the pathophysiology and treatment of depression[END_REF][START_REF] Bowman | Chronic stress effects on memory: sex differences in performance and monoaminergic activity[END_REF][START_REF] Belmaker | Major depressive disorder[END_REF].

Outre la dérégulation de l'axe HPS, le stress perturbe aussi les rythmes circadiens et la sécrétion de mélatonine par la pinéale [START_REF] Konakchieva | Chronic melatonin treatment and the hypothalamo-pituitary-adrenal axis in the rat: attenuation of the secretory response to stress and effects on hypothalamic neuropeptide content and release[END_REF][START_REF] Mcclung | Circadian rhythms and mood regulation: insights from pre-clinical models[END_REF].

Aussi, des niveaux réduits de mélatonine (MT) ont été rapportés chez des patients dépressifs (Claustrat et al. 1984) [START_REF] Lapmanee | Anxiolytic-like actions of reboxetine, venlafaxine and endurance swimming in stressed male rats[END_REF][START_REF] Lapmanee | Beneficial effects of fluoxetine, reboxetine, venlafaxine, and voluntary running exercise in stressed male rats with anxiety-and depression-like behaviors[END_REF].

Cependant, les effets modulatoires de l'exercice de roue volontaire sur la réponse à un stress chronique ne sont pas encore bien compris, et les effets préventifs de cet exercice sur les troubles de l'humeur et les perturbations de la mémoire induits par le stress restent à préciser. Enfin, les mécanismes cellulaires des effets protecteurs de l'agomélatine, de la venlafaxine et de l'exercice physique dans les neurones 5-HT et NA vis-à-vis des changements comportementaux induits par le stress ne sont pas encore connus.

Ainsi, les objectifs de ce travail étaient 1. d'établir un modèle de stress de contention chronique chez le rat pour des études comportementales et des analyses biochimiques des changements d'expression de protéines cibles dans le cerveau.

2. de déterminer les bénéfices de l'activité de roue volontaire sur l'axe hypothalamo-pituito-surrénalien (HPS) et sur l'expression de protéines cibles dans diverses régions cérébrales chez le rat.

3. de comparer l'efficacité de l'Agomélatine (Ago), d'un inhibiteur de la recapture de la 5-HT et de la NA (Venlafaxine, Vlx) et de l'exercice physique volontaire dans la prévention de l'anxiété et de la dépression, ainsi que des perturbations de la mémoire induits par le stress.

4. de déterminer la localisation des récepteurs MT1 et MT2 chez des souris transgéniques rapportrices afin d'identifier des sites potentiels d'action de l'Agomélatine, en particulier dans le contexte du stress.

Les hypothèses centrales de ce travail étaient :

1. Les modulateurs de la neurotransmission monoaminergique et l'activité de roue volontaire peuvent prévenir les comportements induits par le stress chez le rat mâle en modulant la neurogenèse hippocampique et en altérant l'expression de protéines cibles de la neurotransmission monoaminergique centrale dans des régions associées avec la réponse au stress.

2. Des neurones exprimant les récepteurs de la mélatonine MT1 et MT2 localisées dans des régions associées à la réponse au stress pourraient être des cibles de l'action antidépressive et anxiolytique du traitement pharmacologique par l'Agomélatine.

B. Matériel et Méthodes

Partie 1 : Effets du stress de contention, des modulateurs pharmacologiques (Agomélatine, Venlafaxine) et de l'activité de roue volontaire chez des rats mâles

Animaux

Des rats mâles de souche Wistar (âgés de 8 semaines, 180200 g) étaient obtenus du Centre National d'Animaux de Thaïlande (Université Mahidol, Thaïlande).

A la fin des expériences, tous les rats étaient évalués pour des comportements de type anxiété, dépression ou perturbations de la mémoire.

Stress de contention

Les rats ont été placés dans des tubes de contention modifiés pendant 2 heures/jour, 5 jours/semaine pendant 1, 4 ou 8 semaines. Les tubes de contention avaient une ouverture d'1 cm de diamètre à un bout pour permettre aux animaux de respirer.

Études comportementales

Les comportements anxieux ont été évaluées par le labyrinthe élevé en croix (elevated plus maze EPM), le labyrinthe élevé en Tableau 2. Cinétique des changements comportementaux et biochimiques après un stress de contention pendant 1, 4 ou 8 semaines chez le rat male. Le signal MT2-LacZ pouvait être détecté dans de multiples structures à travers le cerveau telles que le bulbe olfactif, les ilots de Calleja, le SCN, le noyau paraventriculaire de l'hypothalamus (PVN), le cortex cérébral, l'hippocampe, le tectum optique, l'amygdale et le noyau arqué de l'hypothalamus. Les marquages les plus forts ont été observés surtout dans le PVN, la CA2 de l'hippocampe et une population de petits interneurones horizontaux dans la couche moléculaire externe du gyrus dentelé de l'hippocampe (Figure 3). Most mouse strains (including the C57BL/6 strain) do not produce MT due to loss of function mutations in the N-acetyltransferase (NAT) and the hydroxyindole O-methyltransferase (HIOMT) genes [START_REF] Kasahara | Genetic variation of melatonin productivity in laboratory mice under domestication[END_REF]. Both of these enzymes are necessary to produce MT from its precursor 5-HT. The MT1-LacZ and MT2-LacZ reporter constructs were initially introduced into C57BL/6 mice that do not produce MT. To test whether the presence or the absence of MT influences the expression pattern of MT1 or MT2 receptors, we crossed the MT1-LacZ or MT2-LacZ C57BL/6 mice with a MT-proficient mouse strain, the C3H strain. In the F1 generation of this cross, animals possess 1 set of chromosomes from the C57BL/6 parent, with mutated non-functional NAT and HIOMT alleles, but potentially with the recombinant MT1-LacZ or MT2-LacZ allele. The second set of chromosomes comes from the C3H parent, thus containing intact NAT and HIOMT alleles. Because of these intact NAT and HIOMT alleles, the C57BL/6 x C3H F1 mice are able to synthesize MT. Among this F1 offspring, we then selected animals containing the recombinant MT1-LacZ and MT2-LacZ alleles to obtain reporter mice able to produce MT.

3. 7

 7 Mouse primers in the PCR experiments for cloning probe. and techniques in this study.

  : luteinizing hormone; PGE: prostaglandin; PRL:

  Figure Page 2.5 Physiological effects of physical exercise: Adaptive mechanisms are responsible for a decreased perception of chronic stress. BDNF: Brain-derived neurotrophic factor; DA: Dopamine; HPA axis: hypothalamic-pituitary-adrenal axis; iNOS: Inducible nitric oxide synthase; IL-1: Interleukin 1; IL-6: Interleukin 6; IL-10: Interleukin 10; NE; Norepinephrine; ROS: Reactive oxygen species; TNF-: Tumor necrosis factor; 5-HT: Serotonin; VEGF: Vascular endothelial growth factor (Modified from Sanches et al., 2016).

4. 1

 1 Figure Page 4.5 Time-dependent effects of the 1-, 4-, and 8-week restraint stressinduced anxiety-like behavior as evaluated by OFT, (A) number of lines crossed in the first 30 seconds, (B) total lines crossed, (C) inner zone time, (D) outer zone time, (E) number of rearing, and (F) number of grooming. Numbers of animals are noted in parentheses.*P < 0.05 compared to non-stressed control group.

  Figure Page 4.24 Protective effects of agomelatine (Ago), venlafaxine (Vlx) and voluntary wheel running (Rw) on stress-induced anxiety-like behavior as determined by EPM, (A) percent open arm entry, (B) percent open arm time, (C) percent closed arm entry, (D) percent closed arm time, (E) percent center of arm time, (F) total arm entries, (G) numbers of grooming, and (H) numbers of rearing.Numbers of animals are noted in parentheses. † P < 0.05 compared to vehicle (Veh)-treated group.

  effects of agomelatine (Ago), venlafaxine (Vlx) and voluntary wheel running (Rw) on stress-induced learning and memory impairment-like behavior as determined by MWM and NOR, (A) escape latency, (B) correct quadrant time, (C) percent correct quadrant time, and (D) discrimination ratio. Numbers of animals are noted in parentheses. † P < 0.05, † † P < 0.01, † † † P < 0.001 compared to vehicle (Veh)-treated group. 101 4.29 Protective effects of agomelatine (Ago), venlafaxine (Vlx) and voluntary wheel running (Rw) on the BDNF protein levels.

  Figure Page 4.31 Protective effects of agomelatine (Ago), venlafaxine (Vlx) and voluntary wheel running (Rw) on the NET protein levels. Numbers of animals are noted in parentheses. † † P < 0.01 compared to vehicle (Veh)-treated group.

  with LacZ-Immunohistochemistry and nonradioactive in situ hybridization for MT2 (green) and GAD (red) colocalization. The MT2 positive cells in the CA2 of the hippocampus are GAD negative. A few non-pyramidal cells of the CA2 are MT2 + GAD positive (arrowheads). Diagram showing the summary results in this present study. Generation of MT1-/MT2-LacZ knock-in mice. The mutant MT1 (Mtnr1a) or MT2 (Mtnr1b) alleles contain a loxP-hUbC-Neomycin-polyA stop cassette. This Neomycin resistance cassette used for the selection of recombinant embryonic stem cells is then deleted from the mutant locus using Cre-mediated recombination to avoid interference of these sequences with the expression profile of mood disorders are common psychiatric illness and are

  determine the effects of the 4 week-voluntary wheel running on the HPA axis under two conditions, non-stress condition or after the 4-week exposure to restraint stress, and changes in the expression of selected target proteins in brain regions associated with stress responses.3. To evaluate the effectiveness of agomelatine, venlafaxine, and voluntary wheel running exercise in the prevention of stress-induced anxiety-, depression-, and memory impairment-like behaviors and changes in the expression of selected target proteins in brain regions associated with stress responses. 4. To characterize the phenotype of MT1-/MT2-LacZ expressing neurons in knock-in mice by analyzing co-expressed neuropeptides and neuronal markers.The hypothesizes of this study were:1. Monoaminergic modulators and voluntary wheel running could prevent stress-related behaviors in male rats by modulating hippocampal neurogenesis and altering the expression of target proteins of central monoamine neurotransmission in the brain regions associate with stress response. 2. MT1/MT2-LacZ expressing neurons in brain regions associated with stress responses were characterized in knock-in mice and might be the sites action of pharmacological treatment. Stress is a state of threat exposure that can disturb homeostasis and thus induces physiological and behavioral adaptations. Both sympathetic and parasympathetic activities are responsible for the rapid acute stress response via neural innervation. The hypothalamic-pituitary-adrenal (HPA) axis also plays a role in the stress response (Figure 2.1). After HPA axis activation, corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) synthetized by hypophysiotropic neurons in the hypothalamic paraventricular nucleus (PVN), are secreted into the hypophyseal portal circulation in the median eminence. CRH activates the anterior pituitary corticotrope cells to release adrenocorticotropic hormone (ACTH), whileAVP provides a synergistic effect on ACTH release. ACTH binds to the melanocortin type 2 receptor (MC2R) in the zona fasciculata of the adrenal cortex to stimulate synthesis and secretion of stress hormones, i.e., glucocorticoids (GCs) (cortisol in humans, or corticosterone in rodents) and catecholamines, i.e., norepinephrine(NE) and epinephrine (Epi). GCs affect the structure and excitability of neurons by binding to the GC receptor (GR) and the mineralocorticoid receptor (MR), whereas catecholamines promote vigilance, arousal, and attention. Positive and negative feedbacks involved in the regulation of the HPA axis act at various brain regions depending on the levels of stress. Therefore, the sympathoadrenal system and HPA axis are important centers for maintaining homeostasis during stress. Changes in hormonal responses to stress (i.e., CRH, ACTH, and GR) affect physiological processes and behavior that can be used to indicate clinical status and therapeutic targets (as reviewed by Ulrich-Lai and[START_REF] Ulrich-Lai | Neural regulation of endocrine and autonomic stress responses[END_REF].

Figure 2 . 1

 21 Figure 2.1 The hypothalamic-pituitary-adrenal (HPA) axis in stress responses showing positive feedbacks (+) and negative feedbacks (). Elevated glucocorticoids (GCs) are induced by the release of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) from the hypothalamic paraventricular nucleus (PVN) and anterior pituitary, respectively. Negative feedback inhibition by GCs acts on the pituitary gland and the hypothalamus. The release of GCs is also regulated by circadian signals () from the suprachiasmatic nucleus (SCN) in the hypothalamus.

  signals to the PVN to stimulate the release of CRH and AVP, whereas the hippocampus, prefrontal cortex (PFC) and ACC control the feedback regulation of the PVN. This limbic structure restrains HPA activity indirectly via stimulation of inhibitory gamma-aminobutyric acid (GABA)ergic neurons located in the ventrolateral septal region and the BNST. These neurons project to CRH-containing parvocellular neurons of the PVN. At the level of hypothalamus-brain stem system, the hippocampus stimulates the locus coeruleus (LC) in the brain stem to release NE to produce alertness. For, the brain-to-adrenal gland system, the hypothalamic nuclei, the parabrachial nuclei in the brain stem control both the sympathetic (NE and Epi) and parasympathetic (acetylcholine, Ach) nervous systems via the spinal cord to preganglionic neurons or to the adrenal glands.

