Thomas Couroussé

Alexandre Bacq

Catherine Belzung

Bruno Guiard

Laure Balasse

Franck Louis

Anne-Marie Le Guisquet

Alain M Gardier

Alfred H Schinkel

Bruno Giros

Sophie Gautron

Brain organic cation transporter 2 controls response and vulnerability to stress and GSK3β signaling

Keywords: organic cation transporter, stress, HPA axis, vulnerability, UCMS, depression

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The etiology of depression and other mood-related disorders is still poorly understood, yet it is increasingly evident that interactions between genetic and experience-based risks factors play a decisive role in the development of these disorders. [1][START_REF] Krishnan | The molecular neurobiology of depression[END_REF][START_REF] Caspi | Genetic sensitivity to the environment: the case of the serotonin transporter gene and its implications for studying complex diseases and traits[END_REF] A fundamental physiological process at the crossroads of these genetic-environment interactions is the activation in stressful conditions of the hypothalamic-pituitary-adrenocortical (HPA) axis, which engenders a spectrum of neurochemical and behavioral modifications over different time scales. In higher organisms, the response to stress ensures an important function in optimizing the reactions and behavioral strategies in the face of potential threats, in terms of perception, risk assessment and decision-making. However, the repeated solicitation or disruption of the underlying biological systems also imposes a negative burden on mental health by promoting disorders such as major depressive disorder (MDD) or post-traumatic stress disorder (PTSD). [START_REF] Mazure | Adverse life events and cognitive-personality characteristics in the prediction of major depression and antidepressant response[END_REF][START_REF] Lupien | Effects of stress throughout the lifespan on the brain, behaviour and cognition[END_REF][START_REF] Herbert | Cortisol and depression: three questions for psychiatry[END_REF] At the central level, the response to stress solicit several interconnected circuits implicating the paraventricular nucleus (PVN) of hypothalamus as well as upstream neural pathways in the forebrain and hindbrain. [START_REF] Ulrich-Lai | Neural regulation of endocrine and autonomic stress responses[END_REF] Among these circuits, specific monoaminergic pathways are activated within minutes after stress, participating in the early steps driving the synaptic and hormonal responses. [START_REF] Ziegler | Neurocircuitry of stress integration: anatomical pathways regulating the hypothalamo-pituitary-adrenocortical axis of the rat[END_REF][START_REF] Kvetnansky | Catecholaminergic systems in stress: structural and molecular genetic approaches[END_REF] Previous findings have revealed the role of low-affinity organic cation transporters (OCTs) in the brain as important postsynaptic determinants of aminergic tonus and mood-related behaviors, in complement to the classical high-affinity monoamine reuptake transporters. [START_REF] Vialou | Altered aminergic neurotransmission in the brain of organic cation transporter 3-deficient mice[END_REF][START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF][START_REF] Horton | Decynium-22 enhances SSRI-induced antidepressant-like effects in mice: uncovering novel targets to treat depression[END_REF] Contrasting with the high-affinity reuptake transporters, OCT transport activity can be inhibited by micromolar concentrations of the stress hormone corticosterone in vitro. [START_REF] Grundemann | Molecular identification of the corticosterone-sensitive extraneuronal catecholamine transporter[END_REF][START_REF] Hayer-Zillgen | Expression and pharmacological profile of the human organic cation transporters hOCT1, hOCT2 and hOCT3[END_REF] Recent evidence also suggests that their activity in the brain can be impaired directly or indirectly by corticosterone, [START_REF] Gasser | Corticosterone-sensitive monoamine transport in the rat dorsomedial hypothalamus: potential role for organic cation transporter 3 in stress-induced modulation of monoaminergic neurotransmission[END_REF][START_REF] Baganz | Repeated Swim Impairs Serotonin Clearance via a Corticosterone-Sensitive Mechanism: Organic Cation Transporter 3, the Smoking Gun[END_REF] an effect which could mediate some of the biological effects of stress.

In this paper, we addressed whether organic cation transporter 2 (OCT2) controlled the hormonal response to stress. We identify OCT2 as a central modulator of the hormonal response to acute stress in mice and explore the underlying neuroendocrine and molecular mechanisms. Importantly, we show that OCT2 activity contributes as a genetic determinant which attenuates the extent of the noxious consequences of repeated stress on the onset of stress-related behaviors.

Material and methods

Animals

OCT2 -/-mice were generated previously by homologous recombination. [START_REF] Jonker | Deficiency in the organic cation transporters 1 and 2 (Oct1/Oct2 [Slc22a1/Slc22a2]) in mice abolishes renal secretion of organic cations[END_REF] Heterozygous animals with 10 backcross generations into C56BL6/J were bred to generate wild-type and knockout littermates, which were genotyped as described previously. [START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF] Mice were maintained on a 12:12 h light/dark cycle (lights on at 0800) and 8-16 weeks-old animals were used for the experiments. Animal care and experiments were conducted in accordance with the European Communities Council Directive for the Care and the Use of Laboratory Animals (86/609/EEC) and approved by local ethical committees.

Immunohistochemistry

Adult male mice were anesthetized and perfused intracardially with 4% (wt/vol) paraformaldehyde/phosphate-buffered saline (PBS). Brains were removed, post-fixed by immersion overnight in 4% paraformaldehyde/PBS and coronal sections (20 μm) were cut on a vibratome and processed for free-floating immunohistochemistry. For immunofluorescent immunohistochemistry, brain sections were preincubated in a blocking buffer containing 0.3% (wt/vol) triton and 4% (wt/vol) bovine serum albumin. OCT2 was detected using affinity-purified rabbit polyclonal antibodies previously validated. [START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF] After washing, sections were incubated with Alexa 488-conjugated secondary antibodies (Molecular Probes, Eugene, Or, USA). For double labeling experiments, OCT2 antibodies were incubated with antibodies against either glial fibrillary acidic protein (GFAP, 1/500) from Abcam (Cambridge, UK) or adrenocorticotropic hormone (ACTH, 1/1000) from Sigma-Aldrich (St Louis, MO, USA) and revealed with Alexa 555-conjugated secondary antibodies (Molecular Probes).

Swim stress, subchronic corticosterone treatment and corticosterone dosage

Naive aged-matched male mice were left undisturbed several days before the experiment. For swim stress, the mice were placed individually in a glass beaker filled with tap water at 25 ± 1°C to a depth of 12 cm for 15 min. For subchronic corticosterone treatment, mice were administered corticosterone (35 μg/ml; Sigma-Aldrich) dissolved in 0.45% (wt/vol) hydroxypropyl-β-cyclodextrin (Sigma-Aldrich) or vehicle (0.45% hydroxypropyl-βcyclodextrin) in drinking water during 4 days. For ACTH stimulation, mice were injected with dexamethasone (100 µg/kg i.p.; Sigma-Aldrich) 2 h prior to saline or cosyntropin injection (50 µg/kg i.p.; Synacthen, Sigma-Tau, Issy-Les-Moulineaux, France). Tail blood samples were collected into EDTA-containing tubes (Microvette, Sarstedt, Numbrecht, Germany) in the morning (0800-1100) before and at different time points after swim stress or ACTH injection, centrifuged and plasma was stored at -80°C until assayed. Plasma corticosterone concentrations were determined using radioimmune assay kits (MP Biomedicals, Illkirch, France) and expressed as ng/ml of plasma. For the dexamethasone suppression test, mice were injected with dexamethasone (100 µg/kg i.p.; Sigma-Aldrich) 30 min prior to swim stress.

Unpredictable chronic mild stress (UCMS) and behavioral assessment

Mice were subjected to various unpredictable stressors for 8 weeks as described earlier. [START_REF] Nollet | Models of depression: unpredictable chronic mild stress in mice[END_REF] Alterations of the bedding (repeated sawdust changing, removal of sawdust, damp sawdust, substitution of sawdust with 21°C water), cage-tilting (45°), predator sounds (15 min), cage shift (mice placed in the empty cage of another male), food deprivation and alterations of the light/dark cycle were used as stressors. Several parameters were evaluated before the start and at different time points during the UCMS procedure. The coat state of each animal was assessed weekly as a measure of UCMS-induced alterations in motivation towards self-care behaviors such as grooming behaviors. The total score of the coat state resulted from the sum of scores from eight different body parts: head, ears, neck, dorsal and ventral coat, tail, forepaws and hindpaws. For each area, a score of 0 was given for a well-groomed coat and 1 for an unkempt coat. [START_REF] Surget | Drugdependent requirement of hippocampal neurogenesis in a model of depression and of antidepressant reversal[END_REF] The social interaction test was performed in a white open-field (42 x 42 cm) containing an empty wire mesh cage (10 x 6.5 cm) located at an extremity of the field in a low luminosity environment (25 lux). Each test mouse was allowed to explore the openfield for two consecutive sessions of 2.5 min. During the second session, an unfamiliar mouse was introduced into the cage. Between the two sessions, the test mouse was placed back into its home cage for approximately one minute. The time spent by the test mouse in the interaction zone, defined as an 8-cm-wide region surrounding the mesh cage, was measured in both sessions by video tracking (Viewpoint, Lyon, France). For the object location test (OLT), the mice were habituated during two successive days to an open-field containing an intra-field cue (one wall covered with black and white stripes). Each mouse was allowed to freely explore the open-field for a 30-min period on day 1 and for two 10-min sessions separated by 5 h on day 2. On the third day, the test mouse was allowed to explore for 5 min two identical objects (5 x 2.5 cm) positioned in two adjacent corners of the open-field (acquisition phase) then returned to its home cage for 1 h. For the sample phase trial, one of the 2 objects was displaced to the opposite corner of the open-field and the time spent exploring displaced and non-displaced objects was measured over a 5-min session by video tracking. For nest building, a square cotton nestlet (5 x 5 cm, 2-3 g; Serlab, Montataire, France) was placed in the cage 1 h before the dark phase. The nests were assessed 5 h later using the following scoring system: 1, the cotton nestlet was intact; 2, the cotton nestlet was partially scattered; 3, the cotton nestlet was scattered but there is no form of nest; 4, the cotton was gathered but there is no nest ("flat nest"); 5, the cotton nestlet was gathered into a hollow sphere with one opening for entry. For the sucrose preference test, singled-house mice were first habituated for 48 h to drink water from two bottles. On the following 3 days, the mice could choose between a water bottle and a 1% (wt/vol) sucrose solution bottle, switched daily. Sucrose solution intake for 24 h was measured during the last 2 days and expressed as a percentage of the total amount of liquid ingested.

