Nabil El Bitar

Bernard Pollin

Gan Huang

André Mouraux

Daniel Le Bars
email: daniel.le_bars@upmc.fr

RVM control of cutaneous vasomotion

Keywords: rat, thermoregulation, ambient temperature, skin temperature, environmental impact, blood pressure, heart rate, infrared camera, rostral ventro-medial medulla, RVM, rostral Medullary Raphe, rMR

Thermal neutrality in rodents is achieved by large cyclic variations of the sympathetic drive of the vasomotion of the tail and paws, the most widely used target organs in current acute or chronic animal models of pain. Given the pivotal functional role of rostral ventro-medial medulla (RVM) in nociception and rostral Medullary Raphe (rMR) in thermoregulation, two largely overlapping brain regions, we aimed at circumscribing the brainstem regions that are the source of premotor afferents to sympathetic preganglionic neurons that control the vasomotor tone of the tail and hind-paws. A thermometric infrared camera recorded indirectly the vasomotor tone of the tail and hind-paws. During the control period, the rat was maintained in vasoconstriction by preserving a stable, homogeneous and constant surrounding temperature, slightly below the core temperature. The functional blockade of the RVM/rMR by the GABA A receptor agonist muscimol (0.5 nmol, 50 nl) elicited an extensive increase of the temperature of the paws and tail, associated to slight decrease of blood pressure and heart rate. Both the increased heat loss through vasodilatation and the decrease heart-induced heat production elicited a remarkable reduction of the central temperature. The effective zones were circumscribed to the parts of the RVM/rMR facing the facial nucleus. They match very exactly the brain regions often described as specifically devoted to the control of nociception. Our data support and urge on the highest cautiousness regarding the interpretation of results aimed at studying the effects of any pharmacological manipulations of RVM/rMR with the usual tests of pain. * P < 0.05 ** , P < 0.01 with respect to the corresponding value in group 1.

INTRODUCTION

Numerous studies have explored the descending systems that control the spinal transmission of nociceptive messages, notably through the Rostral Ventromedial Medulla (RVM) in the brainstem. The RVM includes the nucleus raphe magnus and the neighboring part of the reticular formation, which extends under the gigantocellular reticular nucleus [START_REF] Fields | Central nervous system mechanisms of pain modulation[END_REF]. Two main categories of neurons, so-called "on-" and "off-" cells, were highlighted that: (1) exhibit irregular spontaneous activities in opposition of phase [START_REF] Barbaro | Putative nociceptive modulatory neurons in the rostral ventromedial medulla of the rat display highly correlated firing patterns[END_REF]) and ( 2) are activated and inhibited by nociceptive stimuli, respectively [START_REF] Fields | The activity of neurons in the rostral medulla of the rat during withdrawal from noxious heat[END_REF][START_REF] Vanegas | Tail-flick related activity in medullospinal neurons[END_REF]. These two neuronal types were proposed to belong to a double spino-bulbospinal positive feedback loop that facilitates the spinal transmission of nociceptive messages, either directly or through a mechanism of disinhibition, the "on-" and "off-" cells being supposed to produce an excitatory and an inhibitory drive on the spinal neurons, respectively. However, RVM neurons could play a much less specific role than that of a descending pathway modulating nociception at spinal level (Le [START_REF] Bars | Animal models of nociception[END_REF][START_REF] Lefler | Bulbospinal neurons of the rat rostromedial medulla are highly collateralized[END_REF][START_REF] Lovick | The medullary raphe nuclei: a system for integration and gain control in autonomic and somatomotor responsiveness?[END_REF][START_REF] Mason | Contributions of the medullary raphe and ventromedial reticular region to pain modulation and other homeostatic functions[END_REF]2005a;2005b;[START_REF] Mason | Descending pain modulation as a component of homeostasis[END_REF]2011;[START_REF] Mason | Medullary circuits for nociceptive modulation[END_REF][START_REF] Thurston | Effects of intravenous phenylephrine on blood pressure, nociception, and neural activity in the rostral ventral medulla in rats[END_REF][START_REF] Thurston | Effects of vagal afferent stimulation on ON and OFF cells in the rostroventral medulla: relationships to nociception and arterial blood pressure[END_REF]1995). For example, we described recently the probable involvement of "on-" and "off-" cells in autonomic regulation, notably thermoregulation (El Bitar et al. 2014b).

