Florence Netzer

Caroline Sévoz-Couche
email: caroline.sevoz-couche@upmc.fr

Rostral cuneiform nucleus and the defence reaction: Direct and indirect midbrain-medullary 5-HT mechanisms in baroreflex inhibition

Keywords: BRS, baroreflex sensitivity, HR, heart rate, LPGi, lateral paragigantocellular nucleus, MBP, mean BP, NTS, nucleus tractus solitarius, PAG, periaqueductal grey matter

Background and Purpose: Activation of the defence reaction inhibits the baroreflex response via the intermediate rostro-ventromedial medulla (B3 raphé) and nucleus tractus solitarius (NTS). Our aim was to determine whether and how baroreflex inhibition, induced by the disinhibition of the rostral cuneiform nucleus (part of the defence pathway), involves 5-HT neurons in B3 and 5-HT 3 receptors in the NTS.

Experimental Approach: We performed immunohistochemistry and anatomical experiments to determine whether raphé 5-HT cells expressing Fos were directly targeted by the rostral cuneiform nucleus. The effect of blocking raphé 5-HT neurotransmission and NTS 5-HT 3 receptors on cuneiform-induced inhibition of the baroreflex cardiac response were also analysed.

Key Results: Bicuculline, microinjected into the rostral cuneiform nucleus, induced an increase of double-labelled Fos-5-HT-IR cells in both the lateral paragigantocellular nucleus (LPGi) and raphé magnus. The anterograde tracer Phaseolus vulgaris leucoaggutinin injected into the rostral cuneiform nucleus revealed a dense projection to the LPGi but not raphé magnus. Cuneiform-induced baroreflex inhibition was prevented by B3 injection of 8-OH-DPAT, a selective 5-HT 1A receptor agonist. Cuneiform disinhibition also failed to inhibit the baroreflex bradycardia after NTS microinjection of the 5-HT 3 receptor antagonist granisetron and in 5-HT 3 receptor knockout mice.

Conclusion and Implications:

The rostral cuneiform nucleus participates in the defence inhibition of the baroreflex bradycardia via direct activation of the LPGi and via a projection to the raphé magnus to activate NTS 5-HT 3 receptors and inhibit second-order baroreflex neurons. These data bring new insights in primary and secondary mechanisms involved in vital baroreflex prevention during stress.

| INTRODUCTION

Arterial baroreflex represents a powerful negative feedback control mechanism to maintain BP homeostasis. Activation of the major arterial baroreceptors (aortic and carotid origin) following an increase in BP causes immediate parasympathetic cardiovagal excitation. The rapid (<1 s) decrease in heart rate (HR) will secondarily, through a decrease in cardiac output, cause a decrease in BP after a few seconds (baroreflex loop) [START_REF] Vaschillo | Characteristics of resonance in heart rate variability stimulated by biofeedback[END_REF]. Under specific circumstances, such as during the defence reaction, blood flow is redistributed between organs, increasing blood supply to the skeletal muscles, heart and brain. This redistribution is the consequence of vessel constriction, increase in venomotor tone [START_REF] Martin | Acute stress increases venomotor tone in conscious rats[END_REF] and increase in cardiac output, mainly due to an increase in HR [START_REF] Crestani | Emotional stress and cardiovascular complications in animal models: A review of the influence of stress type[END_REF].

Stroke volume was not affected [START_REF] Schulte | Hemodynamics during emotional stress in borderline and mild hypertension[END_REF][START_REF] Wilson | Effect of thermal stress on cardiac function[END_REF] or only slightly increased [START_REF] Anderson | Pulmonary vasoconstriction elicited by stimulation of the hypothalamic integrative area for the defense reaction[END_REF][START_REF] Lindvall | Stress-induced changes in blood pressure and left ventricular function in mild hypertension[END_REF] during stress. Altogether, these factors produce an increase in BP [START_REF] Nosaka | Modifications of arterial baroreflexes: Obligatory roles in cardiovascular regulation in stress and poststress recovery[END_REF]. This would trigger a baroreflex bradycardia. However, this reflex response would counteract the vital increase in cardiac output necessary for muscles oxygenation and the body's reactions during stress. Several studies found that the cardiovagal effect on the heart (bradycardia) is suppressed during the defence reaction [START_REF] Comet | 5-HT-mediated inhibition of cardiac baroreceptor reflex response during defense reaction in the rat[END_REF][START_REF] Comet | Functional interaction between nucleus tractus solitarius NK1 and 5-HT 3 receptors in the inhibition of baroreflex in rats[END_REF][START_REF] Nosaka | Modifications of arterial baroreflexes: Obligatory roles in cardiovascular regulation in stress and poststress recovery[END_REF][START_REF] Sévoz-Couche | Role of nucleus tractus solitarius 5-HT 3 receptors in the defense reactioninduced inhibition of the aortic baroreflex in rats[END_REF].

The nucleus tractus solitarius (NTS) plays a major role in the reduction in the baroreflex cardiovagal response during the defence reaction. Specific blockade of local 5-HT 3 receptors, the only ligand-gated ion channel among 5-HT receptors [START_REF] Hoyer | Serotonin 5-HT 3 , 5-HT 4 , and 5-HT-M receptors[END_REF], prevents the reflex cardiac inhibition induced by activation of the dorsolateral periaqueductal grey matter (PAG, Comet et al., 2004, 2005), a key region involved in the defence reaction [START_REF] Bandler | Integrated defence reaction elicited by excitatory amino acid microinjection in the midbrain periaqueductal grey region of the unrestrained cat[END_REF]. The possible source of 5-HT released into the NTS to activate these 5-HT 3 receptors, following dorsolateral PAG activation, is the intermediate rostro-ventromedial medulla region (intermediate B3 region), including the lateral paragigantocellular nucleus (LPGi) and the raphé magnus (RMg), as 5-HT neurons in the LPGi and the RMg (but not other raphé nuclei) express Fos protein after dorsolateral PAG activation [START_REF] Bernard | Critical role of B3 serotonergic cells in baroreflex inhibition during the defense reaction triggere by dorsal periaqueductal gray stimulation[END_REF].

The rostral cuneiform nucleus may also be part of the defence reaction as suggested by observations made by [START_REF] Netzer | Brain circuits mediating baroreflex bradycardia inhibition in rats: An anatomical and functional link between the cuneiform nucleus and the periaqueductal grey[END_REF];

(i) the baroreflex inhibition induced by stimulation of the dorsomedial nucleus of the hypothalamus, a key region of the defence reaction [START_REF] Dimicco | Role of the dorsomedial hypothalamus in the cardiovascular response to stress[END_REF], was prevented by the chemical inhibition of the rostral cuneiform following local administration of muscimol and (ii) disinhibition of the latter nucleus through blockade of GABAergic inputs (bicuculline), which caused an inhibitory effect on baroreflex response which could be prevented by dorsolateral PAG blockade.

These observations suggested that, as for the dorsolateral PAG, B3 activation could play a key role in the baroreflex inhibition obtained during cuneiform disinhibition. To answer this question, we performed double immunolabelling in the B3 region (LPGi and RMg), as well as in other raphé nuclei (raphé dorsalis [RDr], raphé pallidus

[RPa] and raphé obscurus [ROb]). We analysed the expression of Fos protein in 5-HT immunoreactive (Fos-5-HT-IR) cells in these nuclei after injection of the GABA A antagonist bicuculline into the rostral cuneiform nucleus. Despite the fact that activation of 5-HT neurons was detected in the entire B3 intermediate region following the dorsolateral PAG stimulation [START_REF] Bernard | Critical role of B3 serotonergic cells in baroreflex inhibition during the defense reaction triggere by dorsal periaqueductal gray stimulation[END_REF], there is a direct link from the latter to the LPGi but not the RMg [START_REF] Babic | Medullary and spinal cord projections from cardiovascular responsive sites in the rostral ventromedial medulla[END_REF]. This raised the possibility that activation of passing fibres, like those from the cuneiform nucleus [START_REF] Netzer | Brain circuits mediating baroreflex bradycardia inhibition in rats: An anatomical and functional link between the cuneiform nucleus and the periaqueductal grey[END_REF], may have been at the origin of dorsolateral PAG-induced 5-HT activation in the RMg.