Iontophoretic electrophysiology

Extracellular recordings of pyramidal neurons in the CA3 region of the hippocampus were carried out using multi-barreled glass micropipettes as previously described. [START_REF] Guiard | Cross-talk between dopaminergic and noradrenergic systems in the rat ventral tegmental area, locus ceruleus, and dorsal hippocampus[END_REF] The central barrel used for extracellular unitary recording and one side barrel used for automatic current balancing were filled with a 2 M NaCl solution. The three other side barrels were filled with serotonin (5-HT, 25 mM in 0.2 M NaCl; pH 4) or norepinephrine (NE, L-NE-HCl, 25 mM in 0.2 M NaCl; pH 4) and quisqualate (1.5 mM in 0.2 M NaCl; pH 8). 5-HT and NE were ejected as cations and retained with currents of -10 to -8 nA. Quisqualate was ejected as an anion and retained with a current of + 1 to + 3 nA. The impedance of the central barrel was 2-5 MΩ and those of the balance barrel and side barrels were 20-30 MΩ and 50-100 MΩ, respectively. The five-barreled glass micropipettes were positioned in mm relative from bregma: A/P, -2.5 to -3; M/L, + 2.3 to 2.8; D/V, -2 to -3. Quisqualate stimulated pyramidal neurons were identified by their high amplitude (0.5-1.2 mV), high frequency and long duration (0.6-1.0 ms) action potential and by their characteristic "complex spike" discharge. Venlafaxine was used as a dual inhibitor to block both 5-HT and NE reuptake. [START_REF] David | Effects of acute treatment with paroxetine, citalopram and venlafaxine in vivo on noradrenaline and serotonin outflow: a microdialysis study in Swiss mice[END_REF] In agreement with this action, venlafaxine at the dose of 16 mg/kg, subcutaneous, produced a reduction of the firing rate of CA3 pyramidal neurons in wild-type and OCT2 -/-mice. The excitatory current of quisqualate was thus increased for both groups to maintain a firing activity rate similar to that before venlafaxine injection. The time required for the firing activity to recover 50% of the initial firing rate after microiontophoretic application (recovery time (RT50) value) was used as an index of the capacity of neurons of the dorsal hippocampus to remove 5-HT or NE from the synaptic cleft in the presence or absence of venlafaxine. [START_REF] David | Effects of acute treatment with paroxetine, citalopram and venlafaxine in vivo on noradrenaline and serotonin outflow: a microdialysis study in Swiss mice[END_REF]

Western blots

Whole tissue extracts were prepared from bilateral punches ( System and Application Software version 3.0 (LI-COR, Biosciences, Lincoln, NE, USA). For analysis of the effect of WAY100635 on Akt/GSK3β signaling, this compound was injected (1 mg/kg i.p.) 3.5 h prior to sacrifice.

[ [START_REF] Li | In vivo regulation of glycogen synthase kinase-3beta (GSK3beta) by serotonergic activity in mouse brain[END_REF]

S]GTP γS binding

The functional activity of 5-HT receptors was assessed as described previously [START_REF] Meller | 5-Hydroxytryptamine(1A) receptor-stimulated [(35)S]GTPgammaS binding in rat brain: absence of regional differences in coupling efficiency[END_REF] with the following modifications. Cryosections were preincubated 30 min at 20°C in HEPES buffer

Statistics

PRISM (GraphPad Software, San Diego, CA, USA) and Statview 5.0 (SAS Institute Inc., Cary, NC, USA) were used for statistical calculations. Student's t-test was used for comparisons of mean values for the two genotypes in the GTP-γS binding experiments and nest building. For corticosterone dosage, coat state, object location test, social interaction, sucrose preference, microiontophoretic electrophysiology and Western blot experiments, data were analyzed using two-way or three-way analysis of variance (ANOVA) followed by Fisher's Protected LSD test. Statistical significance was set at P < 0.05.

Results

OCT2 is expressed in stress-related circuits in the brain

In a previous study, frontal cortex, hippocampus and amygdala were identified as OCT2expressing sites. [START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF] We further investigated by fluorescent immunohistochemistry the distribution of this transporter in the adult mouse brain and found notable expression in several regions implicated in the response to stress. In the telencephalon, OCT2 was prominently expressed in the prelimbic, infralimbic and anterior cingulate cortices, as shown for layers II-III to VI of prelimbic and infralimbic cortices (Figure 1a). In the hippocampal formation, OCT2 was expressed in pyramidal cells of CA1, CA2 and CA3 as previously described [START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF] (Figure 1b), in neurons of the polymorph layer of the DG and diffusely in the lacunosum molecular layer of CA1-3 (Figure 1b). Notable OCT2 levels were found in ventral subiculum (Figure 1c andd), amygdalohippocampal area (Figure 1c) and medial amygdaloid nucleus (Figure 1e). OCT2 was also expressed in several nuclei of the diencephalon, with intense labeling in dorsomedial, ventromedial and arcuate nuclei of the hypothalamus (Figure 1f) and in paraventricular nucleus of the thalamus (Figure 1g). Finally, OCT2 immunolabeling was detected in the median eminence (Figure 1h) and in pituitary (Figure 1i and j). In this last structure, OCT2 was expressed in a non-corticotrope subpopulation of the anterior pituitary, as shown with co-labeling with ACTH antibodies (Figure 1i) and uniformly distributed in the posterior pituitary, apposed to pituicytes labeled with GFAP (Figure 1j). No labeling was detected in OCT2 -/-mice brain, as shown for cingulate and motor cortices (Figure 1k) and hippocampus CA1 (Figure 1l).

OCT2 in the brain controls the hormonal response to acute stress

A number of the above-mentioned circuits expressing OCT2 have been shown to trigger, through direct and indirect connections with the PVN and periPVN regions, the activation of the HPA axis, which culminates in corticosteroid secretion. [START_REF] Herman | Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary-adrenocortical responsiveness[END_REF] We thus investigated in OCT2 -/- mice the neuroendocrine response to acute stress. The circulating corticosterone levels under basal conditions and following a 15-min swim stress were evaluated in wild-type and knockout mice. Basal plasma corticosterone levels were increased by 87% in OCT2 -/-mice compared to wild-type mice (P = 0.0058, Figure 2a). More importantly, swim stress-induced corticosterone release was also strongly enhanced in OCT2 -/-mice compared to wild-type mice, with an increase of 56% of peak levels after stress (P < 0.001, Figure 2a). Besides central circuits, peripheral processes involving circulating catecholamines or paracrine control within adrenal glands may also modulate locally corticosterone response during acute stress. [START_REF] Nussdorfer | Paracrine control of adrenal cortical function by medullary chromaffin cells[END_REF][START_REF] Brede | Differential control of adrenal and sympathetic catecholamine release by alpha 2-adrenoceptor subtypes[END_REF] In particular, OCT2 labeling was found in the adrenal cortex and in some of the norepinephrine transporter (NET)-positive secretory cells of the adrenal medulla, as shown by immunohistochemical labeling (Supplementary Figure 1a). In agreement, significant OCT2specific uptake was detected ex vivo in adrenal cellular extracts (Supplementary Figure 1b). Therefore, to tease apart the respective contributions of central and peripheral processes in corticosterone oversecretion, we performed an ACTH stimulation test after dexamethasone suppression. Dexamethasone at low doses is known to inhibit the HPA axis at the level of pituitary with little or no penetration in the brain and routinely used to evaluate adrenal sensitivity. [START_REF] Rush | The dexamethasone suppression test in patients with mood disorders[END_REF] We found a comparable sensitivity of OCT2 -/-and wild-type mice adrenal glands to physiological doses of ACTH, precluding a local effect of OCT2 (Figure 2b). These results demonstrate that the enhanced hormonal response to stress of OCT2 -/-mice is a direct consequence of the absence of OCT2 in the brain, at pituitary or suprapituitary sites.

OCT2 is implicated in vulnerability to UCMS

We made the hypothesis that their increased hormonal response to acute stress could confer to OCT2 -/-mice vulnerability to repeated stressful conditions. To challenge this possibility, we used UCMS, a depression model with high translational value for the investigation of interactions between genetic factors and environmental stress. [START_REF] Surget | Drugdependent requirement of hippocampal neurogenesis in a model of depression and of antidepressant reversal[END_REF][START_REF] Gourley | Regionally specific regulation of ERK MAP kinase in a model of antidepressant-sensitive chronic depression[END_REF] Mice of both genotypes were subjected to UCMS for 8 weeks and their behavioral assessment was carried out throughout the procedure. OCT2 -/-mice showed increased vulnerability to UCMS for the development of several anomalies reflecting distinctive symptoms of depression, involving grooming, spatial memory, social interaction and stress-sensitive spontaneous behavior. Coat state, an indicator of self-grooming behavior extremely sensitive to chronic stress, [START_REF] Surget | Drugdependent requirement of hippocampal neurogenesis in a model of depression and of antidepressant reversal[END_REF][START_REF] Denmark | The effects of chronic social defeat stress on mouse self-grooming behavior and its patterning[END_REF] deteriorated progressively throughout the UCMS procedure in both genotypes. This deterioration occured however significantly more intensely (P < 0.05 and P < 0.001) in OCT2 -/-mice than in wild-type mice (Figure 3a). The effect of UCMS on short-term spatial memory, a cognitive function implicating hippocampus [START_REF] Bird | The hippocampus and memory: insights from spatial processing[END_REF] often impaired in stress-related disorders, was assessed by measure of performance in the object location test (OLT). We found that UCMS induced notable spatial memory deficits much earlier in OCT2 -/-mice than in wild-type mice (Figure 3b). Significant differences in the exploration time of the displaced object compared to the non-displaced object at basal state were found before UCMS for both genotypes (wild-type, P < 0.0001; OCT2 -/-, P = 0.0003). UCMS affected spatial memory performance of both genotypes, albeit in a different manner. Strikingly, after 3 and 5 weeks of UCMS, only wild-type mice maintained the ability to discriminate the relocated object (P = 0.0039 and P = 0.0453, respectively) while OCT2 -/-mice had lost this ability (Figure 3b), indicating increased vulnerability in the early stages of the stress procedure. Finally, at later stages during the procedure, both genotypes lost the capacity to distinguish between the displaced and non-displaced objects. Next, spontaneous social interaction, an indicator of diminished appeal for social interaction, another anomaly found in MDD, was assessed before and after UCMS (Figure 3c). At basal state, both genotypes showed a comparable time of interaction with target. At 3 and 4 weeks after the start of UCMS, OCT2 -/-mice showed a significant decrease in interaction time with target compared to wild-type mice (P = 0.0248 and P = 0.0457, respectively), while at later stages time of interaction with target became undistinguishable between the two genotypes (Figure 3c). These results reveal the increased vulnerability of OCT2 -/-mice as regards social behavior, again at early stages of the UCMS procedure. Finally, decreased performance in nest building, a hippocampus-dependent task, [START_REF] Deacon | Assessing nest building in mice[END_REF] was detected in OCT2 -/-mice after 8-week UCMS (P = 0.0198), indicating a stress-driven inhibition of spontaneous behavior (Figure 3d). In contrast, sucrose preference was significantly decreased in both OCT2 -/-and control mice by the UCMS procedure (P < 0.0001), without leading to a full anhedonic state i.e., loss of preference at the end of the procedure, but no significant differences were detected between genotypes (Figure 3e). To determine the state of the negative feedback of the HPA axis in OCT2 mutants, the mice were submitted to a dexamethasone suppression test under swim stress conditions, before and after the complete UCMS procedure (Figure 3f). This evaluation showed a significant suppression (63-70%) of corticosterone secretion by dexamethasone for both genotypes, indicative of normal HPA negative feedback control in the mutants. HPA feedback in either genotype was poorly sensitive to UCMS, since no significant modifications in dexamethasone suppression were found after completion of the procedure (Figure 3f). Interestingly, after UCMS, corticosterone secretion induced by swim stress was decreased in OCT2 -/-mice, leading to levels comparable to those of wild-type mice (Figure 3g). Finally, genotype differences in immobility time in the forced-swim test, an acute behavioral despair paradigm, also leveled off after UCMS (Supplementary Figure 2).