On the basis of three animal models used in thermoregulation studies, namely the tail of the rat, the ear of the rabbit and the interscapular brown adipose tissue (BAT) of the rat, it became apparent that neurons in the midline region of the ventral medulla play a critical role in responding to cold [START_REF] Blessing | Raphe pallidus and parapyramidal neurons regulate ear pinna vascular conductance in the rabbit[END_REF][START_REF] Mcallen | Multiple thermoregulatory effectors with independent central controls[END_REF][START_REF] Morrison | RVLM and raphe differentially regulate sympathetic outflows to splanchnic and brown adipose tissue[END_REF], 2011;[START_REF] Morrison | GABA-mediated inhibition of raphe pallidus neurons regulates sympathetic outflow to brown adipose tissue[END_REF][START_REF] Morrison | Central control of thermogenesis in mammals[END_REF][START_REF] Morrison | Central control of brown adipose tissue thermogenesis[END_REF][START_REF] Nakamura | Central circuitries for body temperature regulation and fever[END_REF][START_REF] Nakamura | Central circuitries for body temperature regulation and fever[END_REF]Morrison 2008, 2011;[START_REF] Rathner | Cold-activated raphe-spinal neurons in rats[END_REF][START_REF] Rathner | Differential control of sympathetic drive to the rat tail artery and kidney by medullary premotor cell groups[END_REF][START_REF] Tanaka | Role of the medullary raphe in thermoregulatory vasomotor control in rats[END_REF]. Broadly, cooling the animal results in (1) reduced heat loss through increased skin vasoconstriction of the ear in the rabbit or the tail in the rat and (2) increased heat production by BAT in the rat. These phenomena are triggered by activation of sympathetic preganglionic neurons in the spinal intermediolateral cell column, themselves under the control of premotor neurons located in the rostral Medullary Raphe (rMR), a region immediately rostral to the rostral pole of the inferior olivary complex, that includes the raphe pallidus, the raphe magnus and the laterally extending parapyramidal nucleus [START_REF] Blessing | Lower brainstem pathways regulating sympathetically mediated changes in cutaneous blood flow[END_REF][START_REF] Mcallen | Multiple thermoregulatory effectors with independent central controls[END_REF][START_REF] Morrison | Carl Ludwig Distinguished Lectureship of the APS Neural Control and Autonomic Regulation Section: Central neural pathways for thermoregulatory cold defense[END_REF][START_REF] Nakamura | Central circuitries for body temperature regulation and fever[END_REF][START_REF] Nakamura | Central circuitries for body temperature regulation and fever[END_REF]Morrison 2008, 2011), where the expression of Fos immunoreactivity is increased following cold exposure [START_REF] Bonaz | Induction of Fos immunoreactivity in the rat brain after cold-restraint induced gastric lesions and fecal excretion[END_REF][START_REF] Cano | Anatomical substrates for the central control of sympathetic outflow to interscapular adipose tissue during cold exposure[END_REF][START_REF] Morrison | GABA-mediated inhibition of raphe pallidus neurons regulates sympathetic outflow to brown adipose tissue[END_REF][START_REF] Nakamura | Identification of sympathetic premotor neurons in medullary raphe regions mediating fever and other thermoregulatory functions[END_REF]). Yet a possible discrepancy was noted. Studies focusing on the descending systems postulated to control nociception emphasize a region which includes the nucleus raphe magnus and the gigantocellular reticular nucleus pars alpha, mainly at the level of the facial nucleus (e.g. [START_REF] Brink | Raphe magnus neurons help protect reactions to visceral pain from interruption by cutaneous pain[END_REF][START_REF] Carlson | Sensitization of pain-modulating neurons in the rostral ventromedial medulla after peripheral nerve injury[END_REF][START_REF] Fields | Dorsal horn projection targets of ON and OFF cells in the rostral ventromedial medulla[END_REF][START_REF] Heinricher | GABA-mediated inhibition in rostral ventromedial medulla: role in nociceptive modulation in the lightly anesthetized rat[END_REF][START_REF] Heinricher | Interference with GABA transmission in the rostral ventromedial medulla: disinhibition of off-cells as a central mechanism in nociceptive modulation[END_REF][START_REF] Kaplan | Hyperalgesia during acute opioid abstinence: evidence for a nociceptive facilitating function of the rostral ventromedial medulla[END_REF][START_REF] Morgan | Pronounced changes in the activity of nociceptive modulatory neurons in the rostral ventromedial medulla in response to prolonged thermal noxious stimuli[END_REF][START_REF] Neubert | Nociceptive facilitating neurons in the rostral ventromedial medulla[END_REF][START_REF] Vanegas | Tail-flick related activity in medullospinal neurons[END_REF][START_REF] Thurston | Effects of intravenous phenylephrine on blood pressure, nociception, and neural activity in the rostral ventral medulla in rats[END_REF][START_REF] Thurston | Responses of on and off cells in the rostral ventral medulla to stimulation of vagal afferents and changes in mean arterial blood pressure in intact and cardiopulmonary deafferented rats[END_REF][START_REF] Xu | NMDA receptor-mediated activation of medullary pronociceptive neurons is required for secondary thermal hyperalgesia[END_REF], while studies focusing on thermoregulation emphasize a slightly more caudal region including the raphe pallidus (e.g. [START_REF] Blessing | Raphe magnus/pallidus neurons regulate tail but not mesenteric arterial blood flow in rats[END_REF][START_REF] Cao | Disinhibition of rostral raphe pallidus neurons increases cardiac sympathetic nerve activity and heart rate[END_REF][START_REF] Cao | Medullary pathways mediating specific sympathetic responses to activation of dorsomedial hypothalamus[END_REF][START_REF] Cao | Inhibition of brown adipose tissue thermogenesis by neurons in the ventrolateral medulla and in the nucleus tractus solitarius[END_REF][START_REF] Cerri | Cutaneous vasodilation elicited by disinhibition of the caudal portion of the rostral ventromedial medulla of the freebehaving rat[END_REF][START_REF] Fan | Thermogenesis activated by central melanocortin signaling is dependent on neurons in the rostral raphe pallidus (rRPa) area[END_REF]Madden andMorrison 2003, 2005;[START_REF] Morrison | RVLM and raphe differentially regulate sympathetic outflows to splanchnic and brown adipose tissue[END_REF][START_REF] Morrison | Raphe pallidus neurons mediate prostaglandin E2-evoked increases in brown adipose tissue thermogenesis[END_REF][START_REF] Morrison | Activation of 5-HT1A receptors in raphe pallidus inhibits leptin-evoked increases in brown adipose tissue thermogenesis[END_REF][START_REF] Morrison | GABA-mediated inhibition of raphe pallidus neurons regulates sympathetic outflow to brown adipose tissue[END_REF][START_REF] Morrison | Reduced rearing temperature augments responses in sympathetic outflow to brown adipose tissue[END_REF][START_REF] Nakamura | Central circuitries for body temperature regulation and fever[END_REF]Morrison 2007, 2011;[START_REF] Nakamura | Identification of sympathetic premotor neurons in medullary raphe regions mediating fever and other thermoregulatory functions[END_REF][START_REF] Ootsuka | Interactive drives from two brain stem premotor nuclei are essential to support rat tail sympathetic activity[END_REF][START_REF] Rathner | Cold-activated raphe-spinal neurons in rats[END_REF][START_REF] Rathner | Central pathway for spontaneous and prostaglandin E 2 -evoked cutaneous vasoconstriction[END_REF][START_REF] Salo | Control of cardiac rate, contractility, and atrioventricular conduction by medullary raphe neurons in anesthetized rats[END_REF][START_REF] Tanaka | Role of the medullary raphe in thermoregulatory vasomotor control in rats[END_REF][START_REF] Tanaka | Independent vasomotor control of rat tail and proximal hairy skin[END_REF][START_REF] Yoshida | Neurons of the rat preoptic area and the raphe pallidus nucleus innervating the brown adipose tissue express the prostaglandin E receptor subtype EP3[END_REF].

The aim of the present study was therefore to map exactly the brainstem regions that contain the premotor sympathetic neurons controling the vasomotor tone of the tail and hind-paws of the rat, and to compare these locations with that of nociception-related brainstem areas. This choice was made specifically because many behavioral models of pain/nociception are based on the assessment of the response of a rodent to a thermal stimulus applied to the tail or a hind-paw (reviewed in Le [START_REF] Bars | Animal models of nociception[END_REF][START_REF] Bars | Current animal test and models of pain[END_REF]. Specifically, we measured in 160 adult male Sprague-Dawley rats, the dynamic changes in vasomotor tone induced by microinjections of muscimol within these regions. During the injection, the rats were maintained in a stable state of tail and hind-paw vasoconstriction. Microinjection of muscimol within the RVM/rMR was expected to elicit vasodilation of the tail and hind-paw. When a vasomotor response was present, the latency at which vasodilation occurred was used as an index of the distance between the injection site and the structure generating the response.

GLOSSARY

METHODS

Ethic statement

Animal experiments were performed with permission of the Board of the Veterinarian Services of the French Ministry of Agriculture (permit number 75-151) in accordance with the National Institute of Health's "Guide for the care and use of Laboratory animals", the European Communities Council Directive 86/609/EEC regulating animal research, and the ethics committee of the International Association for the Study of Pain [START_REF] Covino | Ethical standards for investigations of experimental pain in animals. The Committee for Research and Ethical Issues of the International Association for the Study of Pain[END_REF][START_REF] Zimmermann | Ethical guidelines for investigations of experimental pain in conscious animals[END_REF]. The Committee of Ethics for the Animal Experiment of our Institution approved the procedures.

Animals

Experiments were performed on 160 adult male Sprague-Dawley rats (Janvier Labs, Saint-Berthevin, France) weighing 320-370 g. They were housed in groups of 3-4 per cage, allowed free access to food and water with a 12 h alternating light-dark cycle, and acclimatized to the laboratory for at least one week before the experiment. The experiments were conducted between 9 AM and 5 PM.

Experimental procedure

The animals were deeply anesthetized with 2.5% halothane in 100% oxygen. A tracheal cannula was inserted and the ventilation was controlled mechanically with an open circuit respirator equipped with a

scavenging system, at a rate of 50 breaths/min. The tidal volume was adjusted to maintain a normal end-tidal CO 2 (ETCO 2 ). The expiratory halothane level and ETCO 2 were assessed with a capnometer (Capnomac II, Datex Instruments, Helsinki, Finland), recorded each ten seconds and under control of alarms throughout the experiment. The mean arterial blood pressure (MAP) and heart rate (HR) were monitored continuously via a catheter inserted into the common carotid artery and connected to a computer via a transducer. MAP and HR were calculated and recorded using the NOTOCORD ® blood pressure analyzer system.