Figure 2 . 2

 22 Figure 2.2 Neuroanatomical organization of stress responses consist of three levels,

  through a system of noradrenergic projections. Terminals in the cerebellum and cortex could be derived from collateral branches of a single neuron's axon. It plays a major role in the function of the sympathetic nervous system, during states of agitation or stress. The LC and the adrenergic projections are known as the LC-noradrenergic system. Activation of the LC induces the fight or flight reaction in the stress response. NE is synthesized by adrenergic neurons in LC in the brain stem while the major site of Epi synthesis is adrenal medulla. The catecholamine neurotransmitters are synthesized from tyrosine. The biosynthetic pathways of catecholamines involve several enzymes, i.e., tyrosine hydroxylase (TH), pteridine reductase, aromatic amino acid decarboxylase, dopamine β-hydroxylase, and phenylethanolamine-N-methyl transferase. TH in the LC is upregulated in response to depletion of NE in normal and

  DA and NE are stored in the storage vesicles that are located in the nerve terminal. Since the metabolizing enzyme monoamine oxidase (MAO) is present in the cytosol, concentrations of both catecholamines in the cytosol are low. Catecholamines are released by exocytosis from the nerve terminal. Both DA and NE are inactivated by MOA and catechol-Omethyltransferase (COMT) after binding to the receptors. Some catecholamines are transported back into the presynaptic nerve terminal via their respective selective reuptake transporters such as the DA reuptake transporter or the norepinephrine transporter (NET).

Figure 2 . 3

 23 Figure 2.3 Effects of MT1 and MT2 melatonin receptor agonists and antagonists in mammals. FSH: follicle-stimulating hormone; GnRH: gonadotropin-releasing hormone; KLH: Keyhole limpet hemocyanin; LH: luteinizing hormone; PGE: prostaglandin; PRL: Prolactin (Modified from Dubocovich and Markowska, 2005).

2. 3 . 1

 31 SSRIs selectively block the reuptake of 5-HT, leading to increased 5-HT levels in the synaptic cleft and thus greater postsynaptic neuronal activity. SSRIs such as fluoxetine, sertraline, paroxetine, and escitalopram are generally recommended as first line agents. SSRIs are also used in the acute and long-term treatment of patients with major depression, anxiety disorders. SSRIs have less risk of overdose with fewer side effects when compared with TCAs and MAOIs.

  Figure 2.4 Chemical structures of monoamine modulators used in the present study.

  Benefits of physical exercise as an alternative to psychological interventions have been demonstrated in several animal models. The mechanisms of action of exercise probably involve increased expression of neurotrophic factors, increased neurogenesis and increased monoamine neurotransmission in various brain regions. The possible mechanism underlying of beneficial effects of physical exercise are summarized in Figure 2.5. However, some investigators found controversial results, presumably due to the use of different exercise programs and intensities.

Figure 2 . 5

 25 Figure 2.5 Physiological effects of physical exercise: Adaptive mechanisms are responsible for a decreased perception of chronic stress. BDNF: Brain-derived neurotrophic factor; DA: Dopamine; HPA axis: hypothalamic-pituitary-adrenal axis; iNOS: Inducible nitric oxide synthase; IL-1: Interleukin 1; IL-6: Interleukin 6; IL-10: Interleukin 10; NE; Norepinephrine; ROS: Reactive oxygen species; TNF-: Tumor necrosis factor; 5-HT: Serotonin; VEGF: Vascular endothelial growth factor (Modified from Sanches et al., 2016).

  (2006) grouped animal models of anxiety into the conditioned response test or conflict test (e.g., exposure to electric foot shock, punished drinking, defensive burying tests) and the unconditional response test (e.g. exploration test or social test) that includes ethologically based paradigms and involves the animal's natural reactions such as fight, avoidance and freezing. The behavioral paradigms are generally probed as the influencing parameter of anxiety. The methods such as, open field can simultaneously measure locomotion, exploration, and anxiety when exposed to an open field arena, while the EPM test is based upon the conflict between an innate fear to exposed spaces and the elevation of the maze. These studies are designed to indicate anxiety related behaviors that parallel anxiety behavior in humans.

  EPM consists of 4 arms in a crossed arrangement, where 2 open arms represent the unsafe places and the 2 closed arms represent the safer places. Anxiety related behaviors include increases in closed arm activity, grooming, rearing, freezing, defecation and urination. A greater number of these behaviors indicate a greater level of anxiety or fear. Risk assessment behaviors, e.g., stretches-attend postures-forward elongation of the head and shoulders followed by retraction to the normal position, are an index of a greater level of anxiety. Decreased open arm activities and increased risk assessment behaviors in locomotion and exploration indicate increased anxiety in the EPM test. Behavioral and pharmacological validation of the EPM has reported that BDZs and 5-HT 2A/C R antagonist (e.g., mianserin, ritanserin, and ketanserin) increase

  . Based on 2 strategiesavoidance of open arms when the rat is in one of the closed arms, and escape from an open arm to enter a safer (closed arm) the constructing ETM has been succeeded. The maze consists of 2 elevated arms that are 1 enclosed arm and 2 open with equal dimensional arms. Conditioned fear results from inhibitory avoidance that is measured by recording the time taken to leave the enclosed arm, whereas unconditioned fear is measured by recording the time to escape from the open arm. An open arm seems to generate an unpleasant feeling with its elevation and the openness of the maze, such that an animal has an innate or unconditioned fear. This will allow the rats to learn inhibitory avoidance if frequently placed within the enclosed arm to explore the maze. Therefore, learning is indicated by the increase of the withdrawal latency along the trials. When the rat is placed at the end of the enclosed arm, it does not see the open arms until it extends its head further from the walls of the closed arm. On the other hand, when the rat is placed at the end of one of the open arms, it can shift to the closed arm, which is like performing an escape response. Behavioral and pharmacological validation of the ETM has reported

1. 4

 4 Assessment of emotionality in the anxiety of the animal In the assessment of changes in emotion, the number of fecal pellet is related to the affective or anxiety state of the animal. An increased number of fecal pellets are correlated with other measures of anxiety. The excretion of corticosteroid metabolites in fecal pellets is correlated with serum corticosterone levels. The validity of defecation as a measure of anxiety remains clear, i.e., increased defecation indicates increased anxiety. Risk assessment behavior seems to be defensive behavior and is indicative of anxiety when the animal faces an unknown or threatening situation. Animals express stretched-attend postures which when occurring a high frequency indicate a higher level of anxiety. Grooming includes licking the fur, beginning from the muzzle, going to the ears, and down to the rest of the body.

  hippocampus and perirhinal cortex). In 1988, Ennaceur and Delacour developed the NOR test to evaluate the differences in the exploration time of novel and familiar objects. The NOR test is a simple behavioral assessment of memory that relies primarily on a rodent's innate exploratory behavior in the absence of externally applied rules or reinforcement, motivation, reward, or punishment[START_REF] Antunes | The novel object recognition memory: neurobiology, test procedure, and its modifications[END_REF]. However, training or habituation is required. The exploration is the orientation of animal's snout toward the object, sniffing or touching with the snout. Running around the object, sitting or climbing on it is not included as exploration. If animals showed a lack of exploration activity, they are excluded from the test. The NOR test consists of the habituation phase (exploring the empty open field arena), the familiarization phase (exploring only one object) and finally the test phase (exploring the familiar object and a novel one). The objects used in this test vary in shape, size, texture, material, color and appearance. From the familiarization to the test phase, object features change when a novel object that is different from the familiar one is presented. The discrimination index (DI) indicates discrimination between the novel and familiar objects as the difference in exploration time for familiar objects divided by the total amount of exploration of the novel and familiar objects. A positive score indicates more time spent with the novel object, a negative score indicates more time spent with the familiar object, and a zero score indicates a no preference

(

  EPM), elevated T-maze (ETM), and open-field (OFT) tests, while the depressant-like behavior was evaluated by weekly sucrose preference and forced swimming tests (FST). In addition, novel object recognition (NOR) and Morris water maze (MWM) tests were used to evaluate learning and memory. Urine samples were collected from metabolic cages between 11:00 a.m. and 17:00 p.m., prior to the last day of restraint stress induction to determine urinary corticosterone levels. Rats were sacrificed 24 h after the last behavioral test. Blood and adrenal glands were collected for measurement of serum corticosterone levels and adrenal weights, respectively. Brains were removed and the target proteins were analysed in the regions associated with stress responses by Western blot analysis. The experimental protocol and the descriptions of animal groups are presented in Figure3.1 and Table3.1.

Figure 3 . 1

 31 Figure 3.1 Schematic diagram showing the time-course of restraint stress induction and sample collections in the experimental protocol 1.

Figure 3 . 2

 32 Figure 3.2 Schematic diagram showing the time-course of restraint stress induction, voluntary wheel running, and sample collections in the experimental protocol 2.

Figure 3 . 3

 33 Figure 3.3 Schematic diagram showing the time-course of treatments and sample collections in the experimental protocol 3.

4. 1

 1 Elevated plus-maze test (EPM) The EPM was made of black painted wood and consisted of two open arms (50 cm long × 10 cm wide) at right angles to two closed arms (50 cm long × 10 cm wide × 40 cm high). The maze was elevated to 50 cm above the floor. To prevent the rats from falling, the open arms were surrounded by a 1 cm high Plexiglas rim (Figure 3.4).

Figure 3 . 4

 34 Figure 3.4 Schematic showing, the EPM apparatus that consisted of two open arms (50 cm long × 10 cm wide) at right angles to two closed arms (50 cm long × 10 cm wide × 40 cm high). The whole apparatus was elevated to 50 cm above the floor.

Figure 3 . 5

 35 Figure 3.5 Schematic showing, the ETM apparatus that consisted of three arms of equal dimensions (50 cm long ×10 cm wide) and elevated 50 cm above the floor. One arm, enclosed by walls (40 cm high) was perpendicular to the two opposed open arms.

Figure 3 . 6

 36 Figure 3.6 Schematic showing, A. The OFT was made from a black painted wooden box (76 cm long × 57 cm wide × 35 cm high) with a 48 square grid floor (6 × 8 squares, 9.5 cm per side). B. The arena of maze was divided into two zones, i.e., inner (white area) and outer zones (gray area).

  Time exploring novel object (s) -Time exploring familiar object (s)Total exploration time (s)

  and Carman and Mactutus, 2001. The water was made opaque with 200 ml of milk. Each rat was gently placed in one of the four quadrants (North, South, East, and West) at the start of each trial. A stainless steel platform (10 cm diameter and 30 cm high) was placed 1 cm beneath the surface of the water. Lighting in the room was arranged to provide even illumination in all quadrants. The spatial visual cues consisting of different shapes and colors (i.e., black/white circle and cross and black grid) were visible on each wall of the room to provide orientation during the navigational learning trials and memory probe test.

Figure 3 . 7

 37 Figure 3.7 Schematic showing the MWM pool that consisted of a circular aluminum pool (150 cm diameter and 60 cm deep) that was filled with water (25 °C temperature) up to 31 cm. The pool was divided into four quadrants of equal area. An aluminum platform (10 diameter and 30 cm high) was submerged 1 cm below the water surface.

Figure 3 . 8

 38 Figure 3.8 Brain cutting block illustrating orientation of brain and placement of razor blades to obtain coronal brain sections. The numbers on the right refer to brain sections. Coronal brain sections from which brain regions were dissected. Dotted lines indicate borders of brain regions. FC, frontal cortex; S, septum; aH, anterior hypothalamus; pH, posterior hypothalamus; A, amygdala; VTA, ventral tegmentum area; and H, hippocampus[START_REF] Heffner | A rapid method for the regional dissection of the rat brain[END_REF].

Figure 3 . 9

 39 Figure 3.9 Coronal brain sections at the levels of periaqueductal grey and dorsal raphe nuclei (A) and locus coeruleus (B) (Paxinos and Watson, 2005).

  galactosidase (LacZ) reporter into melatonin (MT) receptor 1A (MTNR1A, MT1) and 1B (MTNR1B, MT2) gene locus of MT-proficient C3H mice and MT-deficient C57BL/6 mice (1.518 months, 2060 g) were obtained from the Institute of Cellular and Integrative Neurosciences, University of Strasbourg, France. Mice were housed in the control room of the conventional zone of the Chronobiotron, UMS3415 with 12:12-h light:dark cycle with food and water ad libitum. All efforts were made to minimize the number of animals used. Experimental procedures were approved by the Animal Care and Use Committee of Institute of Cellular and Integrative Neurosciences, University of Strasbourg, France (Figure 3.10).

Both

  MT1 and MT2 transgenic knock-in mice had their genotypes confirmed by polymerase chain reaction (PCR) technique. Wild-type and transgenic mice of different age groups were euthanized by CO 2 inhalation and intracardially perfused with fixative solutions. The free-floating sections were stained either for immunocytochemistry or LacZ enzymatic activity detection. To characterize the phenotype of MT1/MT2-LacZ expressing neurons in transgenic knock-in mice by determination of co-expressed neuropeptides and neuronal markers.Polyethylene glycol (PEG)-embed brains were cut and subjected to double staining with LacZ immunocytochemistry and non-radioactive in situ hybridization using a digoxigenin-labeled neuropeptideor neuronal markers i.e., arginine vasopressin (AVP), corticotropin-releasing hormone (CRH), gastrin releasing peptide (GRP), glutamate decarboxylase (GAD1/2), LacZ and vasoactive intestinal polypeptide (VIP).

Figure 3 .