Exogenous corticosterone can modify both high-affinity transporter-mediated and OCT2-mediated 5-HT and NE clearance in hippocampus in vivo

Previous studies raised the possibility that during the stress response corticosterone might impair clearance of 5-HT or other monoamines through inhibition of OCTs in vivo. To explore this possibility, we investigated the effect of subchronic corticosterone treatment on 5-HT and NE clearance mediated by the high-affinity and low-affinity transporters in vivo. 4day corticosterone oral administration raised circulating concentrations to 115.9 ± 14 ng/ml and 112.8 ± 18 ng/ml for wild-type and OCT2 -/-mice, respectively. The activity of postsynaptic neurons in hippocampus CA3, a region of high OCT2 density, was investigated in vivo by a combined microiontophoretic and electrophysiological approach, before and after subchronic corticosterone treatment. Injection of the dual inhibitor venlafaxine was used to inhibit the 5-HT and NE transporters and reveal OCT-mediated clearance. As previously reported, [START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF][START_REF] Gobbi | Modifications of the serotonergic system in mice lacking serotonin transporters: an in vivo electrophysiological study[END_REF] microiontophoretic applications of 5-HT (Figure 4a, left) or NE (Figure 4b, left) resulted in the suppression of the firing activity of CA3 pyramidal neurons. In pyramidal neurons of both control and corticosterone-treated hippocampus, the firing gradually recovered upon the cessation of the application of monoamines (Figure 4a-b, left). The recovery time for pyramidal neuron firing, with or without administration of corticosterone, and before and after acute systemic administration of venlafaxine, was expressed as RT 50 values (Figure 4a-b, right). In wild-type mice, RT 50 values before treatment with venlafaxine were significantly higher for corticosterone-treated mice than for vehicle-treated controls (5-HT, P = 0.0205; NE, P = 0.0394), indicating a notable decrease of monoamine clearance after subchronic corticosterone when the high-affinity 5-HT and NE transporters are fully active.

Venlafaxine at 16 mg/kg had a main significant effect on RT 50 for 5-HT (F 1,35 = 14.052; P = 0.0006, Figure 4a) and for NE (F 1,22 = 82.900; P < 0.0001, Figure 4b) reflecting partial blockade of the high-affinity transporters, and the differences between control and corticosterone-treated mice were maintained after its administration (5-HT, P = 0.0339; NE, P = 0.0342). In contrast, OCT2 -/-mice showed a similar increase of RT 50 values after corticosterone treatment in absence of venlafaxine (5-HT, P = 0.0021; NE, P < 0.0001), but this effect of corticosterone was no more detectable after venlafaxine treatment (Figure 4a-b, right). This last observation indicates that subchronic corticosterone treatment inhibits 5-HT and NE clearance in vivo when the NET and SERT are partially pharmacologically inactivated in wild-type mice, but not in OCT2 -/-mice. Taken altogether these experiments suggest that subchronic corticosterone administration at physiological concentrations may influence both high-affinity transporter-mediated and OCT2-mediated uptake.

OCT2 -/-mice show dysregulation of Akt/GSK3β signaling in the hippocampus

Glycogen synthase kinase-3β (GSK3β) is a serine/threonine kinase with multiple cellular functions. [START_REF] Cole | GSK3 as a Sensor Determining Cell Fate in the Brain[END_REF] It was recently proposed to be a downstream intracellular mediator of the moodrelated actions of 5-HT, [START_REF] Beaulieu | Role of GSK3 beta in behavioral abnormalities induced by serotonin deficiency[END_REF][START_REF] Beaulieu | Akt/GSK3 signaling in the action of psychotropic drugs[END_REF] an in vivo substrate of OCT2. [START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF] In particular, stimulation of 5-HT release in various brain regions including hippocampus was shown to inhibit considerably GSK3β by Ser-9 phosphorylation through the activation of 5-HT1A receptors, [START_REF] Li | In vivo regulation of glycogen synthase kinase-3beta (GSK3beta) by serotonergic activity in mouse brain[END_REF] an effect mediated by the principal GSK3β regulator, Akt. [START_REF] Frame | A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation[END_REF] We thus searched for anomalies of GSK3β signaling in OCT2 -/-mice, by assessing the functional state of this pathway in stress-related brain regions amenable to quantitative Western blot analysis (Figure 5a). We found that the phosphorylation of GSK3β at Ser-9, a phosphorylation site controlling negatively its activity, [START_REF] Frame | A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation[END_REF] was significantly increased in two regions of OCT2 -/-mice brain compared to wildtype, dorsal hippocampus (P = 0.0037) and PVN (P = 0.038), while total levels of GSK3β were unchanged (not shown), indicating decreased activity selectively in these brain regions. This increase in GSK3β phosphorylation was associated with increased activity of its modulator Akt, which showed increased Threo-308 phosphorylation (P < 0.0001 and P = 0.0194, respectively for hippocampus and PVN). This phosphorylation increase occurred independently of TrkB signaling, the canonical GSK3β-activating pathway activated by neurotrophins [START_REF] Segal | Selectivity in neurotrophin signaling: theme and variations[END_REF] as shown by unchanged phosphorylated to total TrkB ratios (Figure 5a) and total TrkB levels (not shown).

To further study GSK3β dysregulation, we focused on a single region, hippocampus, and explored the functional state of this kinase after acute and chronic stress. Ser-9 phosphorylation of GSK3β was found highly increased after acute swim stress in hippocampus of both wild-type and OCT2 -/-mice (P < 0.0001) while total amounts of GSK3β were unchanged (not shown). This was associated with increases in total amount of phosphorylated TrkB in wild-type mice (P = 0.0006) and of both phosphorylated Akt (P = 0.0007) and phosphorylated TrkB (P = 0.0101) in OCT2 -/-mice (Figure 5b, bottom, pAkt or pTrkB/β-actin ratio) while phosphorylated over non-phosphorylated ratios of these proteins were unchanged or decreased by swim stress (Figure 5b, top), suggesting the implication of neurotrophin/TrkB signaling in acute stress-induced GSK3β inhibition. Interestingly, after swim stress, Ser-9 phosphorylation at GSK3β (P = 0.0021) and Threo-308 phosphorylation at Akt (P = 0.0268) but not total amount of these proteins (not shown) were increased in OCT2 - /-mice hippocampus compared to wild-type with, as at basal state, no genotype-specific differences in TrkB relative activity (Figure 5b) or level (not shown). Thus, the absence of OCT2 potentiates through Akt but independently of TrkB the effects on GSK3β inhibition occurring during acute stress. UCMS in contrast had limited effects on the GSK3β intracellular pathway. This procedure leveled out the differences between genotypes in Ser-9 phosphorylation, which was decreased in OCT2 -/-(P = 0.0005) but not wild-type mice, while decreasing effects on Akt and TrkB activity were detected for both genotypes (Figure 5b).

Link between Akt/GSK3β signaling in OCT2

-/-hippocampus during acute stress and 5-

HT1A receptor activity

As shown above, the levels of phospho-Ser-9-GSK3β and phospho-Threo-308-Akt were significantly increased in certain regions (hippocampus, PVN) of OCT2 -/-mice, revealing a notable perturbation of this intracellular pathway at basal state. Since TrkB was not involved in this dysregulation of Akt/GSK3β signaling, we tested whether it could be linked to altered serotonergic tonus and in particular 5-HT receptor activation. [START_REF] Beaulieu | Role of GSK3 beta in behavioral abnormalities induced by serotonin deficiency[END_REF][START_REF] Li | In vivo regulation of glycogen synthase kinase-3beta (GSK3beta) by serotonergic activity in mouse brain[END_REF] To further identify the mechanisms leading to GSK3β inhibition in OCT2 -/-mice brain, we tested by stimulation of [35S]GTPγS binding on brain sections the functional state of 5-HT1A and 5-HT2A receptors, both highly expressed in hippocampus CA3. [START_REF] Tanaka | Serotonin receptor expression along the dorsalventral axis of mouse hippocampus[END_REF] These experiments indicated that the 5-HT1A receptor was overactivated in hippocampus of OCT2 -/-mice compared to wild-type mice (P = 0.0163), while no significant modification of the activity of 5-HT2A receptors was detected (Figure 6a). To determine whether this increase in 5-HT1A receptor activity in the mutants could participate in modifications of Akt/GSK3β signaling during acute stress, the specific 5-HT1A receptor antagonist WAY100635 was injected prior to swim stress to the mice, and the functional state of the Akt/GSK3β pathway explored in hippocampus. WAY100635 restored in OCT2 -/-mice a ratio of phosphorylated to total GSK3β and Akt comparable to wild-type mice, with an effect in hippocampus of OCT2 -/-mice (GSK3β, P = 0.0058; Akt, P = 0.0036) but not wild-type mice (Figure 6b). These experiments suggest that increased 5-HT tonus and in particular post-synaptic 5-HT1A receptor activation could be responsible at least in part for Akt/GSK3β signaling dysregulation in the mutants during acute stress in the hippocampus.

Discussion

Stress and HPA axis dysregulation constitute important risk factors for psychiatric disorders such as depression and PTSD. [START_REF] Pariante | The HPA axis in major depression: classical theories and new developments[END_REF][START_REF] Pitman | Biological studies of post-traumatic stress disorder[END_REF] Chronic stress in rodents can mimic these noxious effects, inducing gradual neurobiological and behavioral anomalies resembling symptoms of human depression. [START_REF] Surget | Drugdependent requirement of hippocampal neurogenesis in a model of depression and of antidepressant reversal[END_REF][START_REF] Gourley | Regionally specific regulation of ERK MAP kinase in a model of antidepressant-sensitive chronic depression[END_REF] In addition, HPA axis activity has been suggested to play a role in "passive" resilience i.e., resilience mediated by the absence of key neurochemical, molecular or endocrinal responses in certain individuals. [START_REF] Elliott | Resilience to social stress coincides with functional DNA methylation of the Crf gene in adult mice[END_REF][START_REF] Russo | Neurobiology of resilience[END_REF] The major finding of this study was that OCT2 activity profoundly modulates the hormonal response to acute stress and vulnerability to repeated stress. OCT2 -/-mice showed an aggravation of several alterations induced by UCMS (Figure 6c), some of which evocative of the working memory deficits [START_REF] Marazziti | Cognitive impairment in major depression[END_REF] and social withdrawal symptoms that occur in MDD patients. [START_REF] Nestler | Neurobiology of depression[END_REF][START_REF] Nutt | The psychobiology of posttraumatic stress disorder[END_REF] Intriguingly, these genotype-specific differences in social interaction and spatial memory appeared transiently in the first weeks of the UCMS procedure, suggesting a role of OCT2 in the acquisition of this phenotype in the early phases. Contrasting with these behaviors, no significant differential effect of UCMS on anhedonia, evaluated by sucrose preference, was detected in the OCT2 mutants. Anhedonia, a cardinal symptom of MDD, is only present in half of the patients [START_REF] Bogdan | Neurogenetics of depression: a focus on reward processing and stress sensitivity[END_REF] and also prominent in other mental disorders. Since OCT2 has a similar localization in human and rodent brain, [START_REF] Busch | Human neurons express the polyspecific cation transporter hOCT2, which translocates monoamine neurotransmitters, amantadine, and memantine[END_REF] our findings suggest that its activity could be a determinant of individual variability in vulnerability to MDD and contribute to the clinical heterogeneity of this disorder.