To answer this question, we injected an anterograde tracer (Phaseolus vulgaris leucoaggutinin) into the rostral cuneiform nucleus to determine whether descending projections towards the intermediate B3 region (including the RMg) could be found. To definitely establish whether raphé 5-HT neurons are activated by rostral cuneiform disinhibition and that NTS 5-HT 3 receptors are involved in defenceinduced baroreflex inhibition, we performed the following pharmacological experiments. We analysed the baroreflex response reduction obtained normally and during rostral cuneiform disinhibition, before and after (i) local raphé inactivation caused by microinjections of the GABA A receptor agonist muscimol, (ii) raphé 5-HT neuron inhibition using microinjections of 5-HT 1A /5-HT 7 receptor agonist 8-OH-DPAT and (iii), blockade of NTS 5-HT 3 receptors by microinjections of granisetron in rats or by comparing effect of rostral cuneiform disinhibition in wild-type (WT) with those in 5-HT 3 receptor knockout mice.

What is already known

• The baroreflex bradycardia is inhibited during the defence reaction to acute fear.

• This inhibition is a key step for maximal oxygenation of skeletal muscles.

What this study adds

• The rostral cuneiform disinhibition induces activation of 5-HT neurons in the intermediate B3 region only.

• The release of 5-HT targets 5-HT 3 receptors in the NTS.

What is the clinical significance

• Multiple protective medullary pathways are involved in the vital stress-induced inhibition of the baroreflex response.

• 5-HT 3 receptors may be a target for limitation of cardiac events due to stress.

| METHODS

| Animals

All animal care and experimental procedures followed institutional guidelines, which comply with national and international law policies (Council Directive 87-848, October 19, 1987 et al., 2020) and with the recommendations made by the British Journal of Pharmacology [START_REF] Lilley | ARRIVE 2.0 and the British Journal of Pharmacology: Updated guidance for 2020[END_REF].

Experiments were performed using 52 male Sprague-Dawley rats (RRID:MGI_5651135) (330-350 g) and 12 male mice (25 g) homozygous Htr3a KO (n = 6) and WT littermates (n = 6) born from heterozygous mutants on a C57BL/6J (RRID:MGI_5811150) genetic background (>10 generations) and genotyped as described by [START_REF] Zagon | The 5-HT 3 subtype of serotonin receptor contributes to nociceptive processing via a novel subset of myelinated and unmyelinated nociceptors[END_REF]. Animals were kept under controlled environmental conditions (ambient temperature: 21 ± 1 C, 60% relative humidity, unrestricted access to food and water, and alternate 12-h light/12-h dark cycles) for a least 1 week after delivery from the breeding centre (CER Janvier, Le Genest-Saint-Isle, France). Studies were designed to generate groups of equal size, using randomization and blinded analysis, accordingly to recommendations of the British Journal of Pharmacology on experimental design and analysis [START_REF] Curtis | Experimental design and analysis and their reporting II: Updated and simplified guidance for authors and peer reviewers[END_REF].

| Sustained chemical disinhibition of the rostral cuneiform nucleus to induce Fos expression in raphé nuclei

| Rostral cuneiform nucleus long-term disinhibition

Twelve rats were anaesthetized with chloral hydrate (400 mgÁkg -1 , i.p.) for immunohistological experiments. The size of the samples was based on previous experiments [START_REF] Bernard | Critical role of B3 serotonergic cells in baroreflex inhibition during the defense reaction triggere by dorsal periaqueductal gray stimulation[END_REF]. The depth of anaesthesia was assessed regularly by pinching a hind paw and monitoring the stability of the arterial BP and HR recordings, and further injections (i.v.) of chloral hydrate were given as necessary.

The animals were allowed to breathe spontaneously. Rectal temperature was maintained at 37 C with a thermostatically controlled heating blanket. The femoral vein was catheterized for the administration of additional anaesthesia or drugs. Mean BP (MBP) was monitored via a femoral arterial catheter connected to a pressure transducer and DC amplifier (Gould, Courtaboeuf, France). The ECG was recorded using stainless steel pins placed subcutaneously into forepaws and hind paws. Signals were amplified and filtered (Universal Amplifier, Gould). HR was computed from the ECG (R-wave pulses) and displayed as mean frequency per minute (bin size = 1 s). Lidocaine (xylocaine®; 5 mgÁkg -1 ) was injected s.c., at the posterior aspect of the neck at the beginning of all surgery to provide local anaesthesia.

Briefly, anesthetized rats (n = 6) were placed in a stereotaxic frame with the head fixed in the flat skull positionn, two unilateral (left) microinjections (with 10-min interval) at rostral cuneiform sites (-8.2 to -8.5 mm from bregma, 2 mm lateral and 5 mm ventral to the surface of the brain; [START_REF] Netzer | Brain circuits mediating baroreflex bradycardia inhibition in rats: An anatomical and functional link between the cuneiform nucleus and the periaqueductal grey[END_REF] filled with bicuculline methiodide (50 pmol per 50 nl) were necessary to maintain cuneiform activation for 20 min, as confirmed by a sustained and stable increase in MBP and HR. These variables were high but stable after bicuculline administration and started to come back to baseline after 20 min [START_REF] Netzer | Brain circuits mediating baroreflex bradycardia inhibition in rats: An anatomical and functional link between the cuneiform nucleus and the periaqueductal grey[END_REF]. Activation of the defence reaction was identified by the observation of somatic responses such as mydriasis, vibrissae and body movements, and tail erection. Sham animals (n = 6) received vehicle treatment (saline). No somatic, MBP and HR responses were observed.

| Visualization of Fos and/or 5-HT immunoreactive (Fos-IR and/or 5-HT-IR) neurons

Two hours after the end of the second microinjection of bicuculline or saline into the rostral cuneiform, anaesthetized rats were perfused intracardially. The brain was removed and cryoprotected as described above. Coronal frozen sections (50 μm thick) of the whole brainstem were collected in four containers filled with PBS, allowing their parallel processing as serial groups of floating sections.

Processing is described in Data S1. Neurons were observed under bright-field illumination in 50-μm-thick coronal sections (200 μm apart) in regions containing 5-HT neurons (the LPGi, the RMg, the RPa, the ROb and the RDr nuclei). The location of brainstem nuclei containing 5-HT neurons was based on observation of adjacent Nissl-stained sections.

Brain tissue sections were analysed after careful individual examination of each neuron at high magnification (×40). The outline of the section and the main structures were drawn at low magnification (×4 and ×10), the computer providing continuous synchronization between plotting and section location during moving or magnification changes. For each animal, a total of four brain sections for each region of interest were analysed under bright-field illumination. Neurons were plotted using Mercator software (RRID:SCR_014493) (Explora Nova, La Rochelle, France). The computer was connected to (i) a CCD colour video camera that captured images and supplied red, green and blue output and (ii) an XY stage microscope stage controller that sent the micrometric location of the section boundaries to the computer.

| Anterograde tracing of rostral cuneiform nucleus efferents to the B3 region

| Tracer injection

Ten rats were anaesthetized with chloral hydrate (400 mgÁkg -1 , i.p.) for anatomical experiments. The size of the sample was based on previous experiments [START_REF] Netzer | Brain circuits mediating baroreflex bradycardia inhibition in rats: An anatomical and functional link between the cuneiform nucleus and the periaqueductal grey[END_REF]. The depth of anaesthesia was assessed regularly by pinching a hind paw and monitoring the stability of the arterial BP and HR recordings and further injections (i.v.) of chloral hydrate were given as necessary. Rectal temperature was maintained at 37 C with a thermostatically controlled heating blanket.

The animals were allowed to breathe spontaneously. To provide local anaesthesia, lidocaine (xylocaine®; 5 mgÁkg -1 ) was injected s.c. at the posterior aspect of the neck, at the beginning of the surgery, and meloxicam (metacam®; 1 mgÁkg -1 ) at the end of surgery.

Anaesthetized animals were placed in a stereotaxic frame with the head fixed in the flat skull position. Application of Phaseolus vulgaris leucoagglutinin lectin (PHA-L) was made by passing direct current (2-6 μA, electrode tip positive) through a micropipette directed to the left rostral cuneiform nucleus (see above) for 20 s per 30-s period, for 20 min [START_REF] Netzer | Brain circuits mediating baroreflex bradycardia inhibition in rats: An anatomical and functional link between the cuneiform nucleus and the periaqueductal grey[END_REF]. Following a post-operative period of 15 days, animals were deeply anaesthetized with pentobarbitone (200 mgÁkg -1 , i.p.). They were then perfused transcardially and the brain was removed, and cryoprotected in a 20% sucrose solution overnight. Frozen sections (50 μm thick) of the whole brainstem were collected in four containers filled with PBS, allowing their parallel processing as three serial groups of free-floating sections.

| Immunohistochemical processing for anterograde labelling

Processing is described in detail in Data S1.