 3 Figure 3.10 Schematic diagram showing the experimental protocols in the study of part 2.

  Tail tips (23 mm) from new born transgenic mice (four to seven-day-old) were collected and stored frozen at -20 C for DNA extraction. Tissue samples were digested in 0.5 M NaOH, 200 mM EDTA, and H 2 O for 1 h at 95 C. The samples were mixed with 1 M Tris HCL, pH 5 and centrifuged at 13,000 rpm for 3 min at room temperature, and the supernatants were used for PCR genotyping. One (1) µL supernatant was transferred to 15-µL PCR reaction mixture containing 1.5 µl Taq DNA polymerase 10X buffer without MgCl 2 and detergent (Euromedex, Souffelweyersheim, France), 1.5 µl 25 mM MgCl 2 , 0.75 µl DMSO (Hybri-Max, Sigma D2650), 0.5 µl 10 mM dNTP, 0.25 µl 50 µM forward primers, 0.25 µl 50 µM reverse primers, 0.2 µl Taq DNA polymerase (Euromedex), and 9.05 µl molecular biology grade H 2 O. The corresponding primers are shown in Table3.6.

5. 1

 1 Preparation of plasmid DNAFor cloning probe sequences into plasmid, mouse brain complementary DNAs (cDNA) were amplified with target primers (Table3.7) in PCR reaction mixtures. The 1 µL cDNA was transferred to 20-µL PCR reaction mixtures containing 2 µl AmpliTaq® 360 DNA polymerase 10X buffer (Applied Biosystems, CA, USA), 0.1 µl Taq DNA polymerase, 1.6 µl 20 mM MgCl 2 , 0.4 µl 10 mM dNTP, 1.6 µl 20 µM forward primers, 1.6 µl 20 µM reverse primers, and 11.7 µl molecular biology grade H 2 O. Touchdown PCR cycling program was selected that the annealing temperature was gradually reduced. The PCR products were kept at 4 °C after amplification. Ten µl of each PCR product and mass ruler DNA ladder Mix (Euromedex) was electrophoresed through a 1.5% agarose gel in TBE that was then stained with 1g/ml ethidium bromide. DNA fragments were visualized and extracted the correct band using a Zymoclean™ gel DNA recovery kit (Zymo Research Corp, CA, USA). DNA quantity and quality were measured OD260/OD280 ratio by NanoDrop 2000 spectrophotometer. DNA fragments were ligated and cloned into the pT7-Blue 1 vector (Perfectly Blunt TM Cloning Kits, Novagen, Germany). The ligation products were transformed into bacteria (E. coli strains) and colonies were grown overnight at 37 C. Bacterial colonies containing DNA fragments were screened and performed colony PCR. The primer sets of 5'TTTTCCCAGTCACGACGTT3' (primer U19) and 5'ATGACCATGATTACGC3' (primer R20) were used to determine the size and orientation of the inserted cDNA. The expected sizes of PCR products for pT7-Blue 1 vector in both the presence and absence of the 212 bp positive control insert were 468 bp and 256 bp, respectively. The selected colony was then dropped it into Luria-Bertani (LB) broth medium in a 10 mL tube and incubated overnight with shaking at 37 C. Liquid cultures were collected and processed for Sarawut Lapmanee Materials and Methods / 62 plasmid DNA isolation (Qiagen Miniprep kit, Qiagen, Valencia, CA, USA). Bacteria containing the correct plasmid were stored in glycerol stocks and frozen at 80 C.

  To examine the time-dependent effect of the 1-, 4-and 8week stress induction on anxiety-, depression-, and memory impairment-like behaviors and changes in the expression of selected target proteins in the brain regions associated with stress responses.1. Restraint stress induced a change in body weight, adrenal gland weight, food intake, and corticosterone levels.There was no difference in the starting body weight (Figure4.1A).Following the 1-, 4-, and 8-week restraint stress induction, the final body weights of stressed rats were decreased (Figure4.1B), resulting from less body weight gain when compared to non-stress control rats (Figure4.1C). Daily food intake was decreased in the 4-week stressed rats (Figure4.1D). In addition, the adrenal gland weights (dry weight and dry weight-body weight ratio) in the 4-and 8-week stressed rats were increased when compared with non-stress control rats (Figure4.1F and 4.1H). In addition, the results from experimental 2 showed that the 4-week stressed rats had increased serum and urinary corticosterone levels (Figure4.2A-B). These could be correlated with adrenal hypertrophy. Therefore, the 4-weeks restraint stress induction effectively activated the HPA axis in male rats.

Figure 4 . 1

 41 Figure 4.1 Time-dependent effects of the 1-, 4-, and 8-week restraint stress induction on (A) starting body weight, (B) final body weight, (C) body weight gain, (D) daily food intake, (E) wet adrenal gland weight, (F) dry adrenal gland weight, (G) relative wet adrenal gland weights, and (H) relative dry adrenal gland weights. Numbers of animals are noted in parentheses. *P < 0.05, **P < 0.01, ***P < 0.001 compared to non-stress control group.

Figure 4 . 2

 42 Figure 4.2 Time-dependent effects of the 4-week restraint stress induction on (A) basal serum and (B) urinary corticosterone levels. Numbers of animals are noted in parentheses. *P < 0.05 compared to non-stress control group.

  4.7A). The 1-and 4-week stressed rats had spatial memory impairment as shown by an increased time in the correct quadrant (Figure4.7B). Percent correct quadrant was similar among stressed rats and non-stressed control rats (Figure4.7C). Moreover, the 4-week stressed rats showed a lower discrimination ratio than non-stressed control rats in the NOR study(Figure 4.7D). Therefore, the 1-week restraint stress induction produced memory impairment and paradoxical effects on learning in MWM test, whereas the 4-week restraint stress produced learning and memory impairment as evaluated by MWM and NOR tests.

Figure 4 . 3

 43 Figure 4.3 Time-dependent effects of the 1-, 4-, and 8-week restraint stress-induced anxiety-like behavior as evaluated by EPM, (A) percent open arm entry, (B) percent open arm time, (C) percent closed arm entry, (D) percent closed arm time, (E) percent center of arm time, (F) total arm entry, (G) numbers of rearing, and (H) numbers of grooming. Numbers of animals are noted in parentheses. *P < 0.05 and **P < 0.01 compared to non-stressed control group.

Figure 4 . 4

 44 Figure 4.4 Time-dependent effects of the 1-, 4-, and 8-week restraint stress-induced anxiety/learned and innate fear-like behavior as evaluated by ETM, (A) baseline latency, (B) avoidance latency 1, (C) avoidance latency 2, and (E) one-way escape latency. Numbers of animals are noted in parentheses. *P < 0.05 and ***P < 0.001 compared to non-stressed control group.

Figure 4 . 5 Figure 4 . 6

 4546 Figure 4.5 Time-dependent effects of the 1-, 4-, and 8-week restraint stress-induced anxiety-like behavior as evaluated by OFT, (A) number of lines crossed in the first 30 seconds, (B) total lines crossed, (C) inner zone time, (D) outer zone time, (E) number of rearing, and (F) number of grooming. Numbers of animals are noted in parentheses. *P < 0.05 compared to non-stressed control group.

Figure 4 . 7

 47 Figure 4.7 Time-dependent effects of the 1-, 4-, and 8-week restraint stress-induced learning and memory impairment-like behavior as evaluated by MWM and NOR, (A) escape latency, (B) correct quadrant time, (C) percent correct quadrant time, and (D) discrimination ratio. Numbers of animals are noted in parentheses. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to non-stressed control group.

Figure 4 .

 4 12A and E). In contrast, the 4-or 8-week stress induction decreased 5-HT 2C R protein expression in periaqueductal gray and the ventral tegmental area when compared to non-

Figure 4 . 8

 48 Figure 4.8 Time-dependent effects of the 1-, 4-, and 8-week stress induction on the BDNF protein levels. Numbers of animals are noted in parentheses. *P < 0.05 compared to non-stressed control group.

Figure 4 . 9 Figure 4 .

 494 Figure 4.9 Time-dependent effects of 1-, 4-, and 8-week restraint stress induction on the hypothalamic and hippocampal GR protein levels. Numbers of animals are noted in parentheses. *P < 0.05 and ***P < 0.001 compared to non-stressed control group.

Figure 4 . 11

 411 Figure 4.11 Time-dependent effects of the 1-, 4-, and 8-week restraint stress induction on the SERT protein levels. Numbers of animals are noted in parentheses. *P < 0.05 compared to non-stressed control group.

Figure 4 .

 4 Figure 4.12 Time-dependent effects of the 1-, 4-, and 8-week restraint stress induction on the 5-HT 2C R protein levels. Numbers of animals are noted in parentheses. *P < 0.05, and **P < 0.01 compared to non-stressed control group.

  Figure 4.14A-B). Both non-stressed and stressed rats gradually increased running distance through experimental period suggesting that stress did not affect the general locomotor activity in exercise training. Voluntary wheel running increased wet and dry heart weights in both non-stressed sedentary control and stressed rats (Figure 4.14C-F) indicating that the aerobic exercise protocol induced cardiac hypertrophy.In addition, only the non-stressed exercised control rats, but not stressed exercised rats, had an increase in dry adrenal gland weight when compared with nonstressed sedentary control rats (Figure4.15A-D). Therefore, voluntary wheel runninginduced increase in adrenal weights might represent a modulation of sympatho-adrenal systems. Sedentary stressed rats had increased serum corticosterone levels when compared with non-stressed sedentary control rats. The combined stressed and Sarawut Lapmanee Results / 82 exercised rats had a tendency to decrease corticosterone levels when compared to sedentary stressed rats (Figure4.15E). These results suggest that voluntary exercise could partially alleviate and prevent stress-induced increase in serum corticosterone.

Figure 4 .

 4 Figure 4.13 The effects of the 4-week voluntary wheel running on (A) starting body weight, (B) final body weight, (C) body weight gain, and (D) daily food intake. Numbers of animals are noted in parentheses. **P < 0.01, ***P < 0.001 exercise group compared to non-stressed sedentary control. # P < 0.05 sedentary stressed group compared to non-stressed sedentary control group. † P < 0.05 combined stressed and exercised group compared to sedentary stressed group.

Figure 4 .

 4 Figure 4.14 The effects of the 4-week voluntary wheel running on (A) running distance per day, (B) total running distance, (C) wet heart weight, (D) dry heart weight, (E) relative wet heart weight, and (F) relative dry heart weight. Numbers of animals are noted in parentheses. **P < 0.01, ***P < 0.001 exercise group compared to non-stressed sedentary control. † P < 0.05, † † † P < 0.001 combined stressed and exercised group compared to sedentary stressed group.

Figure 4 .

 4 Figure 4.15 The effects of the 4-week voluntary wheel running on (A) wet adrenal weight, (B) dry adrenal weight, (C) relative wet adrenal weight, (D) relative dry adrenal gland weight, and (E) serum corticosterone levels. Numbers of animals are noted in parentheses. **P < 0.01 exercised group compared to non-stressed control sedentary group. ## P < 0.05, ## P < 0.01 sedentary stressed group compared to nonstressed sedentary control group. † P < 0.05 combined stressed and exercised group compared to sedentary stressed group.

Figure 4 . 16

 416 Figure 4.16 Effects of the 4-week voluntary wheel running on restraint stress-induced depression-like behavior as determined by sucrose preference test. Numbers of animals are noted in parentheses. ### P < 0.001 sedentary stressed group compared to non-stressed sedentary control group. † P < 0.05 combined stressed and exercised group compared to sedentary stressed group.

(

  hypothalamus (Figure4.18A-B). In addition, the presence of ACTH receptor in the adrenal cortex was downregulated in the combined stressed and exercised group than in the sedentary stressed group (Figure4.18C). These results suggest that stress exposure and voluntary wheel running can modulate both central and peripheral HPA regulations.3.3 Levels of NETStress decrease in NET protein levels in the locus coeruleus but decreased in the periaqueductal gray. Voluntary wheel running decreased NET protein levels in the locus coeruleus and the ventral tegmental area. Similarly, NET protein levels were decreased in the locus coeruleus in the combined stressed and exercised group (Figure4.19D-F).3.4 Levels of SERTStressed rats had decreased in SERT protein levels in the hippocampus and dorsal raphe(Figure 4.20B and D). Voluntary wheel running increased SERT protein expression in the septum when compared to non-stressed sedentary controls. The combined stressed and exercised rats had reduced SERT protein levels in the ventral tegmentum area when compared to sedentary stressed rats (Figure4.20F-G).

Figure 4 . 18

 418 Figure 4.18 Effects of the 4-week voluntary wheel running on the hypothalamic and hippocampal GR and adrenal MC2R protein levels. Numbers of animals are noted in parentheses. # P < 0.05 sedentary stressed group compared to non-stressed sedentary control group. † P < 0.05 combined stressed and exercised group compared to sedentary stressed group.

Figure 4 .

 4 Figure 4.19 Effects of the 4-week voluntary wheel running on NET protein levels.Numbers of animals are noted in parentheses. *P < 0.05, **P < 0.01 exercised group compared to non-stressed control sedentary group. # P < 0.05 sedentary stressed group compared to non-stressed sedentary control group. † P < 0.05 combined stressed and exercised group compared to sedentary stressed group.

Figure 4 .

 4 Figure 4.20 Effects of the 4-week voluntary wheel running on SERT protein levels.Numbers of animals are noted in parentheses. **P < 0.01 exercised group compared to non-stressed control sedentary group. # P < 0.05, ## P < 0.01 sedentary stressed group compared to non-stressed sedentary control group. † P < 0.01 combined stressed and exercised group compared to sedentary stressed group.

Experiment 3 .