The body of the animal was wrapped up in a water-warming pad connected to an extra-capacity water circulator (TP 220-Kent, scientific corporation) sparing the head, the paws and the tail. The heating blanket was covered with an isothermic metalized polyester film ("survival blanket") to stabilize the space temperature around the body. A two channel OMEGA ® HH506RA digital thermometer and two VIP-T-CT25515 Probes (0.1°C resolution) were used to measure the core temperature T core (throughout a rectal probe inserted 10 cm) and the heating temperature T warm (probe placed between the warming pad and the trunk of the rat). We adjusted T warm in order to maintain (1) T core stable within normal physiological values and (2) the tail and hind-paws in vasoconstriction.

The rat was mounted on a Horsley-Clarke stereotaxic frame. 0.5 ml of xylocaine 2% was injected subcutaneously in the scalp, followed by a 2-cm midline incision. After trepanation, a small incision of the dura-mater was made to introduce the tip of a microinjection glass needle.

After surgery, halothane was decreased to 0.8-0.9% with oxygen being kept at 100%, the tidal volume was adjusted to keep the ETCO 2 around 3.5% and at least 30-min were waited before starting the experimental procedure. After 15-min of control period in steady vasoconstriction, 57 ng (50 nl) muscimol were injected in the RVM/rMR (target zone between -0.8 and -3.3 rostral to inter-aural line) over 60seconds. Since RVM and rMR largely overlap, we will refer to "RVM/rMR" for brain regions that include the raphe pallidus, raphe magnus, parapyramidal nucleus and the reticular formation that extends under the gigantocellular reticular nucleus [START_REF] Mason | Contributions of the medullary raphe and ventromedial reticular region to pain modulation and other homeostatic functions[END_REF](Mason , 2005a(Mason , 2005b[START_REF] Mason | Descending pain modulation as a component of homeostasis[END_REF][START_REF] Mason | From descending pain modulation to obesity via the medullary raphe[END_REF]. Control experiments were conducted with the same procedure, except that muscimol was injected outside the RVM/rMR.

Experimental conditions

The experiments were made in anesthetized rat with unremitting halothane level [0.85 (0.83-0.86)%], while ventilation was controlled in order to achieve a stable and normal acid-base equilibrium during the control period ETCO 2 = 3.65 (3.57-3.73)%. The mean room temperature T amb was stable at 24.2 (24.1-24.4)°C. The paws and tail of the rat were maintained in vasoconstriction during the control period by preserving a stable, homogeneous and constant surrounding temperature [T warm = 37.4 (37.2-37.5)°C], ≈ 0.3°C below T core [37.7 (37.6-37.8)°C], and kept constant till the end of the experiment. In the control period, MAP and HR were 83.9 (77.4-90.4) mmHg and 321 (309-332) bpm, respectively.

Muscimol preparation

We prepared muscimol solution in a concentration of 0.01 nmol/nl, added pontamine sky blue to identify the site of injection, fractioned the solution in 2 µl aliquots for single use and preserved them at -20°C. Muscimol produces a rapid and persistent hyperpolarization of neurons [START_REF] Hikosaka | Modification of saccadic eye movements by GABA-related substances. I. Effect of muscimol and bicuculline in monkey superior colliculus[END_REF][START_REF] Martin | Pharmacological inactivation in the analysis of the central control of movement[END_REF] based on its high affinity and selectivity for the GABA A receptor [START_REF] Beaumont | Muscimol binding in rat brain: association with synaptic GABA receptors[END_REF][START_REF] Enna | Properties of gamma-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fractions[END_REF][START_REF] Gallagher | Effects of glial uptake and desensitization on the activity of gamma-aminobutyric acid (GABA) and its analogs at the cat dorsal root ganglion[END_REF][START_REF] Krogsgaard-Larsen | A new class of GABA agonist[END_REF][START_REF] Nicholson | GABA analogues: conformational analysis of effects on [3H]GABA binding to postsynaptic receptors in human cerebellum[END_REF].

The day of the experiment, we filled the circuit of the glass needle with paraffin to wash out air bubbles, aspirated 0.5 µl of muscimol solution at the tip of the needle using a 1-µl Hamilton syringe and introduced the needle in the brain at the end of the surgery..

Thermographic recordings

Under stable environmental temperature, skin temperature is a reliable indicator of skin blood flow variations (El Bitar et al. 2014a[START_REF] Hertzman | Some relations between skin temperature and blood flow[END_REF]. Heat transmission from deeper tissue to the skin occurs by conduction and vascular convection, and then heat dissipation to the environment through conduction, convection, evaporation and radiation processes. The spatial and temporal evolution of the skin temperature at the level of the tail and paws was monitored using a JADE MWIR (3-5 µm optical bandpass) camera (CEDIP Infrared Systems, Croissy-Beaubourg, France) with a 500 µs integration time, which supplied images of 320x240 pixels at 1 Hz with a sensitivity of 0.02°C at 25°C. The camera was placed 1.5 m upright to the scene and was controlled by the software Cirrus © (CEDIP Infrared Systems, Croissy-Beaubourg, France). It was calibrated by means of a black body as previously described [START_REF] Benoist | Peripheral and central determinants of a nociceptive reaction: an approach to psychophysics in the rat[END_REF].

Recently, we verified the highly significant positive linear correlation between skin temperature and skin blood flow measured by a laser-Doppler probe. The mean time lag between vasodilatation and skin temperature increase was estimated at around half a minute (El Bitar et al. 2014a). Such an approach based on thermal imaging has the advantage of giving accurate measures of temperature at both temporal and spatial levels.

Analysis of the thermographic films was made using the software Altair © (CEDIP Infrared Systems, Croissy-Beaubourg, France). Ten regions of interest (ROI) were defined in the recorded scene, each comprising 10 pixels. Five ROIs were located on the dorsal aspect of the tail. A proximal ROI (tailprox) was placed 3 cm from the root of the tail, an intermediate ROI (tail-mid) was placed at the middle of the tail, and a distal ROI (tail-distal) was placed 3 cm from the tip of the tail. The remaining two zones were distributed equidistantly between tail-prox and tail-mid and tail-mid and tail-dist. Two additional ROIs were located on the plantar aspect of each hind paw; their final designation was defined respective to the side of muscimol microinjection, ipsilateral (paw-ipsi) or contralateral (paw-contra), as determined following histological controls. Finally, a ROI was located over a piece of wood placed in the scene to monitor the ambient temperature (T ambient ). For each time point, the mean of the ten pixels defining each ROI was computed to obtain one single temperature time course for each ROI (T tail-prox , T tail-mid , T tail-dist , T paw-left , T paw- right and T ambient ).

Histological identification of microinjection sites

At the conclusion of the experiment, the rat was deeply anesthetized with 3% halothane and the brain was perfused through the heart with 0.9% NaCl, followed by 10% formaldehyde and removed. The brain was frozen, cut in serial 100-µm thick sections and Nissl-stained with cresyl violet or carmin. Sites of microinjections were determined from microscopic visualization of the serial sections and reported on schemas of frontal sections of the brain [START_REF] Paxinos | The Rat Brain in Stereotaxic Coordinates[END_REF]. Coordinates were then expressed relative to the interaural line: x-axis = latero-lateral; y-axis = ventro-dorsal; z-axis = rostro-caudal. The center of injection was easy to delimit. On average ~1 hour post injection, the overall rostro-caudal diffusion of pontamine blue was 1.1 mm (1.0-1.2 mm), and two times larger than diffusion in the coronal plane.