It is commonly admitted that circulating corticosteroids mediate for a large part the deleterious effects of repeated stress on the brain. Enhanced corticosterone release is thus probably implicated in the differential effects of UCMS in OCT2 -/-mice. Several lines of data argue in favor of this possibility. First, in agreement with our observations, stress and corticosterone have been shown to disrupt hippocampal-mediated learning [START_REF] Lupien | Effects of stress throughout the lifespan on the brain, behaviour and cognition[END_REF][START_REF] Bangasser | The hippocampus is necessary for enhancements and impairments of learning following stress[END_REF][START_REF] Wagner | Homer1 mediates acute stress-induced cognitive deficits in the dorsal hippocampus[END_REF][START_REF] Kaouane | Glucocorticoids can induce PTSD-like memory impairments in mice[END_REF] and spatial memory retrieval. [START_REF] De Quervain | Stress and glucocorticoids impair retrieval of long-term spatial memory[END_REF] Second, in keeping with a transitory action of UCMS, swim stressinduced corticosterone secretion was completely blunted in OCT2 -/-mice at the end of the full UCMS procedure. Finally, chronic administration of corticosterone, another long-term depression paradigm which very rapidly enhances and evens out the levels of circulating corticosterone, was shown to induce in contrast to UCMS a comparable deterioration of the coat state of both genotypes. [START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF] These observations support a causal relationship between increased corticosterone release in OCT2 -/-mice and some of the anomalies found in these mice during the first weeks of the chronic stress procedure.

OCT2 was detected in several brain regions implicated in the response to stress, such as prelimbic and infralimbic cortices, hippocampus, amygdala, dorsomedial and arcuate nuclei of hypothalamus, paraventricular nucleus of the thalamus and pituitary, as well as in adrenals. [START_REF] Ulrich-Lai | Neural regulation of endocrine and autonomic stress responses[END_REF][START_REF] Herman | Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary-adrenocortical responsiveness[END_REF][START_REF] Herman | Neurocircuitry of stress: central control of the hypothalamo-pituitary-adrenocortical axis[END_REF] It is however difficult at this stage to attribute HPA activation in the mutants to the absence of OCT2 within specific brain regions. Investigation of adrenal sensitivity to ACTH allowed us to exclude periphery-based mechanisms involving sympathetic innervation, circulating catecholamines and/or paracrine control within adrenal glands. [START_REF] Nussdorfer | Paracrine control of adrenal cortical function by medullary chromaffin cells[END_REF][START_REF] Brede | Differential control of adrenal and sympathetic catecholamine release by alpha 2-adrenoceptor subtypes[END_REF] Hence, enhanced hormonal response to acute stress in OCT2 -/-mice appears to occur through mechanisms based in circuits controlling HPA activation upstream in the brain, at pituitary or suprapituitary sites, which remain to be determined. Moreover, a normal state of HPA negative feedback was observed in OCT2 -/-mice, consistent with a role for OCT2 in the initial steps of HPA axis activation, rather than in indirect modulation of this response through negative feedback.

Along with increased HPA axis activation during acute stress, OCT2 -/-mice show important dysregulation of brain GSK3β signaling, a pathway previously implicated in the regulation of mood. [START_REF] Kaidanovich-Beilin | Rapid antidepressive-like activity of specific glycogen synthase kinase-3 inhibitor and its effect on beta-catenin in mouse hippocampus[END_REF][START_REF] Polter | Deficiency in the inhibitory serine-phosphorylation of glycogen synthase kinase-3 increases sensitivity to mood disturbances[END_REF][START_REF] O'brien | Glycogen synthase kinase-3beta haploinsufficiency mimics the behavioral and molecular effects of lithium[END_REF] GSK3β is constitutively active in post-mitotic neurons but inhibited by phosphorylation in response to various upstream pathways implicating neurotrophins [START_REF] Segal | Selectivity in neurotrophin signaling: theme and variations[END_REF] and GPCRs. [START_REF] Beaulieu | A beta-arrestin 2 signaling complex mediates lithium action on behavior[END_REF] To investigate the GSK3β modulation during stress, we focused on dorsal hippocampus and found that a profound inhibition of this kinase occurs during acute stress in normal mice, associated with an activation of the neurotrophin receptor TrkB. Remarkably, in OCT2 -/-mice, GSK3β activity was significantly inhibited both at basal state and during acute stress compared to wild-type mice, accompanied by the activation of its main regulator Akt.

As consequence of this modulation of hippocampal signaling, the inhibitory action of acute stress on GSK3β was notably aggravated in mutant mice (Figure 6c).

We excluded the canonical neurotrophin/TrkB pathway as a main mechanism for OCT2mediated GSK3β regulation but found in mutant mice hippocampus a differential coupling with 5-HT receptor activation, another major GSK3β regulatory pathway in neural cells. [START_REF] Beaulieu | A beta-arrestin 2 signaling complex mediates lithium action on behavior[END_REF][START_REF] Polter | Glycogen Synthase Kinase-3 is an Intermediate Modulator of Serotonin Neurotransmission[END_REF] 5-HT deficiency in Tph2 knockout mice was previously shown to activate GSK3β, while the inhibition of this kinase was able to correct the mood-related behavioral anomalies of these depleted mice. [START_REF] Beaulieu | Role of GSK3 beta in behavioral abnormalities induced by serotonin deficiency[END_REF] Reciprocally, elevation of 5-HT level was shown to inhibit considerably GSK3β by Ser-9 phosphorylation in several brain regions including hippocampus, through the activation of 5-HT1A receptors [START_REF] Li | In vivo regulation of glycogen synthase kinase-3beta (GSK3beta) by serotonergic activity in mouse brain[END_REF] and Akt signaling. [START_REF] Polter | Glycogen Synthase Kinase-3 is an Intermediate Modulator of Serotonin Neurotransmission[END_REF] In agreement with this mechanism, our results suggest that increased 5-HT tonus leading to overactivation of 5-HT1A receptor could drive at least in part the anomalies in Akt/GSK3 signaling in OCT2 -/-mice (Figure 6c). Interestingly, post-synaptic 5-HT1A receptor activation was also shown to be involved in corticosterone secretion, [START_REF] Tsuji | Protective effects of 5-HT1A receptor agonists against emotional changes produced by stress stimuli are related to their neuroendocrine effects[END_REF][START_REF] Mikkelsen | Serotonergic stimulation of the rat hypothalamo-pituitary-adrenal axis: interaction between 5-HT1A and 5-HT2A receptors[END_REF] suggesting a potential mechanistic link between 5-HT tonus, corticosterone secretion and GSK3β inactivation.

We do not at this point know whether differential GSK3β inhibition plays a role in the exacerbated vulnerability of OCT2 -/-mice to chronic stress. Nevertheless, several observations support this possibility. GSK3β activation was shown to be important for memory retrieval [START_REF] Hong | GSK-3beta activity in the hippocampus is required for memory retrieval[END_REF] and reconsolidation [START_REF] Kimura | GSK-3beta is required for memory reconsolidation in adult brain[END_REF] and IGFII-dependent memory enhancement [START_REF] Chen | A critical role for IGF-II in memory consolidation and enhancement[END_REF] , while its inhibition was linked to impaired NMDA receptor-dependent LTD, [START_REF] Peineau | LTP inhibits LTD in the hippocampus via regulation of GSK3beta[END_REF] a process required for spatial memory consolidation. [START_REF] Ge | Hippocampal long-term depression is required for the consolidation of spatial memory[END_REF][START_REF] Dong | Hippocampal long-term depression mediates spatial reversal learning in the Morris water maze[END_REF] Other studies suggest that forebrain GSK3β play a role in sociability. [START_REF] Latapy | Selective deletion of forebrain glycogen synthase kinase 3beta reveals a central role in serotonin-sensitive anxiety and social behaviour[END_REF] Interestingly, Akt/GSK3β signaling in the brain was also implicated in susceptibility to social defeat [START_REF] Wilkinson | A novel role of the WNT-dishevelled-GSK3beta signaling cascade in the mouse nucleus accumbens in a social defeat model of depression[END_REF] and to PTSD-like symptoms following inescapable stress. [START_REF] Dahlhoff | AKT/GSK-3beta/beta-catenin signalling within hippocampus and amygdala reflects genetically determined differences in posttraumatic stress disorder like symptoms[END_REF] In agreement with a transitory action of GSK3β during UCMS, our study demonstrates that this kinase was strongly inhibited during acute stress in normal mice hippocampus, while its activation state was unchanged after completion of the UCMS procedure. In this perspective, the potentiation of GSK3β inhibition in OCT2 -/-mice could contribute to their vulnerability during the first weeks of UCMS and constitute at these early stages a marker of vulnerability to chronic stress. Finally, while these results clearly demonstrate that OCT2 deletion has major consequences on corticosterone secretion, a reciprocal action of basal or peak corticosterone on OCT-mediated transport cannot be excluded, as demonstrated here by microiontophoretic electrophysiology. Such an interaction has been demonstrated in vitro [START_REF] Grundemann | Molecular identification of the corticosterone-sensitive extraneuronal catecholamine transporter[END_REF][START_REF] Hayer-Zillgen | Expression and pharmacological profile of the human organic cation transporters hOCT1, hOCT2 and hOCT3[END_REF] and suggested to occur in vivo by recent studies showing that repeated swim stress could reduce 5-HT clearance rate in mice hippocampus through corticosterone release. [START_REF] Baganz | Repeated Swim Impairs Serotonin Clearance via a Corticosterone-Sensitive Mechanism: Organic Cation Transporter 3, the Smoking Gun[END_REF] OCT2 blockade by corticosterone could thus contribute to the neurochemical and molecular adaptative modifications taking place during short-or long-term stress. In this case, corticosterone blockade of OCT2 during the response to stress would be expected to reinforce 5-HT signaling and GSK3β inhibition, providing an additional feed-forward mechanism to modulate this pathway. This possibility is for the time being highly speculative, as this interaction could occur during acute stress in only certain brain regions or neurons, generating radically different consequences than full-blown OCT2 invalidation.

In conclusion, the present study describes a novel mechanism at the core of the geneticenvironment interactions that underlie individual variability in the response to stress. We identify OCT2 as a genetic factor that directly impacts on the molecular and hormonal events taking place during acute stress, accentuating the noxious effects of repeated stress on the development of stress-related phenotypes. This suggests that genetic polymorphisms modifying OCT2 transport activity or long-term administration of drugs inhibiting OCT2, such as certain antidiabetics, cytostatics or antiviral compounds, [START_REF] Takeda | Human organic anion transporters and human organic cation transporters mediate renal antiviral transport[END_REF][START_REF] Kimura | Metformin is a superior substrate for renal organic cation transporter OCT2 rather than hepatic OCT1[END_REF] could have important consequences on the vulnerability to environmental stress.