Labelled fibres in the regions identified in the first set of experiments (identification of double-labelled Fos-5-HT-IR cells) were observed under bright-field illumination in coronal sections. Terminals were differentiated from fibres of passage using classical morphological criteria (ramification, varicosities, thickness and pattern). All immuno-related procedures involved comply with the editorial on immunoblotting and immunohistochemistry [START_REF] Alexander | Goals and practicalities of immunoblotting and immunohistochemistry: A guide for submission to the British Journal of Pharmacology[END_REF].

| Pharmacological experiments

| Acute rostral cuneiform disinhibition in rats and mice

Animals were anaesthetized with urethane (1.5 gÁkg -1 , i.p.) to ensure similar experimental conditions as those performed previously [START_REF] Bernard | Critical role of B3 serotonergic cells in baroreflex inhibition during the defense reaction triggere by dorsal periaqueductal gray stimulation[END_REF][START_REF] Gau | Inhibition of cardiac baroreflex by noxious thermal stimuli: A key role for lateral paragigantocellular serotonergic cells[END_REF][START_REF] Netzer | Brain circuits mediating baroreflex bradycardia inhibition in rats: An anatomical and functional link between the cuneiform nucleus and the periaqueductal grey[END_REF]. The femoral vein was catheterized, and MBP and HR were recorded as mentioned above. Anaesthetized animals were placed in a stereotaxic frame with the head fixed in the flat skull position. The size of the samples was based on previous experiments [START_REF] Bernard | Critical role of B3 serotonergic cells in baroreflex inhibition during the defense reaction triggere by dorsal periaqueductal gray stimulation[END_REF][START_REF] Netzer | Brain circuits mediating baroreflex bradycardia inhibition in rats: An anatomical and functional link between the cuneiform nucleus and the periaqueductal grey[END_REF][START_REF] Zafar | A hypothalamo-midbrain-medullary pathway involved in the inhibition of the respiratory chemoreflex response induced by potassium cyanide in rodents[END_REF]. Briefly, rostral cuneiform disinhibition was caused by left microinjection of bicuculline in rats

| Evaluation of the baroreflex sensitivity in control and experimental conditions in rats and mice

In the control condition, baroreceptors were unloaded using the vasodilator sodium nitroprusside (SNP, 100 μgÁkg -1 , i.v.) followed by baroreceptor stimulation using the vasoconstrictor phenylephrine (PE, 10 μgÁkg -1 , i.v.) [START_REF] Sévoz-Couche | Involvement of the dorsomedial hypothalamus and the nucleus tractus solitarii in chronic cardiovascular changes associated with anxiety in rats[END_REF]. Administration of nitroprusside followed by phenylephrine allowed the generation of baroreceptor function curves (changes in HR as a function of BP changes) by fitting the data to the sigmoid logistic function:

Y = Bottom + (Top -Bottom)∕(1 + exp((V50 -X)∕Slope), using Prism (RRID:SCR_002798) (GraphPad Software Inc., La Jolla, CA, USA),
where Top -Bottom was the maximal baroreflex range. Rectilinear baroreflex slopes were calculated from the baroreceptor curves and considered as reflect of the baroreflex sensitivity (BRS, expressed as bpmÁmmHg -1 ).

In the same animal (rat or mice), after 15 min, left rostral cuneiform disinhibition was performed. When BP increase was stable (2-4 min after bicuculline), SNP + PE was administered again (experimental condition).

To eliminate the influence of defence reaction-induced tachycardia on the cardiac baroreflex responses, all animals used in the following experiments were pretreated with the β-adrenoceptor antagonist atenolol (1 mgÁkg -1 , i.v., 20 min before the first baroreflex induction).

| Microinjections into the B3 region and NTS in rats

A micropipette connected to a Hamilton microsyringe was lowered into the target areas for the first 100-nl injection and refilled with the same volume for the second injection; the time interval between the two microinjections was less than 1 min.

Bilateral microinjections of bodipy TMR-X conjugate muscimol (500 pmol per 100 nl), a selective GABA A receptor agonist [START_REF] Johnston | Central actions of ibotenic acid and muscimol[END_REF], were made in B3 region identified in immunohistological experiments (i.e. between -11.0 and 11.5 mm from bregma, 0 to 1 mm lateral and 9.0 to 9.5 ventral to the surface of the cerebellum).

The injection sites were precisely located owing to the orange light emission (572 nm) of the fluorophore linked to muscimol. 5-HT cells were visualized in coronal sections by the green fluorescent emission (519 nm) of Alexa Fluor goat 488 anti-rabbit (RRID:AB_143165) used after incubation of sections with rabbit anti-5-HT antibody (RRID: AB_1572725) [START_REF] Gau | Inhibition of cardiac baroreflex by noxious thermal stimuli: A key role for lateral paragigantocellular serotonergic cells[END_REF]. Bilateral microinjections of 8-OH-DPAT HBr (1 nmol per 100 nl), a 5-HT 1A receptor agonist [START_REF] Hamon | Alterations of central serotonin and dopamine turnover in rats treated with ipsapirone and other 5-hydroxytryptamine1A agonists with potential anxiolytic properties[END_REF] that binds preferentially to autoreceptors on 5-HT neurons [START_REF] H ; Middlemiss | 8-Hydroxy-2-(di-n-propylamino)tetralin discriminates between subtypes of the 5-HT 1 recognition site[END_REF], were made into the intermediate B3 region [START_REF] Ootsuka | Activation of 5-HT 1A receptors in rostral medullary raphé inhibits cutaneous vasoconstriction elicited by cold exposure in rabbits[END_REF].

Bilateral microinjections of granisetron (250 pmol per 100 nl), a selective 5-HT 3 receptor antagonist [START_REF] Carmichael | A pharmacokinetic study of granisetron (BRL 43694A), a selective 5-HT 3 receptor antagonist: Correlation with anti-emetic response[END_REF]

| Histological localization of microinjection and stimulation sites

Microinjection sites were identified by the location of the micropipette track and pontamine sky blue deposit in 70-μm-thick sections of brain tissue previously fixed in 10% formalin solution and cryoprotected in 20% sucrose solution for 5 days.

| Data and statistical analyses

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology [START_REF] Curtis | Experimental design and analysis and their reporting II: Updated and simplified guidance for authors and peer reviewers[END_REF]. Sample sizes subjected to statistical analysis was at least 6 animals per group (n=6), where n is the number of independent values. Parametric analyses were used as all data sets (raw or log-transformed) successfully passed the Shapiro-Wilk test for normality and Leven's test for equality of variance [START_REF] Niérat | Experimental dyspnea as a stressor: differential cardiovegetative responses to inspiratory threshold loading in healthy men and women[END_REF]). Student's unpaired t-test (GraphPad Prism 6) was used to compare Fos expression in 5-HT cells in control (saline) or experimental (rostral cuneiform disinhibtion) rats. Two-way repeated measures ANOVA (GraphPad Prism 6, San Diego, CA, USA) was used to compare the baroreflex range responses and BRS in control and experimental conditions, before and after local microinjections in B3 and NTS in rats or WT and 5-HT 3 receptor knockout mice. Bonferroni corrections were applied to all ANOVAs only if F in ANOVA achieved P < .05, and differences were considered significant at P < .05.

| Materials

| Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOL-OGY http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 [START_REF] Alexander | The Concise Guide to PHARMACOLOGY 2019/20: G-protein coupled receptors[END_REF]Alexander, Mathie, et al., 2019).

| RESULTS

3

B3 nuclei

An increase in Fos-5-HT-IR cell number in the experimental condition compared to sham was confined to the mid-rostrocaudal extent of the B3 5-HT group, from -10.2 to -11.8 mm from bregma [START_REF] Paxinos | The rat brain in stereotaxic coordinates[END_REF]. Considering absolute values, that is, mean number of neurons per rat, the total number of 5-HT-IR cells was similar in sham and experimental animals in ipsilateral (130 ± 10 vs. 120 ± 8) or contralateral (125 ± 7 vs. 122 ± 6) LPGi, as well as in the RMg (280 ± 5 vs. 250 ± 10). Among total LPGi 5-HT-IR cells, unilateral rostral cuneiform disinhibition (experimental conditions) induced an increase in Fos expression by more than 20-fold in each side compared to sham (Figure 1a). A representative example in ipsilateral LPGi is given in Figure 1b. The number of double-labelled cells in RMg was also significantly higher in experimental than in sham conditions, though the increase (10-fold) was slightly less than in total LPGi (Figure 1a).