Assessing the dynamic effects of muscimol on skin temperature

The skin temperature evolution following muscimol microinjection matched a sigmoid curve. The curves were adjusted to a Boltzmann sigmoid according to the equation T adj = T min + ΔT skin /{1 + exp[-(tt x )/s]}, using the SigmaPlot © software. Time t = 0 corresponded to the end of the microinjection (Fig. 1A).

The following parameters were fitted to the data: T min (adjusted skin temperature during the control period), T max (adjusted maximal skin temperature at the end of the process), ΔT skin = T max -T min (amplitude of the skin temperature variation), tₓ (abscissa of the inflection point of the sigmoid), Tₓ (ordinate of the inflection point of the sigmoid), s = spread of the sigmoid curve. These parameters were calculated for the proximal and distal parts of the tail, and for each hind-paw. The regression coefficients R² of the adjusted curves were always highly significant (> 0.98).

The regulation of the laboratory air-conditioning system was a source of change of ambient temperature in the ∼ 0.5°C range. This represented ∼10% of the magnitude of the ascending phase of muscimol-induced increases in skin temperature at the hind-paws and distal part of the tail and ∼15% of the increases in skin temperature at the proximal tail. Therefore, the beginning (t min ) of the ascending phase of vasodilatation at the hind-paws and distal part of the tail was calculated by considering the 10% percentile of ΔT skin : t min = tₓ -S * Ln(1/0.1-1) (Fig. 1B). A slightly different cutoff (15%) was chosen to estimate the onset of vasodilation at the proximal part of the tail, because of the slower evolution of the process at that location.

The end (t max ) of the ascending phase of vasodilatation was calculated by considering the 90% percentiles of ΔT skin : t max = tₓ -S * Ln(1/0.9-1).

Then, the latency of the vasomotor reactions were divided into three groups according to the distribution of the beginning time t min of the ascending phase of vasodilatation: t min < 7.5 min, 7.5 < t min < 15 min and t min > 15 min. This categorization was justified by the clear bimodal distribution of t min , centered on 3-5 and 10-12 min, respectively (Fig. 1C). Beyond 15 min, the distribution of t min was flat, and the observed effects if any were interpreted as resulting from uncontrolled diffusion of muscimol [START_REF] Edeline | Muscimol diffusion after intracerebral microinjections: a reevaluation based on electrophysiological and autoradiographic quantifications[END_REF].

Together with a small volume of injection (50 nl) and a large number of negative sites of injection, this approach provided an acute delineation of the structures controling sympathetic cutaneous vasoconstrictor activity.

Three-dimensional mapping of response latencies

The large number of microinjection sites provided the opportunity of building a three-dimensional mapping of response latencies as a function of injection site, using interpolation. The interpolation was performed by computing an average of the responses obtained at all injection sites, weighted by the distance between the interpolated voxel and each injection site. This ensured that the interpolated values were most dependent on the responses of the nearest injection sites. In addition, because of the non-homogeneous sampling of the interpolated volume, a mask was used to exclude voxels that were not close to at least one injection point.

For each measurement location (left paw, right paw, proximal tail, distal tail), a 3D volume representing response latencies as a function of injection site was obtained using interpolation. The responses obtained from the left and right paw were merged, by expressing the location of the injection site as ipsilateral (positive x-axis) vs. contralateral (negative x-axis) relative to the paw. The interpolated 3D volume extended from -2 mm to +2 mm along the x-axis, -1.5 mm to +1 mm along the y axis, and -1 mm to +3.5 mm along the z-axis (200 points along each dimension). For each voxel of the volume, an interpolated latency was obtained by computing a weighted average of all latency values: latency( , , ) = ∑ latency The weight assigned for each latency value ( ) was determined by the distance between the voxel ( , , ) and the injection site ( , , ):

= 1 ( -) + ( -) + ( -)
This weight is inversely proportional to the sixth power of the distance. This ensured that at any given location of the interpolated volume, interpolated latencies were more dependent on the latencies of the nearest injection site. Because the 3D volume was not homogeneously sampled (i.e. injection sites were clustered in some regions of the volume), a mask was used to exclude voxels, which were not located at a distance < 0.5 mm of at least one injection point. Finally, because injection at a large amount of sites did not elicit any response, a second mask was computed to distinguish voxels located in regions where injection elicited a response from voxels located in regions where injection did not elicit a response after 55 min of recording. After assigning a latency of 55 min to these trials, the second mask was computed by taking all voxels of the interpolated volume having interpolated latencies above an arbitrary interpolated latency value of 53.5 min.

Heat loss Index

The skin is an interface between core temperature (T core ) and ambient temperature, (T amb ) acting as a radiator. In addition to active changes secondary to vasomotor tone of skin vessels, the temperature T of the skin can be affected passively by either T amb or T core ). To cancel out these passive changes, we computed the Heat Loss Index (HLI) = (T skin -T amb )/(T core -T amb ) as initially proposed by [START_REF] Székely | Skin temperature-skin blood flow: assessment of thermoregulatory changes[END_REF]. The value of HLI can vary between 0 and 1, representing complete vasoconstriction (T = T amb ) and complete vasodilatation (T = T core ), respectively [START_REF] Gordon | Rat tail skin temperature monitored noninvasively by radiotelemetry: characterization by examination of vasomotor responses to thermomodulatory agents[END_REF][START_REF] Romanovsky | Selected contribution: ambient temperature for experiments in rats: a new method for determining the zone of thermal neutrality[END_REF].

Analysis of vital signs

To study the systemic effects of muscimol on MAP, HR, ETCO 2 and T core with respect to the microinjection sites, we divided the experiments in three groups on the basis of the beginning time t min of the ascending phase of vasodilatation. Group 1: (1) t min < 15 min for all considered skin areas either on the tail or the paws. Group 2: t min < 15 min for at least one but not all the areas considered on the tail or the hind-paws.

Group 3: t min > 15 min for all considered skin areas either on the tail or the paws. The mean of each variable during the control period was considered as the reference level, and the temporal evolution was calculated in terms of variation of this level, namely T core (Δ°C), MAP (ΔmmHg), HR (Δ°C) and ETCO 2 (Δ%).

Data processing and statistical analyses

The temporal evolution of the mean skin temperature, MAP and HR were synchronized and measured with a one-second-time resolution. The corresponding time courses were downsampled to a oneminute time resolution by averaging. Data were expressed as means (± confident interval 95%). The comparisons were done using the Mann Whitney U test and the Friedman repeated measures ANOVA on ranks test.

RESULTS

The paws and tail of the rat were successfully maintained in vasoconstriction during the control period by preserving a stable, homogeneous and constant surrounding temperature T warm , adjusted to a few tenths degrees below T cor , and by keeping this temperature constant throughout the experiment. Following a 15-min period of control in steady vasoconstriction, muscimol (0.5 nmol, 50 nl) was injected over 60seconds.

We will first describe in details a typical recording. Then the effects of muscimol will be detailed with reference to the injection sites determined post-mortem. Emphasis will be made on the comparison between hind-paws (ipsilateral vs. contralateral to the injection site). We seized the opportunity of the large number of microinjection sites to build three-dimensional mappings of response latencies as a function of injection site, using interpolation. Results will also be synthesized by taking into account concomitant variations of MAP, HR and T core . Finally, the potential effect of muscimol on a widely used nociceptive test, the tail-flick test, will be assessed by using the present results to compute expected variations in tail-flick latency elicited by the effects of muscimol on both tail temperature and T core .