 3 To evaluate the effectiveness of agomelatine, venlafaxine, and voluntary wheel running exercise in the prevention of stress-induced anxiety-, depression-, and memory impairment-like behaviors and changes in the expression of selected target proteins in brain regions associated with stress responses.1. The 4-week pre-treatment of agomelatine, venlafaxine, and voluntary wheel running induced changes in body weight, adrenal gland weight, food intake, and corticosterone levels.There was no difference in the starting body weight. Following the 4-week pre-treatments, voluntary wheel running animals had lower body weight gain and food intake. Exercising rats gradually increased their running distance throughout the exercise training (Figure4.22A-D). Agomelatine-and venlafaxine-treated rats did not exhibit changes in their body weight gain but their food intake was higher than that of the vehicle-treated sedentary stressed rats (Figure4.22C). Despite gaining body weight after stopping voluntary wheel running (Figure 4.22F), exercising rats subjected to stress induction had a decrease in the final body weight (Figure 4.22D), thus, demonstrating detraining effects of exercise. Therefore, pre-treatments with monoamine modulators and voluntary exercise could prevent the stress-induced decrease in body weight and food intake. Pre-treatment of agomelatine, venlafaxine or voluntary wheel running did not alter adrenal weights (Figure 4.23A-D) but serum corticosterone levels of agomelatine-treated rats was higher than non-stressed controls and vehicle-treated sedentary stressed rats (Figure 4.23E).

Figure 4 .

 4 Figure 4.22 Protective effects of agomelatine (Ago), venlafaxine (Vlx) and voluntary wheel running (Rw) on (A) starting body weight, (B) running distance, (C) body weight, (D) final body weight, (E) daily weight gain in pre-treatment, (F) daily weight gain in stress induction, (G) daily food intake in pre-treatment, and (H) daily weight gain in stress induction. Numbers of animals are noted in parentheses. † P < 0.05, † † P < 0.01, and † † † P < 0.001compared to vehicle (Veh)-treated group

Figure 4 .

 4 Figure 4.23 Protective effects of agomelatine (Ago), venlafaxine (Vlx) and voluntary wheel running (Rw) on (A) wet adrenal weight (B) dry adrenal weight, (C) relative wet adrenal weight, (D) relative dry adrenal weight, (E) serum corticosterone levels in stressed male rats. Numbers of animals are noted in parentheses.

Figure 4 .

 4 Figure 4.24 Protective effects of agomelatine (Ago), venlafaxine (Vlx) and voluntary wheel running (Rw) on stress-induced anxiety-like behavior as determined by EPM, (A) percent open arm entry, (B) percent open arm time, (C) percent closed arm entry, (D) percent closed arm time, (E) percent center of arm time, (F) total arm entries, (G) numbers of grooming, and (H) numbers of rearing. Numbers of animals are noted in parentheses. † P < 0.05 compared to vehicle (Veh)-treated group.

Figure 4 .

 4 Figure 4.25 Protective effects of agomelatine (Ago), venlafaxine (Vlx) and voluntary wheel running (Rw) on stress-induced anxiety/learned and innate fear-like behavior as determined by ETM, (A) baseline latency, (B) avoidance latency 1, (C) avoidance latency 2, and (D) one-way escape latency. Numbers of animals are noted in parentheses. † † † P < 0.001 compared to vehicle (Veh)-treated group.

Figure 4 .

 4 Figure 4.26 Protective effects of agomelatine (Ago), venlafaxine (Vlx) and voluntary wheel running (Rw) on stress-induced anxiety-like behavior as determined by OFT, (A) inner zone time, (B) outer zone time, (C) number of lines crossed in the first 30 seconds, (D) total lines crossed, (E) numbers of rearing, and (F) numbers of grooming.

Figure 4 .

 4 Figure 4.27 Protective effects of agomelatine (Ago), venlafaxine (Vlx) and voluntary wheel running (Rw) on stress-induced depression-like behavior as determined by FST and sucrose preference test, (A) swimming duration, (B) climbing duration, (C) immobility duration, (D) numbers of fecal pellets, and (E) weekly sucrose preference.

Figure 4 .

 4 Figure 4.28 Protective effects of agomelatine (Ago), venlafaxine (Vlx) and voluntary wheel running (Rw) on stress-induced learning and memory impairment-like behavior as determined by MWM and NOR, (A) escape latency, (B) correct quadrant time, (C) percent correct quadrant time, and (D) discrimination ratio. Numbers of animals are noted in parentheses. † P < 0.05, † † P < 0.01, † † † P < 0.001 compared to vehicle (Veh)treated group.

Figure 4 .

 4 Figure 4.29 Protective effects of agomelatine (Ago), venlafaxine (Vlx) and voluntary wheel running (Rw) on the BDNF protein levels. Numbers of animals are noted in parentheses. † P < 0.05, † † P < 0.01, and † † † P < 0.001 compared to vehicle (Veh)-treated group.

Figure 4 .

 4 Figure 4.30 Protective effects of agomelatine (Ago), venlafaxine (Vlx) and voluntary wheel running (Rw) on the hypothalamic and hippocampal GR protein levels. Numbers of animals are noted in parentheses. † P < 0.05 compared to vehicle (Veh)treated group.

  ; BDNF: Brain-derived neurotrophic factor; EPM: elevated-plus maze; ETM: elevated-T maze; FST: forced swimming test; GR: glucocorticoid receptor; LC: locus coeruleus; MWM: Morris water maze; NET: norepinephrine transporter; NOR: novel objective recognition; OFT: open-field test; PAG: periaqueductal gray; SERT: serotonin reuptake transporter; SP: sucrose preference; Vlx: venlafaxine; Rw: wheel running; 5-HT 2C R: serotonin type 2C receptor. Part II: Characterization of melatonin receptor expressing cells in the rodent brain using MT1 and MT2 melatonin receptor LacZ knock-in reporter mice: Identification of potential action sites of agomelatine on brain structures involved in stress.

Figure 4 .

 4 Figure 4.34 Generation of the MT1-/MT2-LacZ knock-in mice. (A) The MT1 or MT2 receptor gene locus organization before and after site-specific insertion. (B) Mouse genotyping by PCR for MT1-/MT2-LacZ transgenic knock-in mice.

Figure 4 .

 4 Figure 4.35 Comparison MT1-LacZ staining between immunocytochemistry (A and C) and X-gal histochemistry (B and D) in the suprachiasmatic nucleus (upper) and pars tuberalis of anterior pituitary (lower) of transgenic knock-in mice.

Figure 4 .

 4 Figure 4.36 MT1-LacZ expressions in neurons as detected by X-gal histochemical staining compared to 125 I-iodomelatonin binding autoradiography by Liu et al., 1997.

Figure 4 .Figure 4 .

 44 Figure 4.37 X-gal histochemical mapping of MT1-and MT2-LacZ expression.

Figure 4 .

 4 Figure 4.37 X-gal histochemical mapping of MT1-and MT2-LacZ expression (cont.).

Figure 4 .

 4 Figure 4.38 Immunocytochemical staining of MT1-LacZ expression in (A) the suprachiasmatic nucleus; SCN and (B) pars tuberalis of anterior pituitary; PT. Nuclear LacZ staining and lysosomal accumulation of LacZ can be seen in the SCN (C). In the PT (D), the cytoplasmic staining is very strong, but sometimes the lysosomal accumulation can also be seen (arrows).

Figure 4 .

 4 Figure 4.39 Immunocytochemical staining of MT2-LacZ expressing neurons in (A) the paraventicular nucleus of the hypothalamus, (B) the arcuate nucleus of the hypothalamus, (C) the dorsal hippocampus, (D) the ventral hippocampus, (E) the CA2 of the hippocampus, and (F) the molecular layer of the dentate gyrus. DG; dentate gyrus of hippocampus.

MT2-

  LacZ expression was present in some GAD2 neurons in the hippocampus(Figure 4.42). GABAergic interneurons are inhibitory neurons. Melatonin signaling through MT1 or MT2 receptors is mostly inhibitory. Thus MT action (and agomelatine action) should result in the inhibition of these inhibitory interneurons, and thus an activation of hippocampal activity. But in the CA2, MT2-LacZ expression is seen in the pyramidal neurons, which are excitatory glutamatergic neurons(Figure 4.43). Thus melatonin (and agomelatine) action should result in the selective inhibition of these CA2 neurons and thus an inhibition of CA2-mediated hippocampal action. The neurons staining most intensely for MT2-LacZ were small horizontal neurons located in the outer molecular layer of the dentate gyrus. No description of this population could be found in the literature and we could not colocalize these neurons with any of the neuropeptide markers we tested (i.e., calbindin, choline acetyltransferase, galanin, neuropeptide Y, and somatostatin). Only a very few of these cells colocalized with GAD.

Figure 4 .

 4 Figure 4.40 Double-labeling with MT1-LacZ-Immunohistochemistry (green) and nonradioactive in situ hybridization for neuropeptides (red). Diagram representing of the cell and fiber distributions of neuropeptides in the SCN (Abrahamson and Moore, 2001) (A) AVP, (B), GRP, and (C) VIP. For AVP, the green dots of the MT1-LacZ cells are clearly located outside of the area of the AVP cells (D). For VIP (E) and GRP (F) some red neuropeptide cells contain the green dot indicating co-expression of the MT1-LacZ transgene (arrows/arrowheads).

Figure 4 .

 4 Figure 4.41 Double-labeling with LacZ-immunohistochemistry for MT2 (green) and non-radioactive in situ hybridization for CRH (red) in the paraventricular nucleus (PVN) of the hypothalamus. Inset shows a group of CRH cells that are MT2 positive. Asterisk shows CRH neurons without MT2 label. Most GRP cells are MT2 positive.

Figure 4 .

 4 Figure 4.42 Double-labeling with LacZ-Immunohistochemistry and non-radioactive in situ hybridization for MT2 (green) and GAD (red) co-localization in the hippocampus (A). Most small horizontal MT2 positive neurons in the external molecular layer (B) are GAD negative (C). Only a few of these horizontal cells are GAD positive (arrow in D). Many GAD positive neurons in the dentate gyrus hilus are also MT2 positive (arrowheads in E).

Figure 4 .

 4 Figure 4.43 Double-labeling with LacZ-Immunohistochemistry and non-radioactive in situ hybridization for MT2 (green) and GAD (red) co-localization. The MT2 positive cells in the CA2 of the hippocampus are GAD negative. A few non-pyramidal cells of the CA2 are MT2 + GAD positive (arrowheads).

Furthermore, voluntary wheel

  running decreased the central NET, SERT, and 5-HT 2C R in brain circuits of the reward system (i.e., LC, VTA, DR, PAG, and NA). Stress promoted activity of HPA axis as indicated by increase in corticosterone, which in turn feedbacked to decrease GR in the hippocampus. Exercise tended to reduce corticosterone possibly by decreasing ACTH (MC2R) receptors in the adrenal glands. The combined exercise and stress partially decreased the basal serum corticosterone levels, upregulated hippocampal BDNF, and downregulated adrenal MC2R and 5-HT 2C R in the PFC. BDNF plays a role in regulating hippocampal

  voluntary exercise should be recommended as an alternative treatment or used in concert with pharmacological treatments (i.e., agomelatine or venlafaxine) for the treatment of anxiety-, depression-, and memory impairment-like symptoms in patients and stressed individuals. Indeed, mapping and phenotyping of MT1 and MT2 receptors expressing neurons indicate possible sites of action for melatonin or melatonin derivatives. MT1-LacZ expressing neurons were found in some studied areas, i.e., suprachiasmatic nucleus (SCN), paraventicular nucleus of thalamus (PVT), and pars tuberalis (PT) of anterior pituitary gland, whereas MT2-LacZ expressing neurons were more widely distributed throughout the whole brain, e.g., SCN, paraventicular nucleus of hypothalamus (PVN), dorsal raphe (DR), amygdala, and CA2 of hippocampus. Further studies are required to clarify the mode of action of agomelatine.Taken together, the 4-week stress caused hyperactivity of HPA axis and increased glucocorticoid/corticosterone levels leading to hippocampal GR and BDNF downregulation. Four-week voluntary wheel running modulated the HPA axis and partially decreased corticosterone levels by downregulating adrenal ACTH receptor and also promoted hippocampal BDNF upregulation resulting in decreased anhedoniclike behaviors in stressed male rats. Furthermore, voluntary exercise prevented stressinduced anxiety, depression, and memory impairment. Pre-treatment with venlafaxine directly inhibited NET to increase norepinephrine (NE) in the synaptic cleft and mediated antidepressant and anxiolytic effects via hippocampal BDNF production. In addition, pre-treatment with agomelatine, a MT receptor agonist, effectively prevented stress-related behaviors. Potential sites of action for these effects through MT1 and MT2 receptors are the SCN, the PVN, the dorsal raphe (DR), and the hippocampus.