Non-5-HT-IR neurons expressing Fos slightly increased in experimental compared to sham conditions in total LPGi, as well as in RMg

Raphé dorsalis (RDr)

The RDr extends from -7.0 to -8.5 mm from bregma. In this region, the total number of 5-HT-IR cells was similar in sham and experimental animals (2,550 ± 110 vs. 2,420 ± 130, Figure 2a). We only observed a slight tendency to increase of 5-HT-IR cells expressing Fos in experimental compared to sham conditions (Figure 1a). In contrast, bicuculline given into the rostral cuneiform nucleus (experimental) induced a large increase in Fos-IR cells in the non-5-HT-IR population compared to sham (Figure 2a), especially in lateral wings at mid-rostrocaudal levels (-7.5 to 8.0 mm from bregma). A representative example in the experimental condition is given in Figure 2b.

RPa and ROb

Analyses were performed between -10.0 and -13.0 mm from bregma. The total number of 5-HT-IR cells was similar in sham and experimental animals in RPa (27 ± 2 vs. 22 ± 3) or ROb (153 ± 8 vs. 138 ± 6). 5-HT-IR cells expressing Fos in these two regions were not affected by rostral cuneiform disinhibition compared to sham (Figure 1a). No increase in Fos expression was seen in the non-5-HT-IR population in the RPa or ROb after bicuculline into the rostral cuneiformis nucleus compared to sham (Figure 2a).

| Anatomical link between the rostral cuneiform nucleus and B3

The anterograde tracer was unilaterally applied in the rostral cuneiform nucleus (Figure 3a). Labelled fine fibres and ramifications with numerous varicosities were highly concentrated in the middle B3 (Figure 3b). The maximum density of labelled fibres was found in the intermediate B3 region, between -10.4 to -11.6 mm from bregma [START_REF] Paxinos | The rat brain in stereotaxic coordinates[END_REF]. The majority of the labelled fibres were found bilaterally in the LPGi and only a few labelled fibres were observed in the RMg (Figure 3c).

| Inhibitory effects of rostral cuneiform: disinhibition on the baroreflex cardiac response in rats and mice

| Involvement of the intermediate 5-HT B3 region: effects of muscimol and 8-OH-DPAT on baroreflex cardiac response induced by rostral cuneiform disinhibition in rats

The results in Figure 1b show that the microinjections of muscimol and 8-OH-DPAT were performed at intermediate B3 coordinates (Figure 4a). The baroreflex response was carried out in the same rat alone (control condition) or during (experimental condition) rostral cuneiform disinhibition, before and after intra-B3 treatment. Interaction between conditions and intra-B3 muscimol or 8-OH-DPAT but not saline was seen over the maximal cardiac range (Table 1) and rectilinear slopes (BRS) derived from baroreflex sigmoid curves (Table 2). 5a-2) was significantly reduced in experimental conditions before (75 ± 1 bpm) but not after (145 ± 3 bpm) intra-B3 treatment, compared with control conditions (165 ± 3 and 155 ± 6 bpm, respectively) (Figure S1). In the same manner, experimental BRS derived from the sigmoid curves was lower before (2.1 ± 0.1 bpmÁmmHg -1 ) but not after (5.1 ± 0.2 bpmÁmmHg -1 ) intra-B3 muscimol than control BRS (6.4 ± 0.1 and 6.2 ± 0.1 bpmÁmmHg -1 , respectively) (Figure S1).

The maximal baroreflex cardiac range obtained from sigmoid curves (Figure 5b) was significantly reduced in experimental conditions before (59 ± 4 bpm) but not after (130 ± 7 bpm) intra-B3 8-OH-DPAT compared with control conditions (160 ± 3 and 147 ± 4 bpm, respectively) (Figure S1). In the same manner, experimental BRS was lower before (1.10 ± 0.05 bpmÁmmHg -1 ) but not after

(5.53 ± 0.09 bpmÁmmHg -1 ) intra-B3 8-OH-DPAT than control BRS

(5.91 ± 0.07 and 6.21 ± 0.19 bpmÁmmHg -1 , respectively) (Figure S1).

Experimental cardiac baroreflex ranges from the baroreflex sigmoid curves before and after intra-B3 saline (52 ± 4 and 54 ± 4 bpm, respectively) were both lower than in controls (156 ± 3 and 163 ± 3 bpm, respectively). Similarly, experimental BRS was lower from control BRS before (1.3 ± 0.1 vs. 5.6 ± 0.1 bpmÁmmHg -1 ) and after (1.4 ± 0.1 vs. 5.7 ± 0.1 bpmÁmmHg -1 ) intra-B3 saline (Figure S2).

| Involvement of 5-HT 3 receptors

Effects of granisetron on baroreflex cardiac response inhibition induced by rostral cuneiform disinhibition in rats

The baroreflex response was performed in the same rat before (control condition) and during (experimental condition) intracuneiform bicuculline, before and after intra-NTS injection. Interaction between conditions and intra-NTS granisetron but not saline was seen for maximal baroreflex range (Table 1) and BRS (Table 2). The maximal cardiac range obtained from the baroreflex sigmoid curves (Figure 5c) in experimental conditions was significantly reduced before (38 ± 3 bpm) but not after (130 ± 5 bpm) intra-NTS granisetron, compared with control conditions (160 ± 5 and 141 ± 4 bpm, respectively) (Figure S1). In the same manner, the experimental BRS derived from the sigmoid curves was lower before

(1.4 ± 0.2 bpmÁmmHg -1 ) but not after (5.4 ± 0.1 bpmÁmmHg -1 ) granisetron than control BRS (5.9 ± 0.1 and 6.0 ± 0.1 bpmÁmmHg -1 , respectively) (Figure S1).

Experimental cardiac baroreflex ranges from the baroreflex sigmoid curves before and after intra-NTS saline (56 ± 3 and 53 ± 3 bpm, respectively) were both lower than in control conditions (163 ± 3 and 157 ± 4 bpm, respectively). Similarly, experimental BRS was lower than control before (1.4 ± 0.1 vs. 5.7 ± 0.1 bpmÁmmHg -1 ) and after (1.4 ± 0.1 vs. 5.8 ± 0.1 bpmÁmmHg -1 ) intra-NTS saline (Figure S2).

Involvement of 5-HT 3 receptors in mice: effects of rostral cuneiform disinhibition on baroreflex cardiac response in WT and 5-HT 3 receptor knockout mice

Injections of bicuculline into the rostral cuneiform were performed in WT or 5-HT 3 receptor knockout mice (Figure 6a). An example of injection at 4.6 mm from bregma is given in Figure 6b. The baroreflex response was performed in the same animal before (control condition) and during (experimental condition), either in WT or in 5-HT 3 receptor knockout mice. A representative example is given in Figure 7a.

Interaction between condition and mice genotype was seen for maximal baroreflex range (Table 1) and BRS (Table 2). The maximal cardiac range obtained from the baroreflex sigmoid curves in experimental conditions was significantly reduced in WT (125 ± 5 bpm) but not in knockout (243 ± 7 bpm) mice (Figure 7b), compared with control conditions (231 ± 7 and 243 ± 5 bpm) (Figure S1). In the same manner, experimental BRS derived from the sigmoid curves was lower in WT (3.3 ± 0.3 bpmÁmmHg -1 ) but not in knockout

| Fos expression in raphé nuclei

The defence reaction is associated with an inhibition of the baroreflex response [START_REF] Nosaka | Modifications of arterial baroreflexes: Obligatory roles in cardiovascular regulation in stress and poststress recovery[END_REF]. We showed in different studies that this effect involved the activation of NTS 5-HT 3 receptors [START_REF] Bernard | Critical role of B3 serotonergic cells in baroreflex inhibition during the defense reaction triggere by dorsal periaqueductal gray stimulation[END_REF][START_REF] Comet | 5-HT-mediated inhibition of cardiac baroreceptor reflex response during defense reaction in the rat[END_REF][START_REF] Comet | Functional interaction between nucleus tractus solitarius NK1 and 5-HT 3 receptors in the inhibition of baroreflex in rats[END_REF][START_REF] Sévoz-Couche | Role of nucleus tractus solitarius 5-HT 3 receptors in the defense reactioninduced inhibition of the aortic baroreflex in rats[END_REF].