A typical example of the effects of muscimol microinjection within the RVM/rMR

An example of a thermographic film is provided as supporting information (Supporting video), from which a series of seven images recorded during 30-min are shown in figure 2A. These images correspond to a typical example of a microinjection centered to the RMg, as determined following histological examination (Fig. 2H). Figure 2B shows the temporal evolution of the temperature of (1) the hind-paws, ipsilateral (T paw- ipsi ) and contralateral (T paw-contra ) to the microinjection site, (2) five sites on the tail and (3) a small piece of wood, indicator of the ambient temperature (T amb ). Figure 2C shows the temporal evolution of the core temperature (T core ). During the control period, T amb and T core were stable while the hind-paws and tail were in vasoconstriction with skin temperatures slightly above T amb . Following the injection of muscimol, a progressive increase of skin temperature is observed, starting at the ipsilateral paw (T paw-ipsi : 4.9 min postinjection), followed by the proximal tail (T tail-prox : 6.6 min), the distal tail (T tail-dist : 11.6 min), and, finally, the contralateral paw (T paw-contra : 12.6 min). The time courses of T paw-ipsi and T paw-contra were similar, increasing by 8.2 and 7.4°C, within 7.7 and 8.0 min respectively, and remaining stable afterwards. The temporal evolution of the temperature of the tail was quite different, taking 26.6 min to achieve a 6.4°C increase for T tail-prox vs.

21.2 min to achieve a 7.8°C increase for T tail-dist . The intermediate parts of the tail showed a similar evolution whose parameters spread out between those of the extreme parts.

Shortly after vasodilatation of the ipsilateral hind-paw, one sees a decrease in core temperature (T core ) (Fig. 2C) that was accentuated by the later vasodilation of the tail and contralateral hind-paw. Overall, T core was reduced by 0.8°C within the 30 min post-injection period despite the active warming that remained stable all over the experiment. The Heat Loss Indexes are shown in figure 2D for each site of T skin skin recording.

MAP (Fig. 2E) and HR (Fig. 2F) were also affected by muscimol. Both parameters dropped and stabilized within 8 min, that is a few minutes earlier than the vasodilatation. At 30 min, the final MAP and HR were 36% and 20% less than during the control period, respectively. These variations were associated with a decrease of ETCO 2 (Fig. 2G).

From such an example, one can infer that microinjection of muscimol centered on the RMg is able to block the drive of the sympathetic control of vasomotion of the hind-paws and the tail, leading to an increase in their skin temperature and a subsequent decrease in T core . The effects were dominant on the part of the body ipsilateral to the microinjection site. These variations were associated with a decrease in MAP, HR and ETCO 2 .

Overall effect of muscimol microinjection within the RVM/rMR on the vasomotion of the hind-paws

During the control period, both hind-paws were vasoconstricted at a stable skin temperature, close to the ambient temperature. The microinjection of muscimol in the RVM/rMR elicited an increase of the plantar skin temperature, indicating increasing blood flow. This reaction always started first in the ipsilateral hind-paw. Following adjustment of each individual curve to a sigmoid by a Boltzmann regression (see Fig. 1AB), the experiments were divided in three groups on the basis of the distribution of the beginning (t min ) of the ascending phase of vasodilatation at the hind-paw ipsilateral to the microinjection site (Fig. 1C): group 1 (acute onset group: t min < 7.5 min); group 2 (intermediate onset group: 7.5 < t min < 15 min); group 3 (late onset or no response group: t min > 15 min). The corresponding temporal evolutions of adjusted skin temperatures are shown in Figure 3 for the 3 groups. Regarding the two groups showing responses with acute or intermediate onset, the ipsilateral and contralateral curves were essentially parallel, but the response elicited in the latter was postponed by 11-12 minutes. The ascending phase of vasodilatation Δt was short lasting (in the 6-8 min range) and large (in the 7-8°C/min range), therefore steep (in the 1°C/min range). The corresponding numerical data are provided in table 1A.

Figure 4 shows the corresponding localization of the muscimol microinjection sites reported on the atlas of the rat brain by [START_REF] Paxinos | The Rat Brain in Stereotaxic Coordinates[END_REF]. For clarity of presentation, the data related to the hindpaws, ipsilateral and contralateral to the microinjection sites, are shown on the right and left parts of the figure, respectively. The black, grey and white circles represent microinjection sites related to experiments belonging to the first, second and third groups, respectively. Regarding the ipsilateral hind-paw, the most effective sites are located between planes -1.3 and -2.8 mm with reference to the inter-aural line, mainly in raphe pallidus, inner layer of raphe magnus and parapyramidal nucleus (Fig. 4B). These points are surrounded laterally and rostro-caudally by the second group (grey symbols), often localized in the outer layer of the raphe magnus. The non-reactive regions are outside these zones. The effects of microinjection on the contralateral hind-paw (Fig. 4A) were very much dependent on its proximity to the midline.

Microinjections distant from the midline by less than 0.1 mm exhibited an acute onset effect, and those by less than 0.3 mm an intermediate onset effect.

We seized the opportunity of the large number of microinjection sites to build a three-dimensional mapping of response latencies as a function of injection site, using interpolation (Fig. 5). Since the effects were clearly lateralized, the responses obtained from the left and right paw were merged, by expressing the location of the injection site as ipsilateral (positive x-axis) vs. contralateral (negative x-axis) relative to the paw. The earliest responses latencies were observed between planes z = -1.3 and z = -2.8 mm both from the midline (raphe pallidus and raphe magnus) and a more lateral part centered on the lateral paragigantocellular nucleus (LPGi) and the Parapyramidal nucleus (PPy).

Overall effect of muscimol microinjection within the RVM/rMR on the vasomotion of the tail

During the control period, the tail was vasoconstricted, with a decreasing thermal gradient from T tail- prox to T tail-dist [27.1 (26.9-27.3) vs. 24.9 (24.7-25.1)°C; P < 0.001]. The microinjection of muscimol within the RVM/rMR elicited an increase of the tail skin temperature. However, a proximal-distal gradient was also seen regarding the reactivity of the tail. Knowing these gradients, we analyzed the proximal and distal parts of the tail, separately, using the type of grouping already made but based on the starting point of the acute ascending phase of vasodilatation on the distal part of the tail (1 st group (acute onset group): 0 < t min < 7.5 min; 2 nd group (intermediate onset group): 7.5 < t min < 15 min; 3 rd group (late or none reactive): t min > 15 min), following sigmoidal adjustment of each individual. The corresponding temporal evolutions of adjusted skin temperatures are shown in Figure 6 for the 3 groups. Regarding the first two groups, the corresponding numerical data are provided in table 1B. By comparison with the paws the process was slower (in the 0.3-0.5°C/min range) and less pronounced (in the 4-6°C range), possibly because we recorded the dorsal facet of the tail (see discussion).

Figure 7 shows the corresponding localization of the muscimol microinjection sites for the proximal (Fig. 7A) and the distal (Fig. 7B) parts of the tail. Again, the black grey and white symbols represent the acute, intermediate and late or none reactive group respectively. The black symbols are located mainly between planes -1.3 and -2.8 mm, close to the midline, in the raphe pallidus and raphe magnus. The grey symbols surround the black ones and are located in the raphe magnus and parapyramidal region. In these two groups, the proximal and distal parts of the tail reacted concomitantly. Overall, the proximal part of the tail was more responsive to the microinjection; in particular, one can see several points from which a vasodilatation was elicited on the proximal but not on the distal part of the tail, at least within the 15-min early period. Most of these points were however located in the raphe magnus and parapyramidal nucleus.