Figure 6 . 1

 61 Figure 6.1 Diagram showing the summary results in this present study.

  Effets du stress de contention, des modulateurs pharmacologiques (Agomélatine, Venlafaxine) et de l'activité de roue volontaire chez des rats mâles Expérience 1 : Dans le groupe des animaux stressés, un stress de contention pendant 1 et 4 semaines induisait une anxiété plus élevée qu'un stress de contention pendant 8 semaines dans les tests EPM, ETM et OFT. Les animaux soumis pendant 1 semaine au stress exhibaient des comportements anxieux forts. Cependant, un stress d'une semaine stimulait la mémoire spatiale dans le test MWM. Les rats stressés pendant 4 semaines présentaient des comportements anxieux et dépressifs, ainsi que des perturbations de la mémoire, en parallèle avec une augmentation des taux de corticostérone dans le sang et les urines. De plus, le stress pendant 4 semaines augmentait l'expression du NET et du 5-HT 2c dans le locus coeruleus et dans l'amygdale, et diminuait l'expression hypothalamique du GR. Ces résultats indiquent une perturbation du rétrocontrôle de l'axe HPS (Smith & Vale 2006). L'augmentation de l'expression du NET et du 5-HT2C dans le cerveau antérieur et le locus coeruleus pourrait causer des comportements liés au stress (Kimura et al. 2009, Chen et al. 2012). Cependant, un stress de 8 semaines induisait moins de comportements liés au stress dans les tests comportementaux. Un stress d'une telle durée pourrait induire des adaptations compensatoires (Dhabar et al. 1997). Ces résultats montrent qu'un stress de 4 semaines est suffisant pour induire de l'anxiété et de la dépression, ainsi que des perturbations de la mémoire. Nous avons donc utilisé ce protocole de stress de 4 semaines pour la suite de nos études.

  , la venlafaxine et l'exercice volontaire de roue peuvent prévenir les comportements anxieux et les perturbations de la mémoire. Les prétraitements par l'agomélatine, la venlafaxine ou l'exercice volontaire n'ont pas changé le poids des surrénales, mais les niveaux de corticostérone sérique chez les animaux traités à l'agomélatine étaient plus élevés que ceux d'animaux contrôles sans exercice de roue et traités uniquement avec le véhicule. Les rats traités par l'agomélatine et la venlafaxine étaient sensible aux niveaux élevés de corticostérone de manière prolongée, ce qui a résulté en une diminution du SERT, du NET, du BDNF et du GR dans les régions contribuant aux réponses autonomiques et comportementales, p.ex. le septum, le locus coeruleus, le gris périacquéducal et l'hippocampe. La venlafaxine et l'activité volontaire de roue ont significativement augmenté les niveaux de BDNF chez des rats stressés. Il est donc possible que les prétraitements par l'agomélatine, la venlafaxine et l'exercice physique volontaire puissent normaliser et coordonner la balance des glucocorticoïdes et du métabolisme des neurotransmetteurs, et induire des adaptations au long terme de la neurotransmission monoaminergique et même de la neurogenèse dans le cerveau (Cotman & Berchtold 2002, Dagyte et al. 2010, Huang et al. 2014), adaptations qui ont été maintenues pendant l'intégralité de la période de stress.

Partie II :

 : Caractérisation des cellules exprimant les récepteurs MT1 ou MT2 dans le cerveau de souris rapportrices LacZ knock-in : Identification de sites d'action potentiels de l'Agomélatine dans des structures impliquées dans le stress Expérience 1 : Validation du modèle transgénique et cartographie des cellules exprimant le MT1-LacZ ou le MT2-LacZ chez des souris transgéniques knock-in Génotypage des souris MT1-LacZ et MT-LacZ Le rapporteur LacZ (beta-galactosidase) a été introduit dans le locus MT1 ou MT2 pour analyser l'expression de ces deux gènes. Pour les deux loci, le transgène LacZ a été inséré en phase avec le codon start ATG original, retenant ainsi toutes les séquences régulatrices en amont et en aval du locus. Seul des séquences régulatrices potentielles situées dans l'intron unique des gènes MT1 ou MT2 ont été délétées par cette stratégie. Ainsi ces souris rapportrices knock-in MT1-LacZ ou MT2-LacZ sont considérées un outil idéal pour investiguer les sites d'expression des récepteurs de la mélatonine MT1 et MT2 en absence d'anticorps spécifiques fiables pour la détection immunocytochimique de ces récepteurs. Nous avons d'abord sélectionné des animaux hétérozygotes MT1-LacZ KI+ ou MT2-LacZ KI+ pour les études initiales. Ces animaux hétérozygotes disposent d'un allèle MT1 ou MT2 intact, réduisant ainsi les risques d'expression ectopique du transgène du fait d'une perte de fonction complète du récepteur MT1 ou MT2. L'étude de Liu et al. (1997) a montré que la délétion d'un allèle du locus MT1 réduisait effectivement le niveau de liaison de la 125 I-iodomélatonine de 50%, mais n'affectait en rien le patron d'expression. Les animaux hétérozygotes ont aussi été croisés entre eux pour produire des animaux homozygotes MT1-LacZ KIKI et MT2-LacZ KIKI , qui sont de fait des animaux invalidés (knockout KO) soit pour le MT1 ou le MT2 comme alors les deux allèles sont remplacés par LacZ. Les animaux transgéniques ont été génotypés par PCR. Chez les animaux MT1-LacZ, un produit de 425 bp indique un allèle MT1 sauvage, donc intact, et un produit de 196 bp indique un allèle MT1-LacZ recombinant. Chez les animaux MT2-LacZ un produit de 143 bp indique l'allèle sauvage intact, tandis qu'un produit de 666 bp indique l'allèle MT2-LacZ recombinant. Cartographie de l'expression MT1-LacZ et MT2-LacZ dans le cerveau Nous avons utilisé deux techniques pour visualiser l'expression de LacZ sous le contrôle du promoteur MT1 ou du promoteur MT2. La première technique est la visualisation histoenzymologique de l'activité enzymatique LacZ en utilisant le substrat chromogénique X-Gal (5-bromo-4-chloro-3-indolyl-beta-Dgalactopyranoside) pour cette enzyme. La deuxième technique est la détection immunocytochimique de LacZ à l'aide d'un anticorps spécifique pour cette enzyme. Dans les deux cas, des animaux sauvages non-transgéniques ont été utilisés comme contrôles négatifs. Les deux techniques ont toujours fourni des résultats identiques quant à la localisation de LacZ, même si l'apparence du signal n'était pas toujours identique. Le signal enzymatique LacZ se présentait sous forme de points très fortement colorés contre un fond pratiquement sans signal. Le signal immunocytochimique présentait aussi les mêmes points très fortement marqués, mais aussi du signal dans le noyau et parfois dans diffus dans le cytoplasme des cellules exprimant MT1-LacZ ou MT2-LacZ. Les points fortement marqués à l'intérieur des cellules par les deux techniques sont probablement des accumulations lysosomiales de LacZ. La détection immunocytochimique présentait aussi plus de bruit de fonds que la détection enzymologique. Nous avons utilisé la détection enzymologique pour la cartographie de l'expression MT1-LacZ et MT2-LacZ du fait du rapport signal/bruit très élevé. Tous les résultats obtenus par détection enzymologique ont été confirmés par détection immunocytochimique. Malheureusement, la détection enzymologique ne permet pas une combinaison avec une détection par hybridation in situ pour le phénotypage des cellules marquées. Nous avons donc utilisé l'immunocytochimie LacZ pour le phénotypage des cellules exprimant MT1-LacZ ou MT2-LacZ en le combinant avec un autre signal immunocytochimique ou d'hybridation in situ. Les cellules MT1-LacZ étaient restreintes à quelques structures bien distinctes : le noyau suprachiasmatique (SCN), le noyau paraventriculaire du thalamus (PVT), le noyau postérieur limitant du thalamus et la pars tuberalis de l'adénohypophyse (PT). Ce marquage est parfaitement superposable à la liaison de la 125 I-iodomélatonine décrite entre autres par Liu et al. (1997), validant ainsi la spécificité de notre modèle transgénique (Figure 1 & 2).

Figure 1

 1 Figure 1 Expression MT1-LacZ détectée par coloration histoenzymologique utilisant le X-gal comme substrat, comparé à la liaison 125 I-iodomelatonine rapportée par Liu et al. (1997).

Figure 2 .

 2 Figure 2. Détection immunocytochimique de l'expression MT1-LacZ dans le noyau suprachiasmatique (SCN, A) et la pars tuberalis de l'adénohypophyse (PT, B). Marquage LacZ dans le noyau et dans des accumulations lysosomiales dans le SCN (C). Dans la PT (D), le signal cytoplasmique est très fort, mais parfois des accumulations lysosomiales peuvent aussi être détectées (flèches).

Figure 3 .

 3 Figure 3. Détection immunocytochimique du MT2-LacZ dans le noyau paraventriculaire de l'hypothalamus (PVN, A), le noyau arqué de l'hypothalamus (B), l'hippocampe dorsal (C), l'hippocampe ventral (D), la CA2 de l'hippocampe (E) et la couche moléculaire du gyrus dentelé de l'hippocampe (F).

  Caractérisation du phénotype des neurones exprimant le MT1-LacZ ou le MT2-LacZ dans des souris transgéniques par détermination de la co-expression de neuropeptides ou de marqueurs neuronauxCo-localisations dans des neurones MT1-LacZAutant la détection enzymatique que la détection immunohistochimique a montré la présence de MT1-LacZ dans des neurones du SCN. Le SCN est subdivisé dans des sous-régions anatomiques qui peuvent être distinguées par leur contenu de neuropeptides tels que la vasopressine (AVP), le gastrin-releasing peptide (GRP) et le vasoactive intestinal peptide (VIP). Nous avons donc sélectionné ces neuropeptides pour le phénotypage des neurones MT1-LacZ. Nous avons pu obtenir des colocalisations pour le GRP et le VIP, mais pas du tout pour l'AVP. Dans le cas du GRP et du VIP, seules certaines cellules GRP ou VIP positives contenaient aussi le MT1-LacZ (Figure4).

Figure 4 .

 4 Figure 4. Double marquages avec du MT1-LacZ par immunocytochimie (fluorescence verte) et de l'hybridation in situ pour des neuropeptides (fluorescence rouge) dans le SCN. Diagrammes représentant la distribution des corps cellulaires et des fibres des neuropeptides dans le SCN (Abrahamson and Moore, 2001) AVP (A), GRP (B) et VIP (C). Les accumulations lysosomiales du MT1-LacZ (points verts) sont clairement situées en-dehors de la zone des neurones à AVP (D). Pour les colocalisations avec le VIP (E) ou le GRP (F), quelques neurones peptidergiques rouges contiennent les points verts des accumulations lysosomiales du MT1-LacZ (flèches).

Figure 5 .

 5 Figure 5. Double marquages avec de l'immunomarquage pour le MT2-LacZ (brun à gauche, fluorescence verte à droite) et de l'hybridation in situ non-radioactive pour le CRH (fluorescence rouge) dans le noyau paraventriculaire de l'hypothalamus (PVN). L'agrandissement montre un groupe de neurones à CRH (rouge) dont la majeure partie exprime aussi le MT2-LacZ (vert). L'astérisque montre un neurone à CRH ne contenant pas de MT2-LacZ.

Figure 6 .

 6 Figure 6. Double marquages avec de l'immunocytochimie pour le MT2-LacZ (brun en A et B, fluorescence verte en C, D et E) et de l'hybridation in situ non-radioactive pour la GAD (fluorescence rouge) dans l'hippocampe. (A) vue générale du marquage MT2-LacZ dans l'hippocampe dorsal. (B) Aspect des petits interneurones horizontaux situés dans la couche moléculaire externe du gyrus dentelé. (C) La plupart de ces petits interneurones n'est pas colocalisés avec la GAD. (D) Seuls quelques rares petits interneurones présentent une colocalisation avec la GAD. (E) Par contre, beaucoup de neurones à GAD dans le hile du gyrus dentelé co-expriment aussi de la GAD.

Figure 7 .

 7 Figure 7. Double marquages avec de l'immunocytochimie pour le MT2-LacZ (brun à gauche, fluorescence verte à droite) et de l'hybridation in situ non-radioactive pour la GAD (fluorescence rouge) dans la CA2 de l'hippocampe. La majorité des cellules exprimant le MT2-LacZ dans la CA2 ne contiennent pas de GAD. Quelques rares cellules non-pyramidales de la CA2 (tètes de flèches) co-expriment le MT2-LacZ avec la GAD.