The NTS receives 5-HT projections from various raphé nuclei, including the RPa and the ROb [START_REF] Palkovits | Innervation of the nucleus of the solitary tract and the dorsal vagal nucleus by thyrotropin-releasing hormone-containing raphe neurons[END_REF], as well as the RDr [START_REF] Schaffar | Central serotonergic projections to the nucleus tractus solitarii: Evidence from a double labeling study in the rat[END_REF] and, in B3, the RMg [START_REF] Schaffar | Central serotonergic projections to the nucleus tractus solitarii: Evidence from a double labeling study in the rat[END_REF][START_REF] Thor | Serotonin-and substance P-containing projections to the nucleus tractus solitarii of the rat[END_REF] and the LPGi [START_REF] Babic | Medullary and spinal cord projections from cardiovascular responsive sites in the rostral ventromedial medulla[END_REF][START_REF] Schaffar | Central serotonergic projections to the nucleus tractus solitarii: Evidence from a double labeling study in the rat[END_REF]. Therefore, these raphé nuclei are potential candidates to be involved in NTS 5-HT 3 receptor activation during the defence reaction. We showed that activation of the dorsolateral PAG, a key [START_REF] Bernard | Critical role of B3 serotonergic cells in baroreflex inhibition during the defense reaction triggere by dorsal periaqueductal gray stimulation[END_REF]. Upstream to the dorsolateral PAG, disinhibition of local cells at specific coordinates (between -8.0 and -8.4 mm from bregma) in the rostral cuneiform nucleus following bicuculline microinjections induced tachypnoea, tachycardia and hypertension associated with the inhibition of the baroreflex response [START_REF] Netzer | Brain circuits mediating baroreflex bradycardia inhibition in rats: An anatomical and functional link between the cuneiform nucleus and the periaqueductal grey[END_REF]. We first examined whether B3 but also other raphé nuclei were activated during left rostral cuneiform disinhibition. As found for dorsolateral PAG activation [START_REF] Bernard | Critical role of B3 serotonergic cells in baroreflex inhibition during the defense reaction triggere by dorsal periaqueductal gray stimulation[END_REF] only raphé nuclei in the intermediate B3 exhibited bilateral double-labelled Fos-5-HT cells following left rostral cuneiform disinhibition, with a higher proportion found in the LPGi than in the RMg. It is unlikely that this result in B3 was a consequence of indirect feedback evoked from the resulting increases in BP and HR. Indeed, no increase of Fos expression was described in the B3 region in response to BP changes [START_REF] Dampney | Functional organisation of central cardiovascular pathways: Studies using c-fos gene expression[END_REF]. No changes were observed in other parts of the 5-HT system, including the RDr. These results differ from those found with social stress. Indeed, a single exposure to social defeat has been found to selectively activate 5-HT neurons in the mid-rostrocaudal and caudal dorsal raphé nucleus [START_REF] Gardner | Early life experience alters behavior during social defeat: Focus on serotonergic systems[END_REF][START_REF] Lkhagvasuren | Distribution of Fos-immunoreactive cells in rat forebrain and midbrain following social defeat stress and diazepam treatment[END_REF]. Similarly, chronic emotional stress (maternal separation or chronic mild stress, [START_REF] Pollano | How does early maternal separation and chronic stress in adult rats affect the immunoreactivity of serotonergic neurons within the dorsal raphe nucleus?[END_REF] increases the number of Fos-5-HT immunoreactive cells in the dorsolateral and ventrolateral part of the RDr. Therefore, at least concerning activation of 5-HT neurons in the RDr, the mechanisms involved in defence reaction (here, related to panic attack; [START_REF] Johnson | Neural pathways underlying lactate-induced panic[END_REF] and social emotional stress (closer from anxiety and/or depression; [START_REF] Blugeot | Vulnerability to depression: From brain neuroplasticity to identification of biomarkers[END_REF][START_REF] Sévoz-Couche | Involvement of the dorsomedial hypothalamus and the nucleus tractus solitarii in chronic cardiovascular changes associated with anxiety in rats[END_REF] seem different.

Rostral cuneiform disinhibition also produced Fos expression in non-5-HT neurons in B3. The RMg and LPGi contain non-5-HT neurons that may be ON and OFF cells responding to noxious tail heat [START_REF] Gao | Serotonergic raphe magnus cells that respond to noxious tail heat are not ON or OFF cells[END_REF]. These cell types have been proposed to play an important role in modulating the alertness evoked by any brief external stimulus, either noxious or innocuous. ON cells may facilitate alertness during waking and OFF cells suppress arousals during sleep [START_REF] Foo | Brainstem modulation of pain during sleep and waking[END_REF]. Therefore, activation of non-5-HT-IR neurons in B3 induced by cuneiform disinhibition may participate in increased alertness during the defence reaction.

T A B L E 1 Statistical analysis of the interaction between the condition and intracerebral microinjections or mice genotyping, on the maximal baroreflex range using two-way repeated measures ANOVA Note: "Effect size" indicates the difference between means ± 95% confidence interval of difference.

Interestingly, non-5-HT-Fos-IR cells have also been found in the caudal part of the RDr, especially in the mid-rostrocaudal lateral divisions of the nucleus. To note, active escape expression in the elevated T-maze recruited non-5-HT neurons within the lateral wings of the RDr [START_REF] Spiacci | Differential involvement of dorsal raphe subnuclei in the regulation of anxietyand panic-related defensive behaviors[END_REF] and systemic injection of sodium lactate in a group of pharmacologically induced panic-prone rats was accompanied by the expression of a greater number of Fos-immunopositive non-5-HT neurons preponderantly within the lateral mid-rostro-caudal RDr [START_REF] Johnson | Neural pathways underlying lactate-induced panic[END_REF], similar to that between defence reaction and panic attack we mentioned above. Activation of RDr non-5-HT neurons during cuneiform disinhibition or panic expression may be involved in descending analgesia because a double fluorescence immunohistochemistry study revealed that RDr neurons expressing Fos after picrotoxin administration were non-5-HT neurons [START_REF] Koyama | Non-serotonergic midbrain neurons are involved in picrotoxin-induced analgesia. An immunohistochemical study in the rat[END_REF].

| Anterograde tracing from the rostral cuneiform nucleus to the intermediate B3

The rostral cuneiform nucleus targets the dorsolateral PAG [START_REF] Netzer | Brain circuits mediating baroreflex bradycardia inhibition in rats: An anatomical and functional link between the cuneiform nucleus and the periaqueductal grey[END_REF] and the dorsolateral PAG sends projections to the intermediate B3 region, at the level of the LPGi [START_REF] Babic | Medullary and spinal cord projections from cardiovascular responsive sites in the rostral ventromedial medulla[END_REF][START_REF] Cameron | The efferent projections of the periaqueductal gray in the rat: A Phaseolus vulgaris-leucoagglutinin study. II. Descending projections[END_REF]. Previous reports using different retrograde tracers including HRP and colloidal gold-labelled wheat germ agglutinin conjugated to HRP showed that neurons labelled from the RMg are distributed throughout the dorsal, lateral and ventrolateral PAG, but not the dorsolateral column [START_REF] Abols | Afferent connections of the rostral medulla of the cat: A neural substrate for midbrainmedullary interactions in the modulation of pain[END_REF][START_REF] Babic | Medullary and spinal cord projections from cardiovascular responsive sites in the rostral ventromedial medulla[END_REF][START_REF] Yin | Neurochemical properties of BDNF-containing neurons projecting to rostral ventromedial medulla in the ventrolateral periaqueductal gray[END_REF]. In the same manner, using anterograde tracing from the left rostral cuneiform nucleus, we show here that extensive projections reach the LPGi, at the area (intermediate level of B3) in which we identified Fos-5-HT-IR cells in the first set of experiments. In addition, as these projections were seen bilaterally, these data explain why disinhibition performed on one side can induce bilateral LPGi activation. However, only scarce labelling from the rostral cuneiform nucleus to the RMg was observed in the present study, though projections exist from the pre-cuneiform nucleus to the caudal RMg [START_REF] Abols | Afferent connections of the rostral medulla of the cat: A neural substrate for midbrainmedullary interactions in the modulation of pain[END_REF][START_REF] Carlton | Supramedullary afferents of the nucleus raphe magnus in the rat: A study using the transcannula HRP gel and autoradiographic techniques[END_REF][START_REF] Zemlan | Nucleus cuneiformis and pain modulation: Anatomy and behavioral pharmacology[END_REF]. Therefore, the origin of RMg 5-HT cell activation after cuneiform disinhibition (and dorsolateral PAG activation) remains unclear. One possibility could be that rostral cuneiform disinhibition partly activates the dorsal and/or lateral PAG along with dorsolateral PAG, even if there was very little Fos expression was observed here T A B L E 2 Statistical analysis of the interaction between the condition and intracerebral microinjections or mice genotyping, on the baroreflex rectilinear slope (BRS) using two-way repeated measures ANOVA and in previous study [START_REF] Netzer | Brain circuits mediating baroreflex bradycardia inhibition in rats: An anatomical and functional link between the cuneiform nucleus and the periaqueductal grey[END_REF] RMg [START_REF] Hermann | Afferent projections to the rat nuclei raphe magnus, raphe pallidus and reticularis gigantocellularis pars α demonstrated by iontophoretic application of choleratoxin (subunit b)[END_REF][START_REF] Zagon | The 5-HT 3 subtype of serotonin receptor contributes to nociceptive processing via a novel subset of myelinated and unmyelinated nociceptors[END_REF].