Figure 8 represents the three-dimensional mapping of response latencies t min as a function of injection site. For both the proximal (Fig. 8A) and the distal (Fig. 8B) parts of the tail, the earliest response latencies were observed between planes z = -1.3 and z = -2.8 mm from the midline (raphe pallidus and raphe magnus) and a more lateral part. Interestingly, the lateral part centered on the Parapyramidal nucleus was restricted to planes z = -2.3 to -2.8 mm without any obvious participation of the lateral paragigantocellular nucleus.

Comparison of Heat Loss Indexes in various vasomotor states

The potential extent of skin temperature variations was physically restricted to the T amb -T core range.

In terms of thermoregulation, these changes are best described using the Heat Loss Index as it eliminates the passive changes and range limitation due to any T amb and T core variations [START_REF] Gordon | Rat tail skin temperature monitored noninvasively by radiotelemetry: characterization by examination of vasomotor responses to thermomodulatory agents[END_REF][START_REF] Romanovsky | Selected contribution: ambient temperature for experiments in rats: a new method for determining the zone of thermal neutrality[END_REF][START_REF] Székely | Skin temperature-skin blood flow: assessment of thermoregulatory changes[END_REF]): HLI = (T skin -T amb )/(T core -T amb ). The HLI values in the control period of the present study were close to those recorded during vasoconstrictions in our previous study and identical in terms of ranking (T tail-dist < T paw-ipsi < T tail-prox ).

However, the HLI values following muscimol microinjection were 20-25% lower than the HLI seen during the maximal physiological vasodilatation achieved during thermo-neutrality. This observation suggests that the ongoing hind-paws and tail fiber sympathetic activity was not completely silenced following the muscimol microinjections, in spite of an apparent ceiling effect.

Overall results, including the effects on vital signs

In order to homogenize the results for further analyzes, the experiments were reorganized in three new groups, again on the basis of the onset time t min of the ascending phase of vasodilatation (Fig. 9). The first includes experiments where t min was < 15 min for all considered skin areas either on the tail or the paws (black circles in figure 9A). They were located between planes -0.8 and -2.8 mm, not more lateral than 0.3 mm from the midline, mainly in the inner part of raphe magnus and raphe pallidus. The second includes experiments where t min was < 15 min for at least one but not all the areas considered on the tail or the hindpaws (grey circles in figure 9A). They were located in the raphe magnus and parapyramidal area, surrounding the preceding group. The third group includes experiments where t min was > 15 min for all considered skin areas either on the tail or the paws (white circles in figure 9A). The corresponding effects of microinjection of muscimol on T paw-ipsi , T paw-contra , T tail-prox and T tail-dist are summarized in figures 9B, C, D and E, respectively.

Figure 9F shows the concomitant effects on T core . There was no statistical difference among the three groups concerning T core during the control period (P = 0.94). After the muscimol injection, the mean temperatures diverged strongly with progressive drops for the 1 st and 2 nd group. The effects of muscimol microinjections on MAP, HR and ETCO 2 are presented in Figures 9G, H andI, respectively. For all three groups, we report a transitory increase of MAP and HR in the first minute following the microinjection of muscimol. Thereafter, the curves declined slowly and slightly till the end of the experiments. Regarding the first two groups, the corresponding numerical data are provided in table 3. These results are in line with previous reports in anesthetized rats following microinjection of muscimol in the RVM/rMR, describing slight or non-significant decreases in MAP, HR and ETCO 2 [START_REF] Bernard | Critical role of B3 serotonergic cells in baroreflex inhibition during the defense reaction triggered by dorsal periaqueductal gray stimulation[END_REF][START_REF] Blessing | Raphe magnus/pallidus neurons regulate tail but not mesenteric arterial blood flow in rats[END_REF][START_REF] Nakamura | Central efferent pathways mediating skin cooling-evoked sympathetic thermogenesis in brown adipose tissue[END_REF][START_REF] Tanaka | Independent vasomotor control of rat tail and proximal hairy skin[END_REF]Zaretsky et al. 2003a;2003b). Note that our recordings were made under an anesthetic regime that preserves withdrawal reflexes while most earlier reports were obtained with deeper regimes that could have masked the effects reported here.

Modeling the effect of muscimol injection within the RVM/rMR on the tail-flick test

In a previous study, we proposed and verified experimentally a simple model to compute the expected tail-flick latency (TFL) of a rat exposed to a source of radiant heat applied onto the tail [START_REF] Benoist | Peripheral and central determinants of a nociceptive reaction: an approach to psychophysics in the rat[END_REF]. The model takes into account the power of the radiant heat source, the initial skin temperature, the core temperature and the site of stimulation on the tail, and has been applied successfully to reconstruct TFLs following a conditioned stress response [START_REF] Carrive | A reassessment of stress-induced "analgesia" in the rat using an unbiased method[END_REF]. Here, the model was used to compute the predictable variations of TFL introduced by muscimol. Decisional and motor latencies were estimated to be 2A in [START_REF] Benoist | Peripheral and central determinants of a nociceptive reaction: an approach to psychophysics in the rat[END_REF]]. The predictive model of TFL was fully verified following variations of the radiant heat source (i.e. α) or the basal temperature of the skin (i.e. T mid-tail here) [see Fig. 8 in [START_REF] Benoist | Peripheral and central determinants of a nociceptive reaction: an approach to psychophysics in the rat[END_REF]]. In the classical tail-flick test, the principal source of variation introduced by experimenters is the power of the electrical bulb used for heating the skin of the animal. The investigator adjusts the radiant heat emission with a rheostat to achieve a predetermined TFL in the control situation, most commonly in the 2-4 seconds range (Le Bars et al. 1999[START_REF] Bars | Current animal test and models of pain[END_REF]). In the model, such latencies are achieved for α in the 0.08-0.2°C 2 /s.

The model was applied to the data of animals in which the beginning of the ascending phase of vasodilatation was < 15 min either on the tail or the paws (black group in figure 9). The model foretells a 30-35% decrease of the TFL, thirty minutes following muscimol administration (Fig. 10). It appears therefore that, regardless of any other additional possible causes, the vasodilatation of the tail is a major source of variation of the TFL following muscimol administration in RVM/rMR.

DISCUSSION

Muscimol was injected in the lower brainstem with the aim of assessing the role of the RVM/rMR on the control of sympathetic drive to the tail and hind-paws. Microinjections within this brainstem region increased the skin temperature of the hind-paws and tail as a result of reduced vasomotor tone. The effects were dominant on the hind-paw ipsilateral to the microinjection site. Increased heat loss through vasodilatation of the tail and paws were associated with a drop of T core and slight decreases of MAP, HR and ETCO 2 .

Following some technical considerations regarding the microinjection procedure, we will discuss the following points: (1) general findings;

(2) lateral localization of effective sites;

(3) rostro-caudal localization of effective sites; (4) pharmacological manipulations of RVM/rMR in pain studies.