Legends to figures

Figure 1 OCT2 is expressed in stress-related circuits in the brain. (a-l) Immunofluorescent histochemistry of coronal mice brain sections. OCT2 is notably enriched in layers II-III, V and VI of the prelimbic (PrL) and infralimbic (IL) cortices (a). In the hippocampal formation, high OCT2 expression is found in the pyramidal cell layer (pyr) of CA1, CA2 and CA3, in the polymorph (po) layer of the DG and diffuse labeling is detected in the lacunosum molecular (LMol) layer of CA1-3 (b). OCT2 positive cells are detected in the ventral subiculum (vSub, c, d) and in the amygdalohippocampal area (AHi, c). OCT2 is also detected in the medial amygdaloid nucleus (MeA, e), dorsomedial (DMH), ventromedial (VMH) and arcuate (Arc) nuclei of the hypothalamus (f), paraventricular nucleus of the thalamus (PVA, g) and median eminence (ME, h). OCT2 is found in a non-corticotrope subpopulation of the anterior lobe of pituitary (AL), as shown by co-labeling with ACTH antibodies (i) and uniformly distributed in the posterior lobe (PL, j). No labeling was detected in OCT2 -/-mice brain, shown for cingulate and motor cortices (Cx, k) and hippocampus (l). Cortical layers are identified by roman numerals. Mol, molecular layer; ec, external capsule; gr, granular layer; Pe, periventricular nucleus of the hypothalamus. Scale bars represent 100 µm (a-c, f, h, k, l) and 20 µm (d, e, g, i, j). Figure 2 OCT2 in the brain controls the hormonal response to acute stress. (a) Plasma corticosterone levels in basal conditions (left) and after a 15-min swim stress (right) are considerably increased in OCT2 -/-mice. Student's t-test, unpaired, two-tailed, t 29 = 3.0, **P = 0.0058, n = 15-16 for basal conditions. Two-way analysis of variance (ANOVA) reveals main significant effects of genotype (F 1,106 = 58.18; P < 0.0001), time (F 7,106 = 49.81; P < 0.0001) and genotype x time interaction (F 7,106 = 2.727; P = 0.0121). Fisher's post-hoc test, **P < 0.01, ***P < 0.001, OCT2 -/-mice versus wild-type mice (n = 4-12) for swim stress.

(b) OCT2 -/-mice adrenal glands respond normally to ACTH stimulation. Plasma corticosterone secretion was induced by a single ACTH injection (solid line) after dexamethasone (Dex) pretreatment in wild-type and OCT2 -/-mice, compared to saline injection (dashed line). Two-way ANOVA (n = 4-6) reveals no main significant effects of genotype (F 1,37 = 3.8; P = 0.0603). Results are given as mean ± s.e.m. Fisher's post-hoc test show differences between wild-type and mice from week 2 to the end of the procedure (*P < 0.05, ***P < 0.001). (b) OCT2 -/-mice show spatial memory deficits during the UCMS procedure. Two-way ANOVA (n = 10) reveals significant main effects of object (F 1,114 = 52.13; P < 0.0001) and object x UCMS interaction (F 3,114 = 5.813; P = 0.0010). Fisher's post-hoc test reveals significant differences in the exploration time of the displaced object compared to the non-displaced object at basal state before UCMS for both genotypes (wild-type, ***P < 0.0001; OCT2 -/-, ***P = 0.0003). At 3 and 5 weeks of UCMS, only wild-type mice retain a significant discrimination of the relocated object (Fisher's posthoc test, P = 0.0039 and P = 0.0453), while OCT2 -/-mice have lost this ability. (c) OCT2 -/- mice show enhanced social interaction deficits during the UCMS procedure. Two-way ANOVA (n = 10) reveals significant main effects of genotype (F 1,86 = 5.087; P = 0.0266).

Fisher's post-hoc test indicate differences in interaction time at 3 weeks (*P = 0.0248) and 4 weeks (*P = 0.0457). (d) Decreased performance in nest building in OCT2 -/-mice after 5week UCMS (Student's t-test, unpaired, two-tailed, t 27 = 2.478, P = 0.0198, n = 14-15). (e) UCMS alters sucrose preference of both OCT2 -/-and wild-type mice. Two-way ANOVA (n = 10) show significant main effects of UCMS (F 2,30 = 16.89; P < 0.0001) but not genotype.

Results are given as mean ± s.e.m. (f) HPA feedback is comparable between OCT2 -/-and wild-type mice before and after UCMS. Two-way ANOVA (n = 10) show significant effect of UCMS (F 1,36 = 4.589; P = 0.039) but not of genotype (F 1,36 = 1.684; P = 0.2026). (g) Swim stress induced corticosterone secretion differences between OCT2 -/-and wild-type mice are blunted after UCMS. Two-way ANOVA (n = 6-10) show significant effect of UCMS (F 1,28 = 24.57; P < 0.0001) and genotype x treatment interaction (F 1,28 = 5.166; P = 0.0309). Fisher's post-hoc test reveals significant differences between OCT2 -/-and wild-type mice before ( # P = 0.0127) but not after UCMS, and significant effects of UCMS for OCT2 -/-mice (***P < 0.0001).

(

  50 mM HEPES, pH 7.4, 100 mM NaCl, 3 mM MgCl 2 , 0.2 mM EGTA, 0.2 mM DTT and 2 mM GDP) for 5-HT1A receptors and in Tris buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 3 mM MgCl 2 , 0.2 mM EGTA, 0.2 mM DTT and 2 mM GDP) for 5-HT2A receptors, then incubated with 25 nM [35S]GTPγS in the same buffers for 2 h in presence of selective agonists. 8-OHDPAT (10 μM) and DOI (5 nM) were used to stimulate [35S]GTPγS binding at 5-HT1A and 5-HT2A receptors, respectively. Receptor subtype-specificity of stimulated [35S]GTPγS binding was verified by the addition of WAY100635 (1 mM) for 5-HT1A receptors and ketanserin (10 μM) for 5-HT2A receptors. Basal binding was determined in the absence of agonists. The sections were washed in cold HEPES buffer for 2 min, briefly dipped in ice-cold water, dried and exposed to Kodak BioMax MR autoradiographic film (VWR, Rochester, NY, USA). Quantification of autoradiograms were carried out with MCID software (Imaging Research Inc, St. Catherines, Ontario, Canada).

Figure 3

 3 Figure 3 OCT2 -/-mice show increased vulnerability to the effects of UCMS. (a) OCT2 -/- mice show enhanced deterioration of coat state during the UCMS procedure. Two-way ANOVA (n = 6-15) reveals significant effects of time (F 8,211 = 17.82; P < 0.0001), genotype (F 1,211 = 77.25; P < 0.0001) and genotype x time interaction (F 8,211 = 3.975; P = 0.0002) and

Figure 4

 4 Figure 4 In vivo OCT2-mediated clearance in hippocampus is sensitive to physiological levels of corticosterone. Recovery of pyramidal neuron firing activity in vivo after 5-HT (a) or NE (b) application in wild-type mice after subchronic corticosterone treatment. (a, b, left) Representative integrated firing histograms show the effects of microiontophoretically applied 5-HT or NE on the spontaneous activity of CA3 pyramidal neurons, before and following systemic administration of venlafaxine (16 mg/kg). White boxes indicate the duration of iontophoretic ejection of 5-HT or NE, respectively. The arrow indicates the time at which the injection of venlafaxine was completed. (a, b, right) Recovery time (RT50) values for pyramidal neuronal activity following microiontophoretic applications of 5-HT (a) or NE (b) are increased by corticosterone treatment in OCT2 -/-mice before but not after venlafaxine injection. Three-way ANOVA of RT50 values (n = 4-6) reveals significant main effects of genotype (5-HT: F 1,35 = 5.880; P = 0.206; NE: F 1,22 = 42.925; P < 0.0001), corticosterone treatment (5-HT: F 1,35 = 14.220; P = 0.0006; NE: F 1,22 = 104.305; P < 0.0001), venlafaxine treatment (5-HT: F 1,35 = 14.052; P = 0.0006; NE: F 1,22 = 82.900; P < 0.0001) and genotype x

Figure 5

 5 Figure 5 OCT2 -/-mice show profound alterations of Akt/GSK3β signaling at basal state and

  (a) Quantitative Western blot analysis show alterations of phosphorylation of GSK3β (pSer9), and Akt (pThr308) independently of phosphorylation of TrkB (pTyr516) in hippocampus (HPC) and paraventricular hypothalamic nucleus (PVN) of OCT2 -/-mice compared to wildtype mice, but not in prefrontal cortex (PFC) and ventral subiculum (vSub). Results are given as mean ± s.e.m of phosphorylated over non phosphorylated protein ratio (Student's t-test, unpaired, two-tailed, GSK3β: HPC, t 33 = 3.122, **P = 0.0037, n = 17-18, PVN, t 24 = 2.196, *P = 0.038, n = 13 ; Akt: HPC, t 33 = 5.288, ***P < 0.0001, n = 17-18, PVN, t 24 = 2.505, *P = 0.0194, n = 13). (b) Quantitative Western blot analysis show alterations of phosphorylation of GSK3β (pSer9), Akt (pThr308) and TrkB (pTyr516) in the hippocampus of wild-type and OCT2 -/-mice exposed to acute swim stress or UCMS. Results are given as mean of phosphorylated over non phosphorylated protein ratio ± s.e.m (top) and mean of phosphorylated protein over β-actin ratio ± s.e.m (bottom). Phospho-TrkB and TrkB are indicated by an arrow. Two-way ANOVA of phosphorylated over non phosphorylated protein ratios (n = 5-18) reveals a significant effect of swim stress for GSK3β (F 1,55 = 116.2; P < 0.0001) and TrkB (F 1,35 = 14.94; P = 0.0005) and of genotype for GSK3β (F 1,55 = 11.52; P = 0.0013) and Akt (F 1,55 = 28.55; P < 0.0001). Fisher's post-hoc test reveals significant increase of phosphorylated over non phosphorylated GSK3β ratios after swim stress in wild-type and OCT2 -/-mice (***P < 0.0001), and of GSK3β ( ## P = 0.0021) and Akt ( # P = 0.0268) phosphorylated over non phosphorylated ratio in OCT2 -/-mice compared to wild-type mice after swim stress. Two-way ANOVA of phosphorylated protein over β-actin ratios reveals a significant effect of swim stress for GSK3β (F 1,55 = 129.8; P < 0.0001), Akt (F 1,55 = 14.57; P = 0.0003) and TrkB (F 1,35 = 20.99; P < 0.0001) and of genotype for GSK3β (F 1,55 = 8.761; P = 0.0045) and Akt (F 1,55 = 26.70; P < 0.0001). Fisher's post-hoc test reveals a significant increase of phosphorylated GSK3β over β-actin ratios after swim stress in wild-type and OCT2 -/-mice (***P < 0.0001). This increase was associated with significant increases in phosphorylated Akt and TrkB over β-actin ratios (wild-type, TrkB: ***P = 0.0006; OCT2 -/-, Akt: ***P = 0.0007, TrkB: *P = 0.0101). Two-way ANOVA of phosphorylated protein over non phosphorylated protein ratios (n = 4-18) reveals a significant effect of UCMS for GSK3β (F 1,51 = 9.522; P = 0.0033), Akt (F 1,51 = 94.97; P < 0.0001) and TrkB (F 1,35 = 17.60; P = 0.0002) and of genotype x UCMS interaction for GSK3β (F 1,51 = 4.839; P = 0.0324) and Akt (F 1,51 = 15.24; P = 0.0003). Fisher's post-hoc test reveals a significant decrease of GSK3β phosphorylation after UCMS in OCT2 -/-(***P = 0.0005) but not wild-type mice, and of Akt and TrkB phosphorylation after UCMS in both genotypes (wild-type, Akt: ***P = 0.0001; TrkB: *P = 0.0125; OCT2 -/-, Akt: ***P < 0.0001; TrkB: **P = 0.0017).