| Pharmacological experiments

Like pentobarbitone [START_REF] Sévoz-Couche | Involvement of the dorsomedial hypothalamus and the nucleus tractus solitarii in chronic cardiovascular changes associated with anxiety in rats[END_REF], urethane anaesthetic was mainly used in our previous studies to successfully evaluate the baroreflex cardiac response during the defence reaction [START_REF] Bernard | Critical role of B3 serotonergic cells in baroreflex inhibition during the defense reaction triggere by dorsal periaqueductal gray stimulation[END_REF][START_REF] Comet | Functional interaction between nucleus tractus solitarius NK1 and 5-HT 3 receptors in the inhibition of baroreflex in rats[END_REF][START_REF] Sévoz-Couche | Role of nucleus tractus solitarius 5-HT 3 receptors in the defense reactioninduced inhibition of the aortic baroreflex in rats[END_REF]. A less cardiovascular depressing anaesthetic, that is, an anaesthetic like isoflurane, could have been used, but that would reduced the baroreflex inhibition induced by dorsolateral PAG activation [START_REF] Bernard | Critical role of B3 serotonergic cells in baroreflex inhibition during the defense reaction triggere by dorsal periaqueductal gray stimulation[END_REF]. We investigated in the present study the possibility that the B3 region could be at least in part the origin of the cuneiform-induced vagal baroreflex inhibition. It should be noted that atenolol was used in all experiments to eliminate the possible influence of the sympathetic mediated tachycardic response evoked by the defence reaction on the baroreflex (see Section 2).

Atenolol would also prevent the sympathetic component of the baroreflex under normal physiological conditions [START_REF] Head | Vagal and sympathetic components of the heart rate range and gain of the baroreceptor-heart rate reflex in conscious rats[END_REF][START_REF] Saad | Effects of middle cerebral artery occlusion on baroreceptor reflex control of heart rate in the rat[END_REF], although, we have shown that None of the data discussed above specifically demonstrated the involvement of 5-HT neurons in the B3 area. The 5-HT 1A receptor is often located presynaptically as an autoreceptor, on the perikaryon and dendritic spines of 5-HT-containing neurons. It is also expressed as a heteroreceptor on neurons receiving input from 5-HT neurons.

8-OH-DPAT is a highly selective and centrally active potent ligand preferentially binding to the somatodendritic 5-HT 1A autoreceptor [START_REF] Hjorth | The 5-HT 1A receptor agonist, 8-OH-DPAT, preferentially activates cell body 5-HT autoreceptors in rat brain in vivo[END_REF][START_REF] H ; Middlemiss | 8-Hydroxy-2-(di-n-propylamino)tetralin discriminates between subtypes of the 5-HT 1 recognition site[END_REF]. Consequently, the release of 5-HT from the 5-HT nerve endings is tors [START_REF] Bernard | Critical role of B3 serotonergic cells in baroreflex inhibition during the defense reaction triggere by dorsal periaqueductal gray stimulation[END_REF][START_REF] Comet | 5-HT-mediated inhibition of cardiac baroreceptor reflex response during defense reaction in the rat[END_REF][START_REF] Comet | Functional interaction between nucleus tractus solitarius NK1 and 5-HT 3 receptors in the inhibition of baroreflex in rats[END_REF][START_REF] Sévoz-Couche | Role of nucleus tractus solitarius 5-HT 3 receptors in the defense reactioninduced inhibition of the aortic baroreflex in rats[END_REF]. Both the microinjection of granisetron into the NTS and the use of mice lacking the 5-HT 3 receptor prevented dorsolateral PAG-induced inhibition of chemoreflex responses [START_REF] Zafar | A hypothalamo-midbrain-medullary pathway involved in the inhibition of the respiratory chemoreflex response induced by potassium cyanide in rodents[END_REF].

In the same manner, the cuneiform-induced baroreflex inhibition was prevented by the blockade of NTS 5-HT 3 receptors with granisetron and was also not observed in 5-HT 3 receptor knockout mice. These results show the role of 5-HT 3 receptors play a role in the cuneiforminduced baroreflex inhibition, presumably at the level of NTS location.

Another elegant and selective approach to confirm these results could be the use of NTS microinjections of a viral vector for 5-HT 3 gene silencing.

In conclusion, our results support and extend data in [START_REF] Netzer | Brain circuits mediating baroreflex bradycardia inhibition in rats: An anatomical and functional link between the cuneiform nucleus and the periaqueductal grey[END_REF] that showed that rostral cuneiform nucleus stimulation produced typical defence responses (an increase in HR and BP associated with a reduction in the baroreflex cardiac response) through activation of the dorsolateral PAG, a key area of the defence reaction [START_REF] Bandler | Integrated defence reaction elicited by excitatory amino acid microinjection in the midbrain periaqueductal grey region of the unrestrained cat[END_REF]. We found here that rostral cuneiform nucleus-evoked baroreflex inhibition involve direct and indirect (at least through the dorsolateral PAG column) activation of intermediate B3 5-HT-containing neurons to release 5-HT into the NTS and activate 5-HT 3 receptors, ultimately inhibiting the baroreflexmediated vagal bradycardia (Figure 8). These data bring new insights in the multiple mechanisms involved in baroreflex prevention during the defence reaction.

Direct and indirect links to B3 appear to be a safety mechanism for baroreflex inhibition, which is vital to allow cardiac output increase and blood flow redistribution when BP increases during stress. On the other hand, as baroreflex failure is a risk factor for ventricular arrhythmia [START_REF] Brouillard | Social defeat: Vagal reduction and vulnerability to ventricular arrhythmias[END_REF], the existence of multiple pathways leading to baroreflex dysfunction may explain why 20% to 40% of sudden cardiac deaths are reported to be precipitated by strong acute stressors [START_REF] Vlastelica | Emotional stress as a trigger in sudden cardiac death[END_REF].

Finally, it is important to note that systemic use of granisetron-a potent anti-emetic [START_REF] Audhuy | A double-blind, randomised comparison of the anti-emetic efficacy of two intravenous doses of dolasetron mesilate and granisetron in patients receiving high dose cisplatin chemotherapy[END_REF] with a highly safe profile [START_REF] Aapro | Granisetron: An update on its clinical use in the management of nausea and vomiting[END_REF])-known to cross the blood-brain barrier [START_REF] Huang | Simultaneous measurement of blood and brain microdialysate of granisetron in rat by high-performance liquid chromatography with fluorescence detection[END_REF], was found in a previous study to prevent baroreflex inhibition during stress [START_REF] Sévoz-Couche | Involvement of the dorsomedial hypothalamus and the nucleus tractus solitarii in chronic cardiovascular changes associated with anxiety in rats[END_REF]. Therefore, treatment with granisetron could be used to restore parasympathetic activity and, thus, reduce the likelihood of adverse cardiac events induced by stress.