Technical considerations regarding the microinjection procedure

The kinetic and volume of efficient microinjection is determined by numerous factors: the concentration, rate of delivery and properties of both the drug -coefficient of diffusion, binding, catabolismand the tissue -volume fraction, tortuosity - [START_REF] Syková | Diffusion in brain extracellular space[END_REF]. In nuclear and cortical homogenous regions, drug distributions are typically spherical or in the shape of drop [START_REF] Bondareff | Intrastriatal spreading of biogenic amines[END_REF][START_REF] Martin | Autoradiographic estimation of the extent of reversible inactivation produced by microinjection of lidocaine and muscimol in the rat[END_REF][START_REF] Myers | Injection of solutions into cerebraltissue: relation between volume and diffusion[END_REF]).

In a "mapping" study aiming at demarcating a functional region in the brain, both positive and negative results are significant. A negative result obtained with a large dose is a very convincing result in this respect. Considering the large volume of the explored brain structure (roughly 3 x 4 x 2 = 24 mm 3 ), a compromise was necessary to both find significant positive results with minimal doses and to avoid useless negative results. Studies on the involvement of these regions in pain/nociception and thermoregulation have used muscimol injections in the 10-1000 pmol / 60-1000 nl ranges [START_REF] Bernard | Critical role of B3 serotonergic cells in baroreflex inhibition during the defense reaction triggered by dorsal periaqueductal gray stimulation[END_REF][START_REF] Blessing | Raphe magnus/pallidus neurons regulate tail but not mesenteric arterial blood flow in rats[END_REF][START_REF] Brink | Raphe magnus neurons help protect reactions to visceral pain from interruption by cutaneous pain[END_REF][START_REF] Cao | Medullary pathways mediating specific sympathetic responses to activation of dorsomedial hypothalamus[END_REF][START_REF] Cao | Inhibition of brown adipose tissue thermogenesis by neurons in the ventrolateral medulla and in the nucleus tractus solitarius[END_REF][START_REF] Cerri | Cutaneous vasodilation elicited by disinhibition of the caudal portion of the rostral ventromedial medulla of the freebehaving rat[END_REF]Fan et al. 2007. Gilbert and[START_REF] Gilbert | GABAergic modulation of descending inhibitory systems from the rostral ventromedial medulla (RVM). Dose-response analysis of nociception and neurological deficits[END_REF][START_REF] Heinricher | GABA-mediated inhibition in rostral ventromedial medulla: role in nociceptive modulation in the lightly anesthetized rat[END_REF][START_REF] Martenson | A possible neural basis for stress-induced hyperalgesia[END_REF][START_REF] Meng | An analgesia circuit activated by cannabinoids[END_REF][START_REF] Morrison | RVLM and raphe differentially regulate sympathetic outflows to splanchnic and brown adipose tissue[END_REF][START_REF] Morrison | Raphe pallidus neurons mediate prostaglandin E2-evoked increases in brown adipose tissue thermogenesis[END_REF][START_REF] Nakamura | Central circuitries for body temperature regulation and fever[END_REF]Morrison 2007, 2011;[START_REF] Ootsuka | Interactive drives from two brain stem premotor nuclei are essential to support rat tail sympathetic activity[END_REF][START_REF] Rathner | Central pathway for spontaneous and prostaglandin E 2 -evoked cutaneous vasoconstriction[END_REF][START_REF] Vianna | Cardiovascular and behavioral responses to conditioned fear after medullary raphe neuronal blockade[END_REF]Zaretsky et al. 2003aZaretsky et al. , 2003b)). Knowing that muscimol does not spread appreciably because it is a potent ligand for neuronal and glial GABA A receptors, we decided to use a small volume of injection (50 nl = 0.05 mm 3 , that is a ~0.5 mm diameter for an ideal sphere) but a relatively high concentration. This was supposed to provide an effective radius of blockade in the ~ 1 mm range [START_REF] Martin | Autoradiographic estimation of the extent of reversible inactivation produced by microinjection of lidocaine and muscimol in the rat[END_REF][START_REF] Martin | Pharmacological inactivation in the analysis of the central control of movement[END_REF][START_REF] Malpeli | Reversible inactivation of subcortical sites by drug injection[END_REF][START_REF] Edeline | Muscimol diffusion after intracerebral microinjections: a reevaluation based on electrophysiological and autoradiographic quantifications[END_REF]. Following similar microinjections using [ 3 H]muscimol, autoradiographic analyses showed a spread ~1.5 mm from the injection site at 15 min [START_REF] Edeline | Muscimol diffusion after intracerebral microinjections: a reevaluation based on electrophysiological and autoradiographic quantifications[END_REF][START_REF] Martin | Autoradiographic estimation of the extent of reversible inactivation produced by microinjection of lidocaine and muscimol in the rat[END_REF]. [START_REF] Martin | Pharmacological inactivation in the analysis of the central control of movement[END_REF] used glucose metabolism to assess the extent of inactivation and observed a central core of blockade (~ 1 mm radius) surrounded by an extensive region of reduced metabolism, possibly due to reduced synaptic activity of neurons receiving projections from the core region. In summary, muscimol binds strongly and diffuses slowly outward at effective concentrations from the region immediately inundated by the bulk flow [START_REF] Malpeli | Reversible inactivation of subcortical sites by drug injection[END_REF].

Since myelinated fiber bundles impede muscimol diffusion [START_REF] Allen | Imaging the spread of reversible brain inactivations using fluorescent muscimol[END_REF], it is likely that the particular architecture of the RVM/rMR with a predominance of rostro-caudal fibers and a low neuronal density favors a rostro-caudal diffusion of the product. In addition, the two paired bundles of myelinated fibers that cover the brainstem floor, namely the pyramidal tract and the medial lemniscus, constitute a strong diffusion barrier. Accordingly, the post-mortem examination of pontamine spread revealed an elongated pattern of diffusion two times longer in the rostro-caudal direction as compared to the coronal plane.

Using these microinjections, we were able to build response maps on the basis of 160 experiments.

Together with the small volume of injection and the large number of negative sites of injection (60%), this approach provided (1) an acute delineation of the structures able to block sympathetic cutaneous vasoconstrictor activity;

(2) the possibility of a temporal analysis of these effects; and (3) the resultant capacity of defining a brain region as the core of effects, surrounded by intermediate and then ineffective zones.

Because of such a large number of injection sites, we introduced a new procedure to compute threedimensional interpolated maps of response latencies and overall sympathetic blockade as a function of injection site. The interpolation was performed by computing an average of the responses obtained at all injection sites, weighted by the distance between the interpolated voxel and each injection site. This ensured that the interpolated values were most dependent on the responses of the nearest injection sites. In addition, because of the non-homogeneous sampling of the interpolated volume, a mask was used to exclude voxels that were not close to at least one injection point. The obtained volumes provide an interesting mean to assess the relationship between the effects of the injection and the anatomical location of the injection.

General findings

Our results are largely in agreement with earlier reports regarding the sympathetic drive of the tail.