  ce travail étaient d'abord d'examiner la cinétique des effets d'un stress de contention sur les comportements d'anxiété et de dépression, ainsi que les perturbations de la mémoire chez des rats mâles. En deuxième lieu, nous voulions déterminer les effets thérapeutiques préventifs d'un traitement pharmacologique par des modulateurs monoamingergiques (agomélatine ou venlafaxine) ou d'un exercice physique volontaire sur ces comportements. Outre des paramètres comportementaux, nous avons aussi investigué des paramètres physiques et physiologiques, ainsi que l'expression de protéines cibles dans les régions associées aux réponses de stress. Enfin, nous avons identifié des sites d'action potentiels pour l'agomélatine dans des souris transgéniques rapportrices MT1-LacZ et MT2-LacZ. Les conclusions sont les suivantes, résumées dans la Figure 8 : Un stress de contention de 4 semaines chez le rat mâle réduit la prise de nourriture et le poids corporel, induit une hypertrophie des surrénales et augmente les niveaux sériques et urinaires de la corticostérone. Ces niveaux élevés de corticostérone pourraient altérer l'expression des protéines cibles, telles que le facteur neurotrophique dérivé du cerveau (BDNF), le récepteur aux glucocorticoïdes (GR), le transporteur de la noradrénaline (NET) et le récepteur 5-HT 2C de la sérotonine dans l'amygdale, l'hippocampe, le locus coeruleus et le gris périacquéducal menant à des manifestations d'anxiété, de dépression et des troubles de la mémoire. Un exercice volontaire d'activité de roue de 4 semaines peut partiellement prévenir l'élévation de la corticostérone sérique et les comportements anhédoniques induits par le stress, probablement par une modulation des récepteurs surrénaliens pour l'hormone adrénocorticotrope (ACTH) et en altérant l'expression de protéines cibles (BDNF et NET) dans des structures impliquées dans la réponse au stress telles que l'hippocampe, le septum, le locus coeruleus et le gris périacquéducal. Ainsi, l'exercice physique volontaire pourrait réduire les effets néfastes d'un stress chronique en modulant l'activité sympathique tant au niveau central qu'au niveau périphérique de l'axe HPS. L'agomélatine, la venlafaxine et l'activité volontaire dans une roue peuvent prévenir les comportements anxieux et dépressifs, ainsi que les perturbations de la mémoire, induits par un stress de contention. L'augmentation de l'expression du BDNF dans l'hippocampe pourrait être un mécanisme des effets protecteurs de la venlafaxine et de l'exercice physique chez le rat mâle stressé. Concernant les applications cliniques de nos observations, l'exercice physique volontaire peut être recommandé comme traitement alternatif ou de concert avec un traitement pharmacologique (p.ex. agomélatine ou venlafaxine) pour le traitement des symptômes d'anxiété, de dépression et de perturbation de la mémoire chez des patients et des individus stressés. La cartographie et le phénotypage des neurones exprimant les récepteurs de la mélatonine MT1 et M2 indique des sites d'action potentiels pour la mélatonine et des agents mélatoninergiques tels que l'agomélatine. L'expression du MT1 est restreinte à quelques structures clés, en particulier le noyau suprachiasmatique (SCN), le noyau paraventriculaire du thalamus (PVT) et la pars tuberalis de la glande pituitaire. Le récepteur MT2 quant à lui est beaucoup plus largement exprimé dans le cerveau, avec notablement le noyau suprachiasmatique (SCN), le noyau paraventriculaire de l'hypothalamus (PVN), l'hippocampe, l'amygdale et le raphé dorsal (DR). Des études supplémentaires seront nécessaires pour clarifier le mode d'action de l'agomélatine. En résumé, un stress de contention de 4 semaines cause une hyperactivité de l'axe HPS et augmente le niveau des glucocorticoïdes menant à une réduction d'expression du récepteur aux glucocorticoïdes et du BDNF dans l'hippocampe. Quatre semaines d'activité de roue volontaire module l'activité de l'axe HPS et diminue partiellement les niveaux de glucocorticoïdes en diminuant l'expression du récepteur surrénalien de l'ACTH. Cette activité augmente aussi l'expression du BDNF réduisant les comportements anhédoniques chez le rat mâle stressé. De plus, l'exercice physique volontaire est capable de prévenir l'anxiété, la dépression et les troubles de la mémoire induits par le stress. Un prétraitement par la venlafaxine inhibe directement le NET pour augmenter la noradrénaline dans la fente synaptique et exerce des effets antidépresseurs et anxiolytiques en promouvant la production de BDNF par l'hippocampe. Un prétraitement par l'agomélatine, un agoniste MT1/MT2, peut prévenir les comportements liés au stress en agissant par les récepteurs MT1 et MT2 dans le SCN, le PVN, le raphé dorsal et l'hippocampe.

Figure 8 .

 8 Figure 8. Résumé graphique des résultats de cette étude.

Figure A. 1

 1 Figure A.1 Generation of MT1-/MT2-LacZ knock-in mice. The mutant MT1 (Mtnr1a) or MT2 (Mtnr1b) alleles contain a loxP-hUbC-Neomycin-polyA stop cassette. This Neomycin resistance cassette used for the selection of recombinant embryonic stem cells is then deleted from the mutant locus using Cre-mediated recombination to avoid interference of these sequences with the expression profile of the LacZ reporter gene.

Table 2 .1

 2 Functional anatomy and physiology of brain regions in stress responses.

Table 2 .2 Summary of localization of selected target proteins in the present study Target protein Study model/Methods Brain regions References Brain-derived neurotrophic factor (BDNF)

 2

	Rat/Western blotting,	Cerebral	cortex,	hippocampus,
	Immunocytochemistry,	forebrain, striatum, hypothalamus,
	In situ hybridization	brainstem, cerebellum, amygdala,
		claustrum, substantia nigra, septum,
		bed nucleus of the stria teminalis,
		preoptic nucleus, olivery pretectal
		nucleus, lateral paragigantocellular
		nucleus, trigeminal nuclei, nuclei
		tractus solitarius, superior colliculus,
		geniculate nucleus, and olivary
		pretectal nucleus.

Table 2 .2

 2 Summary of localization of selected target proteins in the present study (cont.).

	Target protein	Study model/Methods		Brain regions		References
	Norepinephrine	Rat/(3)H-nisoxetine	Cerebral cortex, hippocampus,	Zhao et al.,
	transporter	binding, Western	locus coeruleus, amygdala	2008; Zhu et
	(NET)	blotting , In situ				al., 2002;
		hybridization				Chen et al.,
						2012; Fan et
						al., 2014
	Serotonin	Rat/ Western blotting,	Raphe nuclei, olfactory bulb, frontal	Zhou et al.,
	reuptake	Immunocytochemistry,	cortex, locus ceruleus, hypothalamus,	1996; 2000,
	transporter		frontal cortex, striatal neuroepithelia,	Zhang et al.,
	(SERT)		sensory	thalamic	pathways,	2012
			thalamocortical bundles, reticular
			nucleus, internal capsule bundle and
			form barrels in somatosensory
			cortices, hippocampus, cerebellum,
			hippocampus, and amygdala
	Glucocorticoid	Human/Northern blot,	Prefrontal	cortex,	cerebellum,	Sousa et al.,
	receptor (GR)	In situ hybridization	medulla, putamen, hippocampus	1989
		Rats/ Western blotting	Choroid plexus, hippocampus,	Kitraki et al.,
		In situ hybridization	paraventricular and periventricular	1996
			hypothalamic nuclei, dorsal thalamic
			nuclei, layers II and VI of the
			cerebral cortex, olfactory nucleus,
			olfactory cortex, olfactory bulb.
			mammillary nuclei, subthalamus,
			basal ganglia, rhinencephalon, pons,
			cerebellum, amygdala, subiculum,
			and pituitary gland

Serotonin type 2C receptor (5-HT 2C R)

	Human / RT-PCR, Editing efficiency analyses Rats/microdialysis, In situ hybridization, Immunocytochemistry Alex et al., 2Cerebral cortex, substantia nigra, and cerebellum Striatum, prefrontal cortex, nucleus accumbens, amygdala, hippocampus, hypothalamus, choroid plexus. retrosplenial, piriform and entorhinal cortex, olfactory nucleus, septal Niswender et al., 2001 Pompeiano et al., 1994; Clemett et al., 2000;
	nucleus, subthalamic nucleus,
	subiculum and ventral part of CA3,
	lateral habenula, substantia nigra pars
	compacta, brainstem nuclei,
	geniculate thalamic nuclei, caudate-
	putamen, and dorsal raphe.

.2 Stress-induced mood and memory disorders 1 Stress induced-anxiety disorders

	Kim and Gorman (2005) explained that anxiety is a normal response to
	threat or stress, and it is an unpleasant emotional state consisting of
	psychophysiological responses to the anticipation of an unreal event. According to the
	classification system of the American Psychiatric Association's modified fifth edition
	of the Diagnostic and Statistical manual of Mental Disorder (DSM-5), anxiety
	disorders are divided into 5 subtypes i.e., panic disorder, generalized anxiety disorder,
	social anxiety disorder, post-traumatic stress disorder, and obsessive-compulsive
	disorder (American Psychiatric Association, 2013).
	Anxiety involves multiple brain regions, namely the BNST, the LC and the
	PFC. The activation and regulation of neuroendocrine pathways in fear and anxiety
	involve the stress response in the HPA axis and its related pathways, i.e., the CRH-
	ACTH-cortisol axis and the LC-axis. Anxiety disorders may result from genetic
	factors, threatening environmental factors, life experiences or imbalanced
	neurotransmitters, especially, monoamine neurotransmission. Stress-induced changes
	in monoamines (5-HT and NE) by anxiety such as immobilization could produce
	negative emotions such as anxiety and/or fear and emotional responses such as
	defecation, vocalization and struggling

Table 3 .1

 3 Descriptions of animal groups in the experiment 1.

  ETM, OFT, FST, NOR, and MWM. Twenty-four hours after the last behavioral test, rats were sacrificed. Blood was collected for the serum corticosterone measurement. Adrenal glands were collected and weighed. Brains were removed and analyzed for target proteins in brain regions associated with stress responses by Western blot analysis. The experimental protocol and the descriptions of animal groups are presented in Figure3.3 and Table3.3.

	Sarawut Lapmanee							Materials and Methods / 42
	Non-stressed control Rats were pre-treated for 4 weeks with agomelatine and venlafaxine, or
	Stress underwent voluntary wheel running, followed by 4-week restraint stress induction (2
	Wheel running h/day, 5 days/week). During the restraint stress induction, rats received neither Sacrificed: Blood collection
	and removal of brains and pharmacological treatment nor voluntary wheel running. Anhedonic-like behavior was
	Stress + Wheel running evaluated by a weekly sucrose preference test. At the end of the restraint stress adrenal glands.
	Weekly sucrose preference test induction, all rats were evaluated for anxiety-, depression-, and memory-like behaviors
	-2 using EPM,	-1	0	1	2	3	4	5 weeks
	Acclimatization Pre-exercise
	. Monoamine modulators-treated group were divided into
	3 subgroups
			-Vehicle (Veh, 5 mL/kgBW, p.o., 16 rats/group)
			-Agomelatine (Ago, 10 mg/kgBW, p.o., 16 rats/group)
			-Venlafaxine (Vlx, 10 mg/kgBW, p.o., 16 rats/group)
	2. Voluntary wheel running group (16 rats/group)

Table 3 .

 3 4 Summarizes the parameters and interpretations of behavioral evaluations.

	Test	Parameter	Interpretation Definition	References
	EPM	 Opened arm time/entry  Anxiety-like behavior	Hogg, 1996; Rodgers et
		Closed arm entry	Locomotor activity	al., 1997;
		Total arm entry		Pandaranandaka et al.,
				2006
	ETM	 Inhibitory avoidance	 Conditioned fear/	Graeff et al., 1998;
		latency	Generalized anxiety	Pandaranandaka et al.,
		 Escape latency	 Unconditioned fear/	2009
			Panic-like symptom
	OFT	 Inner zone time	 Anxiety-like behavior	Treit and Fundytus,
		 The first 30 s line	 fear reaction-	1988; McCarthy et al.,
		crossings	thigmotaxis, hyperarousal	1995; Pandaranandaka
		Total line crossings	Locomotor activity	et al., 2009
	NOR	 Discrimination ratio	 Cognitive and memory	Antunes and Biala,
			impairment	2012; Redrobe et al.,
				2010; 2012
	MWM	 Escape latency	 Spatial learning	Morris et al., 1981,
		 Time reach to correct	 Spatial memory	1984
		quadrant

EPM: elevated plus-maze; ETM: elevated T-maze; NOR: novel objective recognition; MWM: Morris water maze; OFT: open-field test.

Table 3 .

 3 4 Summarizes the parameters and interpretations of behavioral evaluations (Cont.).

	Test	Parameter	Interpretation Definition	References
	FST	 Swimming time	 Depression-like behavior Porsolt et al., 1978
		 Immobility time
		 Climbing time
	Sucrose	 % sucrose preference	 Depression-like behavior Willner et al., 1987;
	preference			Papp et al. 1991
	Stress-related behaviors
	 Grooming		Depression-like behavior	Ramos and Mormède,
	 Grooming/Rearing/Defecation + Urinary  Rearing	 Stress/Hyperarousal Depression-like behavior	1998

FST: forced swimming test.

Table 3 .5

 3 Primary antibodies used in Western blotting analysis.

	Antibody	Manufacturer	Catalog #	Host/clonality	Dilution	Reference
	Brain-derived	Santa Cruz	SC546	Rabbit	1:1,000 Seo et al., 2015
	neurotrophic	Biotechnology		polyclonal
	factor (BDNF)
	Glucocorticoid	Abcam	AB2768	Mouse	1:1,000 Taylor et al.,
	receptor (GR)			monoclonal		2011
	Norepinephrine	Alpha	NET11-A	Rabbit,	1:330 Chen et al.,
	transporter	Diagnostic		polyclonal		2012
	(NET)	Intl. Inc
	Serotonin	Santa Cruz	SC33724	Mouse	1:2,000 Zhang et al.,
	transporter	Biotechnology		monoclonal		2012
	(SERT)
	Serotonin type	Abcam	AB133570 Rabbit	1:500 Anastasio et al.,
	2C receptor			monoclonal		2011
	(5-HT 2C R)
	Melanocortin	Millipore	AB5128	Rabbit,	1:1,000 Campbell et al.,
	type 2 receptor	(Chemicon)		polyclonal		2009
	(MC2R)
	-actin	Santa Cruz	SC47778	Mouse	1:2,000 Tan et al., 2015
		Biotechnology		monoclonal

Table 3 .6

 3 The MT1-/MT2-LacZ genotyping and primer designs.

	Genotyping	Primer sequence 5'3' (Forward/Reverse)
	Wild-type	CAAGTTGCTGGGCAGTGGACAGCAG
	MT1-LacZ	CTTCTTGTTGCGGTACACAGACAGG
	Knock-in	CTAGTCTGTTCAGCTGTGTCACACC
		GCGGGAGGGCCATAAAAAGTGGC
	Wild-type	AAGCACCAGCAATTTCCCATTCTCC
	MT2-LacZ	TCTTGCCTGAGGAAGGTCAGAATCC
	Knock-in	ACTTGCTTTAAAAAACCTCCCACA
		ATGGGTCCCTGGAAGTCACTCACC
	Each PCR reaction was performed as follows: cycling touchdown PCR
	program, 94 °C for a 5-min initial strand separation, 94 °C for 30 s, 58°C for 30 s, at
	72 °C for 45 min (repeat for a total of 35 cycles) , and a 5-min final elongation step at
	72°C. Five µl of each PCR product and mass ruler DNA ladder Mix (Euromedex) was
	electrophoresed through a 1.5% agarose gel in Tris-borate-EDTA buffer (TBE) that
	was then stained with 1g/ml ethidium bromide. DNA fragments were visualized
	under ultraviolet light and photographed (Sony camera). DNA fragment phenotypes
	were identified by 425 bp (wild-type) or 196 bp (MT1-LacZ transgene), whereas
	MT2-LacZ DNA fragment phenotypes were identified by 143 bp (wild-type) or 666 bp
	(MT2-LacZ transgene).