(

  50 pmol per 50 nl, n = 30, n = 6 in each of 5 groups: [i] B3 muscimol, [ii] B3 8-OH-DPAT, [iii] B3 saline, [iv] NTS granisetron, and [v] NTS saline) and mice (25 pmol per 25 nl, n = 6 WT and n = 6 5-HT 3 knockout animals).

  , were made into the commissural NTS at the level of the calamus scriptorius, at co-ordinates previously identified (i.e. 0.5 mm lateral and 0.5 mm ventral to the surface of the brain, Sévoz-Couche et al., 2013). Bilateral microinjections of saline for control were made into the intermediate B3 region and the NTS. Baroreflex control and experimental responses (15 min apart) were both elicited in the same rat before and 15 min after microinjections of saline or either bodipy muscimol or 8-OH-DPAT into the B3 region and saline or granisetron into the NTS. At the end of the experiments, animals were killed by administering an overdose of pentobarbitone (200 mgÁkg -1 , i.p.).

  8-OH-DPAT, atenolol, bicuculline, muscimol, phenylephrine and sodium nitroprusside were supplied by Sigma Aldrich Chimie (Saint-Quentin-Fallavier, France). The bodipy TMR-X conjugate muscimol was supplied by Invitrogen (Ref M-23400; Pontoise, France).

  .1 | Functional interaction between the rostral cuneiform nucleus and raphé nuclei in rats 3.1.1 | Fos immunohistochemical labelling after vehicle (sham) or bicuculline (experimental) into the rostral cuneiform nucleus

F

  I G U R E 1 Increase in Fos expression in 5-HT neurons after rostral CnF stimulation. (a) Bar graphs with individual values showing an increased number of 5-HT cells (in four brain sections) expressing Fos protein immunoreactivity in ipsilateral (ipsi) as well as contralateral (contra) lateral paragigantocellular nucleus (LPGi), total LPGi and raphe magnus (RMg), but not in raphe dorsalis (RDors), raphe pallidus (RPall),and raphe obscurus (RObsc), in experimental (intra-cuneiform bicuculline, filled bars) compared to sham (intra-cuneiform saline, open bars) conditions. Each bar is the mean ± SEM. *P < .05, significantly different from sham. (b) Photomicrographs of the LPGi at approximately -11.2 mm from bregma in sham and experimental condition. The high magnification microphotograph is the region framed in the contralateral LPGi of the experimental condition. Arrows with d, s and f indicate examples of Fos + 5-HT double-labelled (black core surrounded by brown staining), 5-HT single-labelled (cytoplasm brown staining) and Fos single-labelled (nucleus black staining) neurons respectively An example of injections of muscimol conjugated to bodipy into B3 targeting the LPGi or the RMg is given in Figure 4b-1,b2 respectively. We pooled our results as no difference between LPGi and RMg microinjections were observed. A representative example of control and experimental baroreflex responses obtained before and after intra-B3 muscimol injection is given in Figure 5a-1. The maximal baroreflex cardiac range obtained from sigmoid curves (Figure

F

  I G U R E 2 Increase in Fos expression in non-5-HT neurons after rostral cuneiform stimulation. (a) Bar graphs with individual values showing an increased number of non-5-HT cells expressing Fos protein immunoreactivity in total LPGi, RMg and RDors, but not in RPall and RObs, in experimental compared to sham conditions. Each bar is the mean ± SEM. *P < .05, significantly different from sham.. Abbreviations as in Figure 1. (b) Photomicrographs of the raphe dorsalis in sham and experimental (Exp) condition. The high magnification microphotograph is the raphe dorsalis region framed in the experimental condition. Aq, aqueduct; dlPAG, dorsolateral column of the periaqueductal grey area; dPAG, dorsal column of the periaqueductal grey area; lPAG, lateral column of the periaqueductal grey area; RDors, raphé dorsalis; vPAG, ventrolateral column of the periaqueductal grey area

F

  I G U R E 3 Projections from the rostral cuneiform nucleus to the B3 region. (a) Photomicrograph displaying the site of Phaseolus vulgaris leucoagglutinin lectin (PHA-L) unilateral injection in the rostral cuneiform nucleus (CnF, arrow), at approximately 8.2 mm caudal to bregma. (b) Camera lucida drawings of anterograde labelling of descending axons and terminal boutons in B3 from injection of PHA-L in the rostral cuneiform nucleus seen in (a). Series of transverse sections. Numbers indicate the distance (in mm) from bregma. (c) Photomicrographs corresponding to approximately -11.4 mm from bregma. PHA-L-immunolabelled fibres and varicosities in the LPGi are shown in framed ×20 magnification photomicrograph. 7, facial motor nucleus; CIC, central nucleus of the inferior colliculus; GiA, gigantocellular reticular nucleus alpha; ml, medial lemniscus; p7, perifacial zone; py, pyramidal tract; sp5, spinal trigeminal tract; VTg, ventral tegmentum. Other abbreviations as in Figures 1 and 2

  (7.9 ± 0.1 bpmÁmmHg -1 ) mice than control BRS (8.3 ± 0.3 and 8.0 ± 0.1 bpmÁmmHg -1 , respectively) (FigureS1).

4 |

 4 DISCUSSIONThis study confirms and extends previous observations on the baroreflex inhibition during rostral cuneiform disinhibition. This negative control involves both LPGi and RMg in the intermediate B3, but direct links were found to reach only LPGi. 5-HT-IR cells in B3 are the source of the 5-HT released into the NTS to activate 5-HT 3 receptors and block second-order baroreceptor neurons.

F I G U R E 4

 4 Microinjection sites of muscimol into the B3 group in rats. (a) Coronal sections at the level of B3 group showing bilateral microinjections of bodipy muscimol (black circle), 8-OH-DPAT (grey circle), or saline (white circle). Abbreviations as in Figure 3. (b) Reconstructive photomicrograph showing the bilateral microinjection of bodipy muscimol (orange fluorescence) within the 5-HT neuron area (green cells) of LPGi (b-1) and RMg (b-2) shown by arrows in (a) at -11.2 and -11.4 mm from bregma, respectively area of the defence reaction, induced a baroreflex inhibition through intermediate B3 raphé nuclei only

  . Another possibility is that the LPGi or the gigantocellular nucleus pars alpha (shown to receive numerous projections from the dorsolateral PAG, Cameron et al., 1995; rostral cuneiform nucleus [present study]) can activated downstream 5-HT neurons in the RMg following either rostral cuneiform disinhibition or dorsolateral PAG activation. In support of this possibility, both regions are known to send projections to the

  not have been the same experimental conditions as in Netzer et al. (2011). The chemical inhibition, by local administration of muscimol, of the intermediate B3 region (including both the LPGi and RMg)

  the defence reaction has no effect on the sympathetic baroreflex[START_REF] Sévoz-Couche | Role of nucleus tractus solitarius 5-HT 3 receptors in the defense reactioninduced inhibition of the aortic baroreflex in rats[END_REF].

F

  I G U R E 5 Inhibition of the baroreflex curves after disinhibition of the rostral cuneiform nucleus in rats. (a-1) Representative recordings illustrating that pharmacological disinhibition of the rostral cuneiform nucleus by local application of bicuculline (Bic CnF, experimental) did not affect the baroreflex tachycardia induced by administration of the vasodilator nitroprusside (SNP) but inhibited the reflex bradycardia induced by administration of the vasoconstrictor phenylephrine (PE) compared to the control condition, before but not after intra-B3 muscimol. (a-2) Sigmoid baroreflex curves corresponding to the animal shown in (a-1) showing that the baroreflex cardiac response to MBP changes was reduced in experimental compared to control conditions, before but not after intra-B3 muscimol. (b, c) Sigmoid baroreflex curves showing that the baroreflex cardiac response to MBP changes was reduced in experimental compared to control conditions, before but not after intra-B3 8-OH-DPAT (b) or intra-NTS granisetron (c) F I G U R E 6 Microinjection sites of bicuculline into the rostral cuneiform nucleus in mice. (a) Coronal sections at the level of the rostral CnF showing unilateral microinjections of bicuculline in wild-type (grey circle) or 5-HT 3 knockout (black circle) mice. (b) Photomicrograph showing pontamine blue deposit (arrow) below the tip of the micropipette used to inject bicuculline into the CnF at -4.6 mm from bregma, as indicated by the arrow in (a). Abbreviations as in Figure 3 F I G U R E 7 Inhibition of the baroreflex bradycardia after disinhibition of the rostral cuneiform nucleus in mice. (a) Representative recordings illustrating that pharmacological disinhibition of the rostral cuneiform nucleus by local application of bicuculline (Bic CnF, experimental) did not affect the baroreflex tachycardia induced by administration of the vasodilator nitroprusside (SNP) but inhibited the reflex bradycardia induced by administration of the vasoconstrictor phenylephrine (PE) compared to the control condition, in wild-type but not in 5-HT 3 knockout mice. (b) Sigmoid baroreflex curves corresponding to the animal shown in (a) showing that the decrease in HR in response to MBP changes was reduced in experimental compared to control conditions, in wild-type but not in 5-HT 3 knockout mice Though a higher proportion of 5-HT cells were activated in the LPGi than in the RMg, there was a marked increase of cFos in both regions. Thus, we microinjected muscimol or 8-OH-DPAT in either LPGi or RMg and we pooled our data. In this context, it is relevant that muscimol-induced inactivation of the intermediate portion of the B3 region suppressed almost completely the inhibitory effect of rostral cuneiform disinhibition on vagal baroreflex response range and BRS.