Figure 6

 6 Figure 6 Enhanced 5-HT1A receptor activity restores Akt/GSK3β signaling in OCT2 -/-mice during swim stress. (a) 5-HT1A receptor activity is selectively increased in OCT2 -/-mice hippocampus. Stimulated [35S]GTPγS binding for the 5-HT1A and 5-HT2A receptors are expressed as mean ± s.e.m. of percentage values of basal level (Student's t-test, unpaired, twotailed, t 9 = 2.947, *P = 0.0163, n = 5-6) and representative sections of specific ligands binding are shown below. (b) Quantitative Western blot analysis of phosphorylation of

Acknowledgments

We thank M. Nosten-Bertrand for advice concerning behavior and statistics and F. Machulka for expert assistance in animal care. T.C. A.B. and L.B. were recipients of fellowships from the French Ministry for Research and the Société Française de Pharmacologie et Thérapeutique. Financial support was provided by the Institut National pour la Santé et la Recherche Médicale (INSERM), the Fondation de France and the Agence nationale de la recherche (ANR-13-SAMENTA-0003-01).

Conflict of interest

The authors declare no conflict of interest.

Supplementary Information accompanies the paper on the Molecular Psychiatry website.

c BRAIN ORGANIC CATION TRANSPORTER 2 CONTROLS RESPONSE AND VULNERABILITY TO STRESS AND GSK3β SIGNALING

Thomas Couroussé, Alexandre Bacq, Catherine Belzung, Bruno Guiard, Laure Balasse, Franck Louis, Anne-Marie Le Guisquet, Alain M Gardier, Alfred H Schinkel, Bruno Giros and Sophie Gautron Supplementary Figures 12Supplementary Figure 1 OCT2 is expressed in the adrenal glands and participates locally in 5-HT and NE uptake. 2 UCMS blunts the genotype-specific differences in the forced-swim test, a behavioral despair paradigm. As previously described, 1 OCT2 -/-mice show increased immobility in the forced-swim test (FST) compared to wild-type mice before (Student's t-test, unpaired, two-tailed, t 14 = 2.724, *P = 0.0165, n = 7-9) but not after UCMS. Results are given as mean ± s.e.m.


William Timothy
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email: ludovic.orlando@univ-tlse3.fr

Early dispersal of domestic horses into the Great Plains and northern Rockies

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

T he spread of domestic horses and their integration into Indigenous societies contributed to profound social and ecological transformations across western North America. However, the mechanisms and timing of this transition are poorly understood. Horses and other members of the genus Equus originated in North America (1,2). Horses and equids formed an important component of human lifeways across the continent during the final Pleistocene (3)(4)(5), which is still encoded in some Indigenous oral traditions, including those of the Lakota [START_REF] Horse | The Relationship Between the Indigenous Peoples of the Americas and the Horse: Deconstructing a Eurocentric Myth[END_REF]. Although Western scholars commonly consider horses to have disappeared at lower latitudes by the early Holocene, environmental DNA suggests their presence in arctic zones as late as 5000 to 6000 years before the present (7,[START_REF] Haile | Proc. Natl. Acad. Sci. U.S.A[END_REF]. Few archaeozoological studies have carefully addressed their possible persistence at lower latitudes during the Holocene.

Viking colonizers brought horses as far as Greenland during the 10th to 14th centuries CE [START_REF] Enghoff | Hunting, Fishing and Animal Husbandry at the Farm Beneath the Sand[END_REF] and settled along areas of the Newfoundland coast during the 11th century CE [START_REF] Kuitems | [END_REF]. There is, however, no direct evidence that Viking horses reached settlements on the mainland (11). Instead, most western scholars accept that horses were first reintroduced into the Americas by Spanish settlers in the late 15th century CE, reaching the mainland in the early 16th century CE with the Spanish colonization of Mexico [START_REF] Mitchell | Horse Nations: The Worldwide Impact of the Horse on Indigenous Societies Post-1492[END_REF]. During the 17th to 19th centuries CE, colonizing European powers, including the British, Spanish, and French [START_REF] Bowen | The starving time at Jamestown: Faunal analysis of pit 1, pit 3, the bulwark ditch, ditch 6, ditch 7, and midden 1[END_REF][START_REF] Jones | [END_REF], and possibly Russian and Chinese merchants (15) imported considerable numbers of horses into western North America.

Whereas horses would generally be categorized as domestic commodities, Indigenous peoples often maintain different relationships with them. Lakota peoples attribute to horses a nationhood status equal to their own. The Lakota-horse relationship is thus one of great reverence, deeply embedded in their identity, spirituality, science, and cosmogony. Lakota peoples do not have concepts for "wild" and "domesticated." In fact, Šungwakaŋ-"the Horse Nation"-was neither controlled behind fences nor forced into breeding. Rather, the Lakota peoples strove to cultivate their environment and adapt their lifeways to ensure that Šungwakaŋ could live aligned with its natural systems. Within this nation-to-nation alliance, the horse enhanced the abilities of the Lakota with regard to hunting, mobility, healing, and more [START_REF] Hunska Tašunke Icu | Standing for Unči Maka (Grandmother Earth) and All Life: An Introduction to Lakota Traditional Sciences, Principles and Protocols and the Birth of a New Era of Scientific Collaboration[END_REF]. Therefore, for the Lakota peoples, saying "our horse" never reflects ownership but rather responsibility for a sacred relative.

European colonization entirely altered Indigenous social dynamics, hierarchy, and lifeways, introducing profound changes to subsistence modes, movement, and warfare [START_REF] Ewers | The Horse in Blackfoot Indian Culture, with Comparative Material from Other Western Tribes[END_REF]. Many Indigenous peoples within the Great Plains and American Southwest developed horsebased pastoral or hunting economies and expanded transcontinental networks of raiding and exchange. Some became militarily dominant polities that maintained autonomy and sovereignty into the end of the 19th century CE, with many maintaining this sovereignty today [START_REF] Hämäläinen | Lakota America: A New History of Indigenous Power[END_REF][START_REF] Hämäläinen | The Comanche Empire[END_REF].

Historical models for the post-Columbian North American dispersal of horses and their integration into Indigenous cultures are almost exclusively derived from textual sources written by European observers dating largely to the 18th and 19th centuries CE [e.g., [START_REF] Wissler | [END_REF]21)]. These sources depict horses first spreading in appreciable numbers north from what is today the American Southwest after the Pueblo Revolt of 1680 CE, when Spanish settlers were temporarily expelled from much of New Mexico [START_REF] Olsen | Wild Equids: Ecology, Management, and Conservation[END_REF]. Given that most of the continent north of New Mexico was terra incognita to European chroniclers, natural and cultural landscapes remained largely uncharacterized until the early 19th century CE [START_REF] Kessell | Whither the Waters: Mapping the Great Basin from Bernardo de Miera to John C. Frémont[END_REF]. Furthermore, these Euro-American historic records are often rife with inaccuracies and strong anti-Indigenous biases, depreciating the fundamental relationship between Indigenous peoples and horses [START_REF] Taylor | [END_REF].

Despite representing a major source for understanding the timing and ways in which horses were managed, ridden, and integrated into early societies, archaeological remains of domestic horses from Indigenous contexts are also overlooked [START_REF] Taylor | [END_REF]. In this study, we extensively surveyed existing archaeological collections to identify early historic horse specimens with potential for reconstructing early human-horse relationships across the American Southwest and Great Plains (Fig. 1). Together, DNA, archaeozoological, and stable isotope data support the introduction of Spanish-sourced domestic horses into Indigenous societies across the plains before the first half of the 17th century CE.

Results

Indigenous societies incorporated horses before the Pueblo Revolt

Of 33 early American equid specimens, we successfully radiocarbon dated 29 and characterized a total of 27 genetically, along with six new specimens from Eurasia (producing nine ancient genomes with an average depthof-coverage of 2.06× to 12.24×, with substantial genome-wide sequence data for seven additional horse specimens, 0.06× to 0.96×, plus one donkey genome, 1.32×) (Fig. 1). Zonkey software analyses (25) confirmed all specimens as horses, except NW36 from Chupaderos, Mexico, which is a donkey jennet (table S1). Although a plateau in the radiocarbon calibration curve prevents easy discrimination between horses dating between 1670 CE and the early 20th century CE, we identified three horses from North American Indigenous contexts conclusively predating the Pueblo Revolt. Near-infrared (NIR) spectrum analysis failed to detect any external contaminants that could have affected radiocarbon dating (materials and methods section 3). The three specimens include a juvenile horse burial from the site of Blacks Fork in southwestern Wyoming, an adult horse cranium from Kaw River, Kansas, and isolated skeletal elements from the site of Paa'ko, New Mexico, along with new analysis of a previously dated specimen from American Falls Reservoir, Idaho, dated to between 1597 and 1657 CE (26), which we also assessed with NIR spectroscopy (materials and methods section 3). Assuming that the historic reintegration of horses was bounded temporally by the first presence of European horses on the North American mainland (1519 CE), Bayesian radiocarbon modeling suggests a date of between 1516 and 1599 CE (2s modeled range) for the initial adoption of horses by Indigenous societies in western North America, with a median boundary date of ~1544 CE (Fig. 1D and materials and methods section 2). Various models provided good measures of agreement (A model and A overall > 80 in all cases), and excluding anomalous values did not meaningfully affect date estimates (materials and methods section 2).

Historic North American horses descend primarily from Spanish genetic sources Molecular phylogeny revealed that historic and modern North American male horses carried Y-chromosomal haplotypes belonging to the "Crown group" (Fig. 2A), which became dominant within the past ~1500 years, following the increasing popularity of oriental stallions at the origin of most non-Asian domestic bloodlines today, including Arabians, Barbs, and Thoroughbreds (27). Mitochondrial phylogenetic inference also rejected maternal continuity from Late Pleistocene horses excavated both north and south of the North American ice sheets (Fig. 2B). Furthermore, BIONJ phylogenetic reconstruction based on autosomal variation at ~7.5 million nucleotide transversions supported a deep divergence between Late Pleistocene North American horses and all present and past lineages identified in Eurasia. This analysis placed both historic and modern North American horses within the genomic variation of modern domestic horses (Fig. 2C). Combined, these phylogenetic reconstructions portray historic and modern North American horses as mainly descending from domestic bloodlines that started spreading outside their native area of the Don-Volga region no earlier than 4200 years ago (28).