Table 3 .7

 3 Mouse primers in the PCR experiments for cloning probe.

	Gene

Name (Abbreviated) Primer sequence 5'3' (Forward/Reverse) Product Length, bp

	Corticotropin-releasing hormone	AGAAACTCAGAGCCCAAGTACG	1,062
	(CRH)	GATGACTCCCATCTGCTTTTTC
	Gastrin-releasing peptide (GRP) CTGAGAACCGTCCTCCGTC	766
		GGAAAAAGCGTTCATTGGAA
	Glutamic acid decarboxylase 1	TGTGCCCAAACTGGTCCT	982
	(GAD1)	TGGCCGATGATTCTGGTT
	Glutamic acid decarboxylase 2	TCTTTTCTCCTGGTGGCG	869
	(GAD2)	TTGAGAGGCGGCTCATTC
	-galactosidas (LacZ)	CCAACGTGACCTATCCCATTAC	1,524
		CAAAGACCAGACCGTTCATACA

Table 3 .8

 3 Summary of parameters and techniques in this study.

	Parameter	Technique	Brief objectives
	Anxiety-like behaviors	EPM	To evaluate stress-induced anxiety.
		ETM
		OFT
	Depression-like behaviors	Sucrose preference	To evaluate stress-induced depression.
		FST
	Learning and memory	NOR	To evaluate stress-induced learning and
	impairment-like behaviors	MWM	memory impairment.
	Serum and urinary CORT	ELISA	To confirm successful stress induction.
	Protein expression	Western blotting	To examine protein expression in the brain
			region and stress-related organs
			contributing to stress responses.
	Riboprobe synthesis for	PCR cloning	To prepare probes for in situ hybridization.
	in situ hybridization
	MT1/ MT2-LacZ	Immunocytochemistry,	To map and phenotype melatonin receptor
	localization	in situ hybridization,	MT1 and MT2 expressing neurons in
		X-gal histochemistry	transgenic mice

CORT: corticosterone; ELISA: enzyme-linked immunosorbent assay; EPM: elevated-plus maze; ETM: elevated T-maze; FST: forced swimming test; NOR: novel objective recognition; MT: melatonin receptor; MWM: Morris water maze; OFT: open-field test; PCR: polymerase chain reaction; X-gal: 5-bromo-4-chloro-3-indolyl-β-d-galactoside.

Table 4 .1

 4 Changes in bio-behavioral parameters of time dependent effects of the 1-, 4, and 8-week restraint stress induction in male rats.

	Parameter	Stress 1 week	Stress 4 weeks	Stress 8 weeks
	Anxiety	EPM	↑↑	↑	↑
	Depression	ETM OFT % SP	↑ ↑ ↑	↑↑↑ ↑ ↑ at the 2 nd week of stress induction	↔ ↑ ↑ at the 2 nd week ↓ at the 5 th to 8 th
					week of stress
					induction
	Learning Memory Locomotion BDNF	FST MWM MWM/NOR EPM/OFT	↔ ↓↓ ↑ ↔ ↓ in the PAG	↑ ↑↑↑ ↑↑↑ ↔ ↔	↔ ↔ ↔ ↔ ↓ in the VTA
	GR		↑ in the	↓↓↓ in the	↔
			hypothalamus	hippocampus
	NET SERT		↔ ↓ in the VTA	↑ in the LC ↔	↔ ↓ in the
					hippocampus
	5-HT 2C R		↑ in the amygdala	↑ in the amygdala, ↓ in the PAG	↑↑ in the LC, ↓ in the VTA

BDNF: Brain-derived neurotrophic factor; EPM: elevated-plus maze; ETM: elevated-T maze; FST: forced swimming test; GR: glucocorticoid receptor; LC: locus coeruleus; MWM: Morris water maze; NET: norepinephrine transporter; NOR: novel objective recognition; OFT: openfield test; PAG: periaqueductal gray; SERT: serotonin reuptake transporter; VTA: ventral tegmental area; SP: sucrose preference; 5-HT 2C R: serotonin type 2C receptor.

Table 4 .2

 4 Changes in bio-behavioral parameters of effects of the 4-week voluntary wheel running under non-stressed condition and/or after the 4-week restraint stress induction in male rats.

	Parameter		Ex	Stress	Stress + Ex
			vs control	vs control	vs stress
	Depression	% SP	↔	↓↓↓	↑
	Serum corticosterone		↔	↑	↔
	BDNF		↓ in the NA	↓↓ in the	↑ in the
				hippocampus and the	hippocampus
				septum	and the septum,
	GR		↔	↓ in the hippocampus	↓ in the NA ↔
	Adrenal MC2R		↔	↔	↓
	NET		↓↓ in the LC and	↑ in the LC, ↓ in the	↓ in the LC
			the VTA	PAG
	SERT		↑↑ in the septum	↓↓ in the	↓ in the VTA
				hippocampus and the
				DR
	5-HT 2C R		↓ in the DR, the	↑ in the amygdala	↓ in the PFC
			PAG, and the NA

BDNF: Brain-derived neurotrophic factor; DR: dorsal raphe; EPM: elevated-plus maze; ETM: elevated-T maze; FST: forced swimming test; GR: glucocorticoid receptor; LC: locus coeruleus; MWM: Morris water maze; NA: nucleus accumbens; NET: norepinephrine transporter; NOR: novel objective recognition; OFT: open-field test; PAG: periaqueductal gray; SERT: serotonin reuptake transporter; VTA: Ventral tegmental area; SP: sucrose preference; 5-HT 2C R: serotonin type 2C receptor.

Table 4 .3

 4 Changes in bio-behavioral parameters of preventive effects of the 4-week agomelatine, venlafaxine, and voluntary wheel running in stressed male rats.

	Parameter	Ago	Vlx	Rw
	Anxiety	EPM	↔	↓	↔
	Depression	ETM OFT % SP	↓↓↓ ↓ ↔	↓↓↓ ↔ ↔	↓↓↓ ↓ ↔
	Learning	FST MWM	↓↓ ↑↑↑	↓↓ ↑↑	↔ ↑↑↑
	Memory	MWM/NOR	↑↑↑	↑↑↑	↑↑↑
	Locomotion	EPM/OFT	↔	↔	↔
	Serum corticosterone	↑	↔	↔
	BDNF		↓↓ in the LC	↑↑↑ in the hippocampus, ↓ in	↑ in the hippocampus
				the septum

  . De ce fait, les deux types de récepteurs de la

	récepteur aux glucocorticoïdes (GR) et de facteur neurotrophique dérivé du cerveau
	(Brain Derived Neurotropic Factor, BDNF) dans l'hippocampe, ainsi qu'une réduction
	de la neurogenèse dans cette structure, le tout conduisant à des troubles de l'humeur
	tels que l'anxiété et la dépression, ainsi que des perturbations de la mémoire (Schmidt
	& Duncan 2007, Autry & Monteggia 2012).
	A côté de ces agents pharmacologiques, l'exercice physique est connu
	pour stimuler la neurotransmission monoaminergique et la production de BDNF
	(Cotman & Berchtold 2002). L'exercice physique volontaire possède des propriétés
	antidépressives et anxiolytiques chez les rongeurs et chez l'homme (Burghardt et al.
	2004, Ströhle et al. 2009, Lapmanee et al. 2013). L'activité locomotrice volontaire
	dans une roue augmente la production de BDNF et la neurogenèse dans l'hippocampe
	mélatonine, MT1 et MT2, sont devenus des cibles thérapeutiques pour le
	développement de nouveaux antidépresseurs. Cependant, la localisation cellulaire
	précise des récepteurs MT1 et MT2 n'est pas encore bien connue du fait de l'absence
	d'anticorps bien caractérisés et parfaitement spécifiques pour l'immunocytochimie.
	Basé sur ses effets anxiolytiques et antidépresseurs (Milan et al. 2005, Papp et al.
	2006, Taylor et al. 2014), l'agomélatine, un agoniste MT1/MT2 et antagoniste 5-
	HT2C pourrait être un candidat nouveau pour la prévention de changements
	comportementaux induits par le stress.
	Outre les perturbations de l'axe HPS et des systèmes monoaminergiques,
	le stress chronique augmente les glucocorticoïdes, ce qui décroit la production de

(Cotman & Berchtold 2002)

, et améliore le fonctionnement cérébral en modifiant la neurotransmission monoaminergique

[START_REF] Dishman | Brain monoamines, exercise, and behavioral stress: animal models[END_REF]

. Certains patients ne répondent pas ou peu aux traitements pharmacologiques ou présentent une longue phase de latence avant de présenter des effets anxiolytiques ou antidépresseurs. L'exercice physique ou d'autres interventions pourraient donc s'avérer bénéfiques pour ces individus. Précédemment, nous avions montré qu'un inhibiteur de la recapture sérotonine-noradrénaline-dopamine, la venlafaxine, ainsi que l'activité locomotrice volontaire dans une roue pouvaient réduire des comportements de type anxiété et dépression chez des rats stressés

  Après, le sang et des urines ont été collectés pour des mesures de la corticostérone. Le coeur, les surrénales et le cerveau ont été prélevés dans toutes les expériences. Des régions cérébrales ont été

	microdisséquées pour étudier l'expression de protéines cibles : BDNF, récepteur au
	glucocorticoïdes (GR), transporteur de la NA (NET), transporteur de la 5-HT (SERT)
	et le récepteur de la sérotonine 5-HT 2c . Les niveaux d'expression des protéines ont été
	évalués par Western blotting dans des régions cérébrales contribuant à la réponse au
	stress : cortex frontal, amygdale, hypothalamus, hippocampe, raphé dorsal, locus
	coeruleus, gris périacquéducal, septum, noyau accumbens et aire tegmentale ventrale.
	Toutes les procédures animales ont été validées par le Comité de Soins et d'Utilisation
	d'Animaux de la Faculté de Médecine de l'Université Thammasat, Thaïlande.

  Effets de 4 semaines d'activité locomotrice volontaire sur l'axe HPS en conditions basales et après 4 semaines d'exposition à un stress de contention.