  diminished. In an in vitro study on dorsal raphé slices, 92% of neurons responding to 8-OH-DPAT contain 5-HT and the application of 8-OH-DPAT inhibits action potential firing in these cells[START_REF] Yasufuku-Takano | Morphological and physiological properties of serotonergic neurons in dissociated cultures from the postnatal rat dorsal raphe nucleus[END_REF]. In line with these studies, 8-OH-DPAT prevents the 5-HT-induced shivering during cooling when administered in the LPGi[START_REF] Brown | Activation of 5-HT 1A receptors in medullary raphé disrupts sleep and decreases shivering during cooling in the conscious piglet[END_REF] and reduces thermoregulatory responses to peripheral thermal challenges when given into the RMg[START_REF] Berner | 8-OH-DPAT-sensitive neurons in the nucleus raphe magnus modulate thermoregulatory output in rats[END_REF]. Here, we observed that 8-OH-DPAT in B3 had a similar preventive action on the baroreflex bradycardia inhibition induced by rostral cuneiform disinhibition to that obtained after muscimol. Even if we cannot exclude the possibility that 8-OH-DPAT could also affect 5-HT 1A heteroreceptors located on non-5-HT-IR neurons, our overall data strongly suggest the key role is played by 5-HT-IR neurons of the LPGi and RMg in the intermediate B3 in baroreflex inhibition during the defence reaction. These cells are most presumably responsible for 5-HT release into the NTS. Previously we showed that the baroreflex inhibition induced F I G U R E 8 Schematic representation of putative medullary pathways producing baroreflex inhibition during the defence reaction. Baroreceptor glutamatergic vagal afferents are known to excite barosensitive NTS cells (A) through the glutamatergic (GLU) activation of metabotropic amino acid receptors (grey square) to induce vagal baroreceptor reflex bradycardia after activation of preganglionic (PG) cells in the nucleus ambiguous (NAmb). Previous and present findings show that, during the defence-like reaction triggered by rostral cuneiform nucleus (rCnF) disinhibition, activation of (i) direct and indirect (through the dorsolateral PAG, dlPAG) projections to the LPGi and (ii) indirect projections to the RMg, both located in the intermediate B3 (iB3) region, produces the release of 5-HT into the NTS. 5-HT downstream stimulates 5-HT 3 receptors (small grey circle) localized presynaptically on gastrointestinal (GI) vagal glutamatergic afferents in the NTS. The resulting facilitation of glutamate release ends with the excitation of NTS GABAergic interneurons, which, in turn, inhibit baroreflex bradycardia through the stimulation of GABA A receptors (small black circle) on barosensitive a cells. "+" indicates excitatory effect; "-" indicates inhibitory effect by the dorsolateral PAG involved the activation of NTS 5-HT 3 recep-

  , Ministère de l'Agriculture et de la Forêt, Service Vétérinaire de la Santé et de la Protection Animale; Permission No. 75-855 to C.S.-C.). Animal studies are reported in compliance with the ARRIVE guidelines (Percie du Sert

Control vs. experimental condition Before vs. after treatment in rats or wild-type vs. KO mice Interaction

	Intra-B3 muscimol	F(1, 5) = 1920	F(1, 5) = 254.01	F(1, 5) = 119.10
		P < .05	P < .05	P <.05
		Effect size:	Effect size:	Effect size:
		50.42 ± 2.95	-29.92 ± 4.81	79.17 ± 18.62
	Intra-B3 8OH-DPAT	F(1, 5) = 72.82	F(1, 5) = 30.70	F(1, 5) = 37.81
		P < .05	P < .05	P <.05
		Effect size:	Effect size:	Effect size:
		58.75 ± 17.7	-29.58 ± 13.72	84.17 ± 35.21
	Intra-B3 saline	F(1, 5) = 4.39	F(1, 5) = 1.71	F(1, 5) = 0.44
		P > .05	P > .05	P > .05
		Effect size:	Effect size:	Effect size:
		107.1 ± 8.70	-4.87 ± 9.01	4.16 ± 16.06
	Intra-NTS granisetron NTS	F(1, 5) = 341.31	F(1, 5) = 48.88	F(1, 5) = 299.40
		P < .05	P < .05	P <.05
		Effect size:	Effect size:	Effect size:
		65.17 ± 9.06	-37.67 ± 13.85	108.70 ± 16.15
	Intra-NTS saline	F(1, 5) = 5.56	F(1, 5) = 2.85	F(1, 5) = 0.07
		P > .05	P > .05	P > .05
		Effect size:	Effect size:	Effect size:
		105.8 ± 6.36	5.00 ± 7.5	1.66 ± 15.8
	Mice	F(1, 5) = 60.36	F(1, 5) = 26.76	F(1, 5) = 10.50
		P < .05	P < .05	P < .05
		Effect size:	Effect size:	Effect size:
		54.17 ± 17.92	-65.83 ± 32.71	105.00 ± 83.31

Experimental vs. control condition Before vs. after treatment in rats or wild-type vs. KO mice Interaction

	Intra-B3 muscimol	F(1, 5) = 653.3	F(1, 5) = 111.0	F(1, 5) = 95.6
		P < .05	P < .05	P < .05
		Effect size:	Effect size:	Effect size:
		2.67 ± 0.27	-1.42 ± 0.35	3.11 ± 0.81
	Intra-B3 8OH-DPAT	F(1, 5) = 445.1	F(1, 5) = 660.3	F(1, 5) = 95.6
		P < .05	P < .05	P < .05
		Effect size:	Effect size:	Effect size:
		2.92 ± 0.35	-2.25 ± 0.11	3.87 ± 0.59
	Intra-B3 saline	F(1, 5) = 3.9	F(1, 5) = 0.02	F(1, 5) = 1.51
		P > .05	P > .05	P > .05
		Effect size:	Effect size:	Effect size:
		4.33 ± 0.36	-0.66 ± 0.15	0.03 ± 0.56
	Intra-NTS granisetron NTS	F(1, 5) = 361.4	F(1, 5) = 119.7	F(1, 5) = 121.0
		P < .05	P < .05	P < .05
		Effect size:	Effect size:	Effect size:
		2.90 ± 0.41	-1.92 ± 0.43	3.31 ± 0.77
	Intra-NTS saline	F(1, 5) = 0.25	F(1, 5) = 0.09	F(1, 5) = 0.59
		P > .05	P > .05	P > .05
		Effect size:	Effect size:	Effect size:
		4.30 ± 0.18	0.04 ± 0.15	0.08 ± 0.95
	Mice	F(1, 5) = 57.7	F(1, 5) = 287.0	F(1, 5) = 64.65
		P < .05	P < .05	P < .05
		Effect size:	Effect size:	Effect size:
		2.53 ± .0.86	-2.18 ± 0.32	4.93 ± 1.57
	Note: "Effect size" indicates the difference between means ± 95% confidence interval of difference.

ACKNOWLEDGEMENT

The authors thank Julie Schneider for proofreading the manuscript.

We thank the Association Sorbonne Université and Institut National de la Santé et de la Recherche Médicale (INSERM) for their support.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

AUTHOR CONTRIBUTIONS

C.S.-C. contributed to the conceptualization, investigation and methodology of the study, writing of the original draft and writing of the review and editing the manuscript and F.N. to the data curation and formal analysis of the study.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC

RIGOUR

SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section at the end of this article.