Admixture graph modeling did not show evidence of gene flow from Late Pleistocene into historic or modern North American horses (fig. S6.2). The individual ancestry profiles of North American horses were consistent with those found in recent domestic Eurasian bloodlines, sporadically including a minor possible contribution from Late Pleistocene North American horses or related lineages (<0.73%) (Fig. 2C). This ancestry was, however, not exclusive to historic or modern North American horses but instead shared across most Eurasian lineages, including a ~4000-year-old horse from Iberia, a ~5100-year-old horse from western Beringia, and several ancient domestic specimens such as a 1447 to 1621 CE sample from Iran (Belgheis). Therefore, the minor ancestry component detected likely reflects multiple ancient contacts between Eurasia and North America through the Beringian land bridge during the past 830,000 years, in line with previously reported studies (26) and also apparent in mitochondrial phylogenies (Fig. 2B).

To further characterize the main genetic sources of North American horses, we implemented the qpAdm modeling rotation scheme (29), considering either single or two-donor sources among 37 populations. These included Late Pleistocene North American horses as well as a representative panel of both modern and ancient domestic horses from around the globe. This analysis rejected Late Pleistocene North American horses as a possible source for both historic and modern North American horses but supported domestic bloodlines with almost exclusively European origins. Moreover, historic North American horses showed strong genetic affinities to three ancient domestic horses from Spain (Jal5885), Iran (Belgheis), and France (Inva22), dated to between the 15th and 18th centuries CE. This was true for all specimens except the one from Fort Boonesborough (Boones1/Kentucky, 1778 CE), which showed greater genetic affinity to British horses from the 18th century CE (Witter Place) (Fig. 3A). Notably, the influence of British bloodlines is also apparent among modern North American horses, which are commonly modeled as mixtures between Spanish-like (Galiceño and Cartujano) and British-like sources (Thoroughbreds and Welsh, Shetland, and Dartmoor Hill ponies) (table S3 and Fig. 3B).

This indicates a temporal shift in the genetic composition of North American horses tracing new inputs from growing British and, later, American presence in eastern North America, and the integration of these horses into Indigenous spheres of interaction. Historic North American horses show greater relatedness to the historic Iberian specimen (Jal5885) than to modern Welsh bloodlines, but modern North American horses show increased Welsh relatedness (Fig. 3, B, C, andE). This is true for all except for feral horses isolated in the Santa Cruz Islands (STCZ3322 and STCZ3327), consistent with their alleged Spanish historical origins. Finally, both historic and most modern North American groups remain closer genetically to Jal5885 than to Icelandic horses (Fig. 3D), and qpAdm modeling revealed ancestry profiles incompatible with genetic contribution from a Viking specimen dating to between the 9th and 11th centuries CE (VHR102) (supplementary materials section 6).

The genomic analyses described above focus on the minute fraction of the genome that is different across all horse lineages investigated. However, the Lakota find key instruction in the >99% of the genome fraction that appears common to all.

Pre-Pueblo Revolt contribution of horses to Indigenous beliefs, trade, and transport networks

Archaeological specimens dated to the early 17th century CE show pathological and osteological evidence of care, management, and use in transport. A horse phalanx from an Indigenous-affiliated context at Paa'ko Pueblo, New Mexico, and a metacarpal from American Falls Reservoir, Idaho, demonstrate the presence of horses among Native communities as far north as Idaho by the first half of the 17th century CE. Furthermore, a foal from Blacks Fork, Wyoming, exhibits entheseal ossification of the nuchal ligament attachment at the rear of the skull at levels typically and exclusively found in horses used for transport or kept in confinement (30,31). It also features a severe, healed cranial fracture that may have resulted from a kick caused by confinement in close were also found in the horse from Kaw River, Kansas, dating to the same period (Fig. 4) (33). Several other pathological features reflect the use of a metal curb bit of the type used by early Spanish colonists and later Mexican and Navajo craftsmen, such as the fracturing of the upper palate caused by the curb (34) and arthritis of the temporomandibular joint. Combined, these analyses indicate that early plains horses were already used for mounted riding.

Strontium isotope values for Blacks Fork, reflecting prenatal (dP4, 87 Sr/ 86 Sr = 0.70970) and postnatal (M1, 87 Sr/ 86 Sr = 0.70969) values for the foal, are directly in line with published values for modern fauna from the Green River Basin, where the specimen was found (0.70950 to 0.71000) (35,36). Although similar values characterize some stretches of the Colorado River tributary system farther to the south (37), the Blacks Fork values rule out most of Wyoming, including the border with Utah (38). Additionally, strontium isotope results from the Kaw horse, Kansas, are consistent with available values from northeastern Kansas (39) but indicate some mobility during the recorded sequence ( 87 Sr/ 86 Sr = 0.70905 near the crown, versus 87 Sr/ 86 Sr = 0.70930 near the root), which spans a 12-to 14-month period across the horse's fourth and fifth year of life, according to mineralization schedules (40). When considered alongside stable isotopes of oxygen (d 18 O) and carbon (d 13 C), strontium isotope values suggest that the Kaw horse spent part of its life farther north, indicating gradual movement from an area matching reference data from South Dakota, Nebraska, and Iowa (Fig. 4 and materials and methods section 5). Therefore, isotope values from both early horses with complete dentition suggest that the animals were either raised and managed (A) Ternary plots showing ancestry combinations of the historic horse genomes from North America. (B) Ternary plots showing ancestry combinations of the modern horse genomes from North America. Ancestry proportions were estimated using qpAdm modeling and rotating 37 possible donor sources. Models with highest P values are shown where multiple models could not be rejected. Asterisks depict two samples for which the ancestry profiles presented correspond to the second best qpAdm model (table S3), while the cross accompanying OGLAKO/Ojiwbe2 indicates that its best qpAdm model involves Galiceño and Icelandic horses as donor populations, instead of British sources (supplementary materials section 6). (C) Relative genetic affinities of historic and modern North American horses to Jal5885 versus Welsh horses. Genetic affinities are estimated using f 4 -statistics in the form of (S-URAL, X; C-PONT, Jal5885 or Welsh), where X represents historic and/or modern horses from North America, and C-PONT horses from the third millennium CE showing the closest genomic affinities to modern DOM2 domesticates. Triangles show the two historic samples that returned modern radiocarbon dates (NW14/Wyoming and SAH7/Argentina). (D) Relative genetic affinities of historic and modern North American horses to Jal5885 versus Icelandic horses. The calculations are the same as in (C), except that modern Welsh horse genomes were replaced by genomes from modern Icelandic horses and the VHR102 Viking sample (850 CE to 1050 CE). An additional qpAdm analysis considering modern Icelandic horses and the sample VHR102 as separate donors of genetic ancestry confirmed that the specimen OGLAKO/Ojibwe2 received introgression from modern Icelandic horses, ruling out genetic contribution from relict populations of Viking horses (supplementary materials section 6). Error bars correspond to twice the standard error estimated from jackknifing. (E) Distribution of Z-scores for f 4 -statistics of the form (Historic North American Horse, Modern North American Horse; Jal5885, Welsh).

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A d 13 C spike toward the end of the sampled section of the Kaw molar shows a strong input of C 4 -pathway plants, potentially reflecting winter foddering with the Indigenous domestic crop maize (materials and methods section 5), a traditional practice among Plains groups, including the Pawnee. Other situations, such as herd movement south and west to rangeland higher in C 4 grasses or during events such as seasonal bison hunts, could account for the enriched value but are not consistent with strontium and oxygen isotope data. Pawnee (Chaticks si Chaticks) villages typically relied on stores of maize to subsist through the winter (41), and the practice of winter foddering horses with maize in the northern Missouri River region is documented ethnographically during the 18th and 19th centuries CE, including among the Pawnee (42,[START_REF] Weltfish | The Lost Universe: Pawnee Life and Culture[END_REF]. Regardless of whether the Kaw specimen was affiliated with ancestral Pawnee or another Central Plains nation, our results indicate the presence of horses in Indigenous cultural and economic systems of the Missouri River drainage during the first half of the 17th century CE.

Discussion

Our archaeological analyses show the dispersal of domestic horses from Spanish settlements in the American Southwest to the northern Rockies and central Great Plains by the first half of the 17th century CE at the latest. They provide evidence of local raising and veterinary care of horses, likely foddering with domestic maize, and use of horses in transport by Indigenous peoples by this time. A directly dated radiocarbon specimen from Paa'ko Pueblo in northern New Mexico shows that horses reached the region via Indigenous groups before Spanish colonization of the American Southwest, as previously hypothesized [START_REF] Forbes | [END_REF][START_REF] Blackhawk | Violence over the Land: Indians and Empires in the Early American West[END_REF]. Moreover, our new temporal framework shows that horses were present across the plains long before any documented European presence in the Rockies or the central plains. Despite their Iberian genetic makeup and earlier arguments attributing one of these horses to Spanish exploration [START_REF] Lockwood | [END_REF], strontium, carbon, and oxygen isotope results suggest that these animals were raised and died locally. Osteological analyses also provide some indication that horse dispersal was tied to broader economic links; the Kaw River horse was indeed controlled with a European-style metal curb bit, and the Blacks Fork horse body was cut with metal tools (32). Therefore, a possible mechanism of horse dispersal was exchange across Indigenous networks at the margins of New Mexico and the American Southwest [START_REF] Cowdrey | Bridles of the Americas: Horses & Bridles of the American Indians[END_REF]. Once acquired, horses could have moved via ancient trade routes based on kinship ties and social networks established throughout the Great Plains and Rockies millennia before European contact [START_REF] Mitchell | Crafting History in the Northern Plains: A Political Economy of the Heart River Region[END_REF]. The dispersal of Puebloan groups toward the northeast, from Spanish New Mexico into western Kansas, provides another mechanism for the transmission of domestic horses into the Central Plains.

Our findings have deep ramifications for our understanding of social dynamics in the Great Plains during a period of disruptive social changes for Indigenous peoples. The area of southwestern Wyoming including Blacks Fork is considered to be a homeland for the Shoshonean-speaking ancestral Comanche (Nu -mu -nu -u -), who migrated to the southern plains of Texas, New Mexico, Colorado, and Oklahoma before the early 18th century CE [START_REF] Mitchell | [END_REF][START_REF] Betty | Comanche Society: Before the Reservation[END_REF]. The drive to acquire horses from Spanish New Mexico is often cited as a likely mechanism for this transcontinental movement [START_REF] Betty | Comanche Society: Before the Reservation[END_REF]. However, our new data-which align with some Comanche and Shoshone oral accounts (51) (materials and methods section 7)-suggest that ancestral Comanche had already integrated horse raising, ritual practices, and transport into their lifeways at least a full half century before their southward migration, effectively moving to the southern plains as horse herders. Once in the southern plains, the Comanche were able to marshal these advantages, along with their herding and equestrian skills, to build an empire on horse and bison trade by the middle of the 18th century CE [START_REF] Hämäläinen | The Comanche Empire[END_REF]. By the time Europeans arrived in southwestern Wyoming, the area was already a critical "secondary diffusion center" for horse transmission to Northern Plains groups [START_REF] Ewers | The Horse in Blackfoot Indian Culture, with Comparative Material from Other Western Tribes[END_REF][START_REF] Binnema | Common and Contested Ground: A Human and Environmental History of the Northwestern Plains[END_REF]. Considering the small body of archaeological data available, our findings raise the possibility of rapid, non-European transmission of horses farther northward, including the Columbia Plateau, the Canadian Rockies, and the middle and upper Missouri regions.