	Animaux	Pour phénotyper les cellules exprimant le LacZ-MT1 ou LacZ-MT2, nous
	Des souris (1,5 à 18 mois d'âge, 20-60 g) mâles et femelles adultes avons pratiqué des doubles immunomarquages, ou combiné la détection
	Les rats ont été séparés en 4 groupes : 1 groupe non-stressé sans roue, 1 transgéniques avec le rapporteur LacZ introduit dans le locus MT1 (MTNR1A) ou immunocytochimique de LacZ avec une détection par hybridation in situ de marqueurs
	groupe non-stressé avec exercice de roue, 1 groupe stressé sans roue et 1 groupe MT2 (MTNR2A) ont été obtenues à la plateforme Chronobiotron (CNRS UMS 3415) cellulaires. Comme marqueurs cellulaires nous avons utilisé des neuropeptides ou des
	stressé avec exercice de roue. Tous les rats ont été évalués pour des comportements de l'Institut des Neurosciences Cellulaires et Intégratives (INCI CNRS UPR 3212) de enzymes impliqués dans la synthèse de neurotransmetteurs.
	anhédoniques par le test de préférence pour le sucrose. Le sang et les urines ont été l'Université de Strasbourg. Ces mutations étaient introduites dans la lignée C57/BL6, Tableau 1. Résumé des paramètres analyses et des techniques utilisées
	collectés pour la détermination du niveau de corticostérone. Le coeur et les glanes qui ne produit pas de mélatonine. Nous avons utilisé des souris hétérozygotes (KI/+) et Paramètre Technique Objectif
	surrénales ont été prélevés et pesés. Les cerveaux ont été prélevés et analysés pour les ETM des souris homozygotes (KI/KI) pour nos études. Des souris non-mutantes (+/+) des Comportements anxieux EPM Evaluation de l'anxiété induite par le stress
	protéines cibles. Les glandes surrénales ont aussi analysées pour le niveau OFT mêmes portées ont été utilisées comme contrôles. Pour obtenir des souris mutantes Comportements dépressifs Préférence Sucrose Evaluation de la dépression induite par le
	d'expression protéique du récepteur aux mélanocortines 2 (MC2R, récepteur de produisant de la mélatonine, nous avons utilisé des souris hétérozygotes (KI/+) issues FST stress
	l'ACTH). Apprentissage et mémoire d'un croisement F1 avec la lignée C3H, qui produit de la mélatonine. L'euthanasie des NOR Evaluation des perturbations de MWM l'apprentissage et de la mémoire induits par
	le stress souris a été effectuée selon les protocoles agrémentés pour la plateforme « Corticostérone sérique et ELISA Confirmer l'induction du stress
	urinaire Chronobiotron » (UMS 3415) par le Comité Régional d'Ethique en Matière Expression protéique Western blot Examiner l'expression de protéines cibles
	d'Expérimentation Animale de Strasbourg.	dans les organes et les structures cérébrales
				impliquées dans le stress
	Localisation MT1-LacZ /	Histochimie X-Gal,	Cartographier et phénotyper les neurones
	Les rats ont été séparés en 4 groupes : 1 groupe contrôle recevant la Immunocytochimie, exprimant les récepteurs MT1 et MT2 dans Analyses histologiques : MT2-LacZ Hybridation in situ des souris transgéniques
	solution véhicule (5 ml/kg de saline 9‰ per os), 1 groupe Agomélatine (10 mg/kg per Les souris ont été euthanasiées par inhalation de CO 2 et perfusées par des
	os), Venlafaxine (10 mg/kg per os) et 1 groupe avec exercice de roue volontaire. Les solutions de fixateur à travers le coeur. Nous avons utilisé le fixateur Periodate-Lysine-
	animaux ont été soumis à ces traitements pendant 4 semaines (administrations per os Paraformaldéhyde (PLP) pour les analyses par immunocytochimie et par hybridation
	tous les jours). Ensuite, ces traitements ont été arrêtés et les animaux ont été soumis in situ. Pour la détection histoenzymologique de l'activité LacZ, les animaux ont été
	pendant 4 semaines au stress de contention (2 h/jour, 5 jours/semaine). Les fixés par du formaldéhyde 4% dans du tampon phosphate 100 mM. Après dissection,
	comportements anhédoniques ont été évalués par un test hebdomadaire de préférence le cerveau a été postfixé par immersion dans le fixateur et soit préparé pour des coupes
	pour le sucrose. A la fin des 4 semaines de soumission au stress, tous les rats ont été flottantes en congélation après cryoprotection au sucrose (Histoenzymologie LacZ) ou
	évalués pour des comportements anxieux ou dépressifs, ainsi que pour l'apprentissage enrobé en Polyéthylène Glycol (PEG) (immunocytochimie et hybridation in situ).
	et la mémoire. Des coupes flottantes (30-40 m d'épaisseur) ont été colorés pour l'activité beta-
	Protocoles expérimentaux galactosidase (LacZ) en utilisant un substrat chromogénique (5-bromo-4-chloro-3-
	Partie 2 : Caractérisation des cellules exprimant les récepteurs MT1 ou MT2 dans le indolyl-beta-D-galactopyranoside, X-Gal).
	Expérience 1 : Cinétique de l'induction par le stress de contention de comportements cerveau de souris rapportrices LacZ knock-in : Identification de sites d'action Pour la détection immunocytochimique de la protéine LacZ, des coupes en
	anxieux ou dépressifs, et de perturbations de la mémoire. potentiels de l'Agomélatine dans des structures impliquées dans le stress PEG ont été pratiquées à 10 m d'épaisseur et montées sur lames. Après une
	réactivation antigénique, la présence de la protéine LacZ a été révélée par un anticorps
	anti-LacZ (Abcam), suivi d'une amplification immunoperoxidase.

T (elevated T maze ETM) et le test « Open Field » (OFT). Les comportements dépressifs ont été évalués par des tests hebdomadaires de préférence pour le sucrose et de nage forcée (forced swimming test FST). La piscine de Morris (Morris water maze MWM) et le test de reconnaissance d'objets nouveaux (novel object recognition NOR) ont été utilisés pour évaluer l'apprentissage et la mémoire.

Les rats ont été séparés en 4 groupes : 1 groupe contrôle (non stressé) et 3 groupes de rats soumis au stress de contention pendant 1, 4 ou 8 semaines Expérience 2 : Expérience 3 : Effets de l'agomélatine, de la venlafaxine et de l'activité locomotrice volontaire dans la prévention de perturbations de la mémoire et de comportements de type anxiété et dépression.

  L'activité de roue volontaire a induit une réduction du poids corporel et une augmentation du poids sec du coeur autant chez les rats contrôles que chez les rats stressés. La présence d'une hypertrophie cardiaque indique le succès du protocole d'exercice physique aérobique comme montré précédemment par[START_REF] Wang | Animal models in the study of exercise-induced cardiac hypertrophy[END_REF] et[START_REF] Lapmanee | Beneficial effects of fluoxetine, reboxetine, venlafaxine, and voluntary running exercise in stressed male rats with anxiety-and depression-like behaviors[END_REF]. L'exercice physique volontaire a aussi augmenté le poids des glandes surrénales sans changement des niveaux de corticostérone sériques chez les rats non-stressés. Ceci est en accord avec une étude précédente qui a montrée que 4 semaines d'activité de roue volontaire n'avaient aucun effet sur les niveaux de corticostérone circulante, que ce soit au niveau basal ou au niveau maximal du rythme circadien chez le rat mâle(Fedicu et al. 2006). De plus, l'activité de roue volontaire à diminué les niveaux du NET, du SERT et du 5-HT 2C dans le circuit de la récompense Changements comportementaux et biochimiques chez des rats mâles contrôles ou après 4 semaines d'activité de roue volontaire et / ou soumis pendant 4 semaines à un stress de contention.

	Expérience 2 : Paramètre	Exercice	Stress	Stress + Exercice
				vs contrôle	vs contrôle	vs stress
	NET			 dans le LC et la	 dans le LC,  dans	 dans le LC
				VTA	le PAG
	SERT			 dans le septum	 dans	 dans la VTA
					l'hippocampe et le
					DR
	5-HT 2C R			 dans le DR, le	 dans l'amygdale	 dans le PFC
				PAG, et le NA
	DR raphé dorsal, LC locus coeruleus, NA noyau accumbens, PAG gris périacquéducal, PFC cortex
	préfrontal, VTA aire tegmentale ventrale
		Paramètre	Stress 1 semaine	Stress 4 semaines	Stress 8 semaines
	Anxiété		EPM			
			ETM			
			OFT			
	Dépression	% Préférence Sucrose		 à la deuxième semaine de stress	 à la deuxième semaine de stress 
						de la 5 ème à la 8 ème
						semaine de stress
			FST			
	Apprentissage MWM			
	Mémoire		MWM/NOR			
	Locomotion	EPM/OFT			
	BDNF			 dans le PAG		 dans la VTA
	GR			 dans	 dans	
				l'hypothalamus	l'hippocampe
	NET SERT	Paramètre	  dans la VTA Exercice vs contrôle	 dans le LC  Stress vs contrôle	 Stress + Exercice  dans vs stress
	5-HT 2C Dépression Corticostérone sérique % Préférence Sucrose	 dans l'amygdale  dans l'amygdale,  dans le PAG    	l'hippocampe   dans le LC,  dans la VTA 
	BDNF			 dans le NA	 dans	 dans
	LC locus coeruleus, PAG gris périacquéducal, VTA aire tegmentale ventrale l'hippocampe et le	l'hippocampe et le
					septum	septum,
						 dans le NA
	GR				 dans l'hippocampe	

dans le système nerveux central (p.ex. locus coeruleus, aire tegmentale ventrale, raphé dorsal, gris périacquéducal et noyau accumbens). La combinaison du stress avec l'exercice physique volontaire a partiellement diminué le niveau basal de corticostérone, a augmenté le niveau de BDNF dans l'hippocampe et réduit le récepteur MC2R dans la surrénale et le récepteur 5-HT 2C dans le cortex frontal. Ces résultats suggèrent que l'exercice physique volontaire peut réduire l'effet négatif du stress chronique en modulant l'activité sympathique de l'axe HPS tant au niveau central qu'au niveau périphérique, ce qui par conséquent a réduit la sécrétion de corticostérone

[START_REF] Droste | Voluntary exercise impacts on the rat hypothalamic-pituitary-adrenocortical axis mainly at the adrenal level[END_REF][START_REF] Patki | Novel mechanistic insights into treadmill exercise based rescue of social defeatinduced anxiety-like behavior and memory impairment in rats[END_REF]

.

Tableau 3.

Table 4 .

 4 Changements comportementaux et biochimiques chez des rats prétraités pendant 4 semaines avec de l'agomélatine, de la venlafaxine ou de l'exercice volontaire de roue, et soumis après pendant 4 semaines à un stress de contention.

	Paramètre	Agomélatine	Venlafaxine	Exercice
	Anxiété	EPM			
		ETM			
		OFT			
	Dépression	% Préférence			
		sucrose
		FST			
	Apprentissage MWM			
	Mémoire	MWM/NOR			
	Locomotion	EPM/OFT			
	Corticostérone sérique			
	BDNF		 dans le LC	 dans l'hippocampe, 	 dans l'hippocampe
				dans le septum
	GR			 dans	
				l'hippocampe
	NET		 dans le PAG	 dans	 dans
				l'hippocampe et le	l'hippocampe et
				PAG	le LC
	SERT		 dans le septum	dans le septum	
	5-HT 2C R				
	LC locus coeruleus, PAG gris périacquéducal

Table 5 .

 5 Localisation des récepteurs MT1 and MT2 dans le cerveau de la souris d'après l'expression des transgènes MT1-LacZ et MT2-LacZ

	Région cérébrale	MT1	MT2
	Cortex cérébral	quelques cellules isolées	 dans la couche III
	Cerveau basal antérieur
	-tubercule olfactif
	-Ilots de Calleja

ACKNOWLEDGMENTS

thankful Dr. Panan Suntornsaratoon, Dr. Jarinthorn Teerapornpuntakit, and Miss Nithipak Thammayon for their technical assistance.

I am thankful for the scholarships and co-funding institutions from the Institutional Strengthening Program, Faculty of Science, Mahidol University, the Royal Golden Jubilee Ph.D. Program, Thailand Research Fund, the Embassy of France in Thailand, the Graduate Furthermore, I would like to express my gratitude to the experimental animals, my appreciation

LIST OF FIGURES (cont.)

TRANSGENIC MOUSE MODELS

Strategies for gene transfer into early embryonic stages have been developed to generate animal models to study human physiology and the pathophysiology of diseases. Technically, the process of generating transgenic mice consists in the purification of a transgenic construct, harvesting donor zygotes, microinjection of the transgenic construct into these zygotes, implantation of the microinjected zygotes into pseudo-pregnant recipient mice, and genotyping and analysis of transgene expression in founder mice [START_REF] Cho | Generation of transgenic mice[END_REF].

Such transgenic models also include knock-out models, where genes are inactivated by the deletion of all or only parts of the coding sequence of a gene to be studied. Similarly, knock-in models allow the introduction of either specific mutations or foreign coding sequences into the locus of a gene. For these knock-in and knock-out models, the transgenic constructs are introduced directly into the locus of the gene of interest by homologous recombination in embryonic stem cells. These modified embryonic stem cells are then introduced into blastocysts, which then generate chimeric animals. These chimeric animals are then bred to generate first heterozygous founder mice, and further into homozygous mutant knock-in or knock-out mice if desired. Classical transgenic animals, knock-in and knock-out animals are tools to study the underlying mechanisms of candidate genes at cellular and molecular levels.

The Cre/LoxP system is an additional tool to generate mutant mice.

Briefly, the Cre recombinase enzyme deletes a part of DNA located between two loxP sites (locus of crossing over of the P1 bacteriophage). The loxP sites can be introduced around an essential exon in a gene by the knock-in technique described above. The mouse strain containing the floxed allele is phenotypically identical to wild type animals. However, when these mice are then crossed with a transgenic mouse line expressing the enzyme Cre recombinase, the Cre recombinase will delete the sequence between the two loxP sites, but only in the cells that express the Cre recombinase.

Thus the Cre/LoxP system allows the targeting of gene deletions to selective cell types, according to the regulatory sequences that drive the expression of the Cre recombinase (reviewed by [START_REF] Kos | Cre/loxP system for generating tissue-specific knockout mouse models[END_REF][START_REF] Hall | Overview: generation of gene knockout mice[END_REF].

In the transgenic animals used in this study, the LacZ reporter gene was is an enzyme converting lactose into glucose and galactose. When presented with the substrate analog X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), the LacZ enzyme activity results in the formation of a blue precipitate.

Heterogeneous (KI +/-) MT1 or MT2 knock-in mice conserve one intact MT1 or MT2 allele. Thus these KI +/-mice still express this one intact allele. Liu and co-workers (1997) reported that the expression of MT1 in Mtnr1a +/-mice is about half that of wild-type animals, as assessed by 125 I-iodomelatonin binding. Except for this reduction in the quantity of 125 I-iodomelatonin binding, the qualitative pattern of 125 Iiodomelatonin binding in the Mtnr1a +/-animals is identical to that of wild type Mtnr1a +/+ animals. Thus deletion of one Mtnr1a allele does not seem to affect the expression pattern of the MT1 receptor, and replacing one of the Mtnr1a alleles with a reporter gene construct should not modify the expression pattern due to the regulatory elements. Deletion of both Mtnr1a alleles in Mtnr1a -/-knockout animals resulted in the complete abolishment of 125 I-iodomelatonin binding, indicating that this 125 Iiodomelatonin binding on tissue sections was entirely due to the MT1 receptor (Liu et al., 1997).

BIOGRAPHY