DNA data from horses currently caretaken and historically protected by the Oglala Lakota were included in this study. All protocols for the transmission of sacred and traditional knowledge were followed, as outlined by our Lakota Elder Knowledge Keeper Internal Review Board. They provide further context of the findings, which clarifies the Lakota culture and their relationship with the horse. Chief Joe American Horse states: "Horses have been part of us since long before other cultures came to our lands, and we are a part of them. The Horse Nation is our relative. We always protect our relatives and the next seven generations. We stand with the horse and we will always do so however it has evolved through its journey.

That is what being Lakota is" (original quote, in Lakota, provided in materials and methods section 7). This study established that Indigenous peoples were living and interacting with the horse before the Pueblo Revolt of 1680 CE, which was the earliest date accepted by Western science. However, current genetic evidence shows that the horses caretaken by Indigenous peoples from as early as the first half of the 17th century CE do not share an excess of genetic ancestry with Late Pleistocene North American horses. Given that the Horse Nation is foundational to Lakota lifeways ( 16), one possible implication of this finding is that relationships of the kind developed by Lakota peoples could have already been in place by the Late Pleistocene. Such life management practices may even have extended to other members of the horse family at that time. Testing these implications requires further paleontological, archaeological, genetic, and ethnographic research. Dr. Antonia Loretta Afraid of Bear-Cook adds: "The Horse Nation always chose their own mates. Bringing in new blood is a replenishment and renewal process of life that we celebrate. It strengthens our wé (the life force from our blood). No matter how our horses may have transformed, or where they are around the world, we will always call to them. Together we are home" (original quote, in Lakota, provided in materials and methods section 7). This study demonstrates that colonization did not just drastically affect Indigenous peoples but also their horses, whose genetics captures an ancestry shift from Spanish to British bloodlines. Dr. Antonia Loretta Afraid of Bear-Cook thus reminds us that the current genetic makeup of the horse does not change the Lakota responsibility toward Šungwakaŋ. In fact, for the Lakota and other Indigenous peoples with deep ancestral relationships with the horse, their evolution was not to be feared or controlled, but rather something to be respected. Protecting horses now, regardless of their origins, is in fact protecting the Lakota and other Indigenous lifeways. A commitment to protecting these is a commitment to protect all life.

8 Fig. 1 .

 81 Fig. 1. Sample spatial and chronological distribution. (A) American samples. Processed archaeological and/or museum specimens are shown as red triangles, with those successfully sequenced shown as a plain circle; modern specimens are indicated with squares. LP, Late Pleistocene [data from Vershinina et al. (26)]; ELEN, Equus lenensis (Siberia). (B) Ancient Eurasian samples in the comparative panel (table S1). Horses from western Beringia are colored in purple, the remaining in blue. Modern genomes are not projected on the map but are provided in table S1. (C) Geographic visualization of Bayesian radiocarbon dates for early horse dispersals. Produced in OxCal assuming a uniform prior, with results transferred and visualized in ArcGIS. For each specimen, point diameter corresponds to the portion of total probability distribution within each time slice. Note that presence or absence of a dot does not necessarily indicate occupation during a given time slice (see materials and methods section 2 for detailed chronological modeling of sites thought to predate 1680 CE). (D) Modeled radiocarbon boundary for introduction of Spanish-sourced domestic horses into Indigenous societies in the western United States, based on analyzed radiocarbon dates.

Fig. 2 .

 2 Fig. 2. Phylogenetic affinities. (A) Y-chromosomal maximum-likelihood tree (N = 110; 5244 nucleotide transversions). (B) Mitochondrial maximum-likelihood tree (N = 340; 16,406 base pairs). (C) Neighbor-joining tree based on ~7.5 million nucleotide transversions (N = 241; left), and the corresponding genetic ancestry profiles (right). The ancestry proportions for K = 7 genetic components were estimated using Struct-f4 (28). Sample names written as "OGLAKO/Apache" represent modern North American horses culturally associated with Apache communities. LP, Late Pleistocene; ELEN, Equus lenensis (Siberia); DOM2, modern domestic lineage descending from a lower Don-Volga genetic source expanding to Eurasia after 4200 years ago.

TaylorFig. 3 .

 3 Fig. 3. Temporal changes in the genomic makeup of American horses. (A) Ternary plots showing ancestry combinations of the historic horse genomes from North America. (B) Ternary plots showing ancestry combinations of the modern horse genomes from North America. Ancestry proportions were estimated using qpAdm modeling and rotating 37 possible donor sources. Models with highest P values are shown where multiple models could not be rejected. Asterisks depict two samples for which the ancestry profiles presented correspond to the second best qpAdm model (tableS3), while the cross accompanying OGLAKO/Ojiwbe2 indicates that its best qpAdm model involves Galiceño and Icelandic horses as donor populations, instead of British sources (supplementary materials section 6). (C) Relative genetic affinities of historic and modern North American horses to Jal5885 versus Welsh horses. Genetic affinities are estimated using f 4 -statistics in the form of (S-URAL, X; C-PONT, Jal5885 or Welsh), where X represents historic and/or modern horses from North America, and C-PONT horses

Fig. 4 .

 4 Fig. 4. Horse herding in the 17 th century CE. (A) Osteological indicators of bridling and riding: (1) palatal damage from curb bit; (2) bit damage to anterior margin of lower second premolar; (3) osteoarthritis at the temporomandibular joint; (4) entheseal changes to the nuchal ligament attachment site; and (5) remodeling of the premaxilla. (B) Osteological indicators of human activity: (1) Healed kick fracture in young foal, suggesting health care. The inset shows healing at 50× magnification. (2) Ossification of the nuchal ligament, indicating use in transport or confinement and/or tethering. (C) Modeled northward dispersal of horses. Model based on archaeological discoveries and consideration of historically documented cultural developments and migrations. (D) Isotopic evidence for foddering. Stable carbon and oxygen and strontium isotope sampling locations for the Kaw River horse, as measured in millimeters from the root (lower-right third molar). Sampling locations are represented on the x axis as the midpoint of a 2-mm section (e.g., the section from 0 to 2 mm is shown as "1" on the graph). Filled triangles show strontium isotope values, with stable carbon values represented by filled circles, and stable oxygen values as open circles. [Kaw horse image credit: E. Scott]

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ACKNOWLEDGMENTS

We are grateful to the scholars, elders, and leaders who make up the Lakota Review Team and who supervised the Oglala Institutional Review Board process for this research, as well as their counterparts from other Indigenous nations who supported this research and interpretation. Wopila Tanka to the Horse Nation and each of the Indigenous nations who served as cultural caretakers for these horses. Special thanks to M. Sims, S. Olsen, and C. Beard of the KU Natural History Museum for facilitating access to the Kaw specimen (KUVP 347; imagery provided courtesy of E. Scott); to B. Peecook and A. Commendador for facilitating access to the American Falls specimen (IMNH 71004/25895, a Bureau of Reclamation specimen under the care of the Idaho Museum of Natural History, Idaho State University); and to D. Walker and the University of Wyoming Archaeological Repository (Office of the Wyoming State Archaeologist, 1000 E. University Ave., Dept. 3431, Laramie, WY 82071, USA) for providing imagery and facilitating

access to collections at Blacks Fork (48SW8319), located in Flaming Gorge National Recreation Area, managed by the USDA-Ashley National Forest. Analysis of Blacks Fork material was completed with permission from the USDA-Ashley National Forest, coordinated through J. Rust, Heritage Program Leader. Additional thanks to B. Britt (the Maxwell Museum of Anthropology), D. Gifford-Gonzalez, and K. Kjaer for facilitating research and access to additional collections. Funding: Research was funded by an award from the National Science Foundation (Collaborative Research: Horses and Human Societies in the American West, Awards 1949305, 1949304, and 1949283) and from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement 681605). Additional funding was provided by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie (grant agreement 890702-MethylRIDE); the French Government "Investissement d'Avenir" FRANCE GENOMIQUE (ANR-10-INBS-09); the Universidad Nacional de la Patagonia Austral (PIP 29/A476); the Researchers Supporting Projects at King Saud University, Riyadh, Saudi Arabia (NSRSP2022R1), Taif University, Taif, Saudi Arabia (NSTURSP2022), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia (NSPNURSP2022), and Almaarefa University, Riyadh 13713, Saudi Arabia (NSTUMA/1); and the Marie Skłodowska Curie programme (101027750-HOPE). P.R. and M.L. thank the Max Planck Society for funding for the isotope analyses. Author contributions: Designed and coordinated the study: W.T.T.T., E.L.J., and L.O. Performed radiocarbon dating: J.S. and G.H. Performed ancient DNA laboratory work: L.Chau., L.T.-C., S.S., A.S.-O., A.F., N.K., C.D.S., X.L., S.W., and Y.R.H.C., with input from L.O. Performed ancient DNA computational analyses: P.L. and L.O. Provided material, reagents, and methods: W.T.T.T., P.R., B.L.M., N.O., J.A.L., E.B.-S., E.B., L.Char., E.R., T.D., K.G., A.O.V., J.W., P.R.Š., M.M., I.S., J.-M.A., A.Perd., S.A., A.H.A., K.A.S.A.-R., T.T.V., M.B., E.S., C.N., J.R.B., C.A.T., W.E.H., C.G., J.B.B., C.I.B.-O., C.R., M.Sp., B.S., J.S., A.O., L.P.R., K.P., M.So., A.W., W.M., G.L., S.B., C.E., C.D., O.B., P.W., G.H., S.T., B.B., E.L.J., Y.R.H.C., and L.O. Interpreted data: W.T.T.T., P.L., J.A., C.W., J.B.-C., J.R.B., C.A.T., V.M., A.Perr., W.E.H., J.L.C., P.L.R., S.G.B., J.B.B., C.R., M.Sp., G.H., S.T., B.B., P.R., E.L.J., Y.R.H.C., and L.O. Wrote the supplementary text: W.T.T.T., P.L., M.M., E.S., V.M., A.Perr., C.N., S.G.B., J.B.B., C.I.B.-O., I.A.H., S.T., B.B., E.L.J., Y.R.H.C., and L.O., with input from all coauthors. Wrote the article: W.T.T.T., B.B., E.L.J., Y.R.H.C., and L.O., with input from all coauthors. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The sequence data generated in this study are available for download at the European Nucleotide Archive (accession no. PRJEB56773). The repository information for each individual sample and all other data used in this study are included in table S1. License information: Copyright © 2023 the authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original US government works. https://www.science.org/about/sciencelicenses-journal-article-reuse