Microinjection of glycine, GABA or muscimol in the RVM/rMR blocks the activation by cold or fever of sympathetic fibers that innervate the tail [START_REF] Blessing | Raphe magnus/pallidus neurons regulate tail but not mesenteric arterial blood flow in rats[END_REF][START_REF] Cerri | Cutaneous vasodilation elicited by disinhibition of the caudal portion of the rostral ventromedial medulla of the freebehaving rat[END_REF][START_REF] Korsak | Rostral ventromedial medulla and the control of cutaneous vasoconstrictor activity following i.c.v. prostaglandin E 1[END_REF][START_REF] Ootsuka | Interactive drives from two brain stem premotor nuclei are essential to support rat tail sympathetic activity[END_REF][START_REF] Ootsuka | Inhibition of rostral medullary raphé neurons prevents coldinduced activity in sympathetic nerves to rat tail and rabbit ear arteries[END_REF][START_REF] Rathner | Central pathway for spontaneous and prostaglandin E 2 -evoked cutaneous vasoconstriction[END_REF][START_REF] Vianna | Cardiovascular and behavioral responses to conditioned fear after medullary raphe neuronal blockade[END_REF]). On the other hand, microinjection of glutamate or bicuculline increases the activity of vasomotor sympathetic nerves of the tail, thus decreasing the blood flow, without affecting the mesenteric vascular bed [START_REF] Blessing | Raphe magnus/pallidus neurons regulate tail but not mesenteric arterial blood flow in rats[END_REF][START_REF] Morrison | Differential regulation of brown adipose and splanchnic sympathetic outflows in rat: roles of raphe and rostral ventrolateral medulla neurons[END_REF][START_REF] Rathner | Differential control of sympathetic drive to the rat tail artery and kidney by medullary premotor cell groups[END_REF]. Several aspects of our contribution should be highlighted.

The experimental conditions were close to those of thermoneutrality (El Bitar et al. 2014a). T warm was adjusted to ~ 0.3°C below T core . Demonstrating their vasoconstricted state, the temperature of the distal part of the tail was very close to the ambient temperature (~ 0.7°C above T ambient ). It then increased by ~ 6°C

following the injection of muscimol in responsive brainstem sites. In comparable experimental conditions, microinjection of GABA or muscimol in RVM/rMR blocks the activity of the sympathetic fibers innervating the rat tail [START_REF] Korsak | Rostral ventromedial medulla and the control of cutaneous vasoconstrictor activity following i.c.v. prostaglandin E 1[END_REF][START_REF] Ootsuka | Interactive drives from two brain stem premotor nuclei are essential to support rat tail sympathetic activity[END_REF].

We have previously discussed (El Bitar et al. 2014a) that the vasomotor tone of the dorsal facet of the tail is less reactive than the ventral facet, notably because the ventral artery is larger than the lateral arteries, while the ventral vein is thinner than the lateral veins [START_REF] Knoppers | La queue du rat, témoin de la régulation thermique[END_REF][START_REF] Rwjr | The biology of rodent tails: a study of form and function[END_REF][START_REF] Wu | A non-uniform three-dimensional perfusion model of rat tail heat transfer[END_REF][START_REF] Young | Evidence for on-off control of heat dissipation from the tail of the rat[END_REF]. It follows that the temperatures of the dorsal and ventral facets of the tail are identical during vasoconstriction but shift by ~ 3°C during full vasodilatation. The recorded effects from the tail were thus underestimated, as compared to those seen on the hind paws.

A major novelty of our study was to include the paws to these basic phenomenological observations. Such as for the tail, the hind-paws were in a stable vasoconstricted state, as demonstrated by the fact that the temperature of the hind-paws was close to the ambient temperature (2-3°C above T ambient ). This increased by ~ 7°C following the injection of muscimol in the active sites. We have already discussed the involvement of the paws in rat thermoregulation (El Bitar et al. 2014a). The feet make up approximately 10% of the total surface area of the body, slightly more than the tail [START_REF] Lin | Studies on thermoregulation in the rat[END_REF]. It follows that the substantial muscimolinduced vasodilatation of the paws contributed significantly to the core temperature drop (~ 1°C within half an hour).

Our purpose was not to confirm well-documented notions. We specifically aimed at mapping the brainstem regions that contain the premotor sympathetic neurons that control the vasomotor tone of the tail and hind-paws because most models of pain/nociception are based on behavioral responses elicited by thermal stimulation (reviewed in Le [START_REF] Bars | Animal models of nociception[END_REF][START_REF] Bars | Current animal test and models of pain[END_REF]. We undertook this large study because studies on pain/nociception have emphasized the Rostral Ventromedial Medulla (RVM), which includes the nucleus raphe magnus and the gigantocellular reticular nucleus pars alpha (mainly at the level of the facial nucleus), while studies on thermoregulation have emphasized a slightly shifted more caudal region centered on the raphe pallidus. In addition, we described in a previous work that neurons involved in pain/nociception located in the brainstem are probably also implicated in autonomic regulation, notably cutaneous vasomotion (El Bitar et al. 2014b).

The results of the present study suggest that the regions described as being involved in pain/nociception modulation and the regions involved in thermoregulation are spatially matched, at least functionally regarding the vasomotor tone of the tail and hind-paws.

Localization of effective sites in coronal planes

The most effective sites were restricted to well-circumscribed regions. Numerous negative microinjections sharpened the delineation. Our observations regarding the tail are in keeping with the mapping by [START_REF] Korsak | Rostral ventromedial medulla and the control of cutaneous vasoconstrictor activity following i.c.v. prostaglandin E 1[END_REF] who reported that GABA microinjections in the region of raphe pallidus and magnus markedly decreased the sympathetic cutaneous vasoconstrictor activity in the dorsal collector nerves of the tail, while injections more dorsal or lateral tended to produce either a smaller decrease or have no effect. [START_REF] Blessing | Raphe magnus/pallidus neurons regulate tail but not mesenteric arterial blood flow in rats[END_REF] also emphasized the raphe magnus, raphe pallidus and parapyramidal nucleus in this respect, although they did not provide a graphical mapping of the effects.

Only microinjections in RMg, RPa and PPy elicited a vasodilatation in the ipsilateral hind-paw within less than 7.5 min and those distant from the midline by less than 0.3 mm induced a vasodilatation in the contralateral hind-paw within the same period. These observations deserve two comments. First, within the coronal plane, the lateral diffusion of muscimol was limited, as expected. Second, projections of RVM/rMR cells are mainly lateralized, predominantly controlling vasomotor tone of the ipsilateral body side. Our results are in keeping with studies showing that RVM/rMR neurons send axons towards the spinal cord mainly through the ipsilateral dorso-lateral funiculus [START_REF] Basbaum | The origin of descending pathways in the dorsolateral funiculus of the spinal cord of the cat and rat: further studies on the anatomy of pain modulation[END_REF][START_REF] Fields | Dorsal horn projection targets of ON and OFF cells in the rostral ventromedial medulla[END_REF][START_REF] Lefler | Bulbospinal neurons of the rat rostromedial medulla are highly collateralized[END_REF][START_REF] Light | The spinal terminations of single, physiologically characterized axons originating in the pontomedullary raphe of the cat[END_REF]. Microinjections close to the midline also elicited early vasodilatation of the tail, the most effective sites being restricted to the same well-circumscribed regions. Neurons in the PPy present morphological, histo-chemical [START_REF] Helke | Chemical neuroanatomy of the parapyramidal region of the ventral medulla in the rat[END_REF][START_REF] Lynn | Thyrotropin-releasing hormone-immunoreactive projections to the dorsal motor nucleus and the nucleus of the solitary tract of the rat[END_REF]) and functional [START_REF] Blessing | Cutaneous vascular bed is not involved in arterial pressure changes elicited by increasing or decreasing the activity of inhibitory vasomotor neurons in caudal ventrolateral medulla in rabbits[END_REF] properties similar to those of the RPa. Some authors consider therefore PPy as part of RPa (e.g. [START_REF] Cano | Anatomical substrates for the central control of sympathetic outflow to interscapular adipose tissue during cold exposure[END_REF].

Interestingly, brainstem neurons activated both antidromically from the lumbar dorso-lateral funiculus and by mild cooling of the animal core temperature, were found in both raphe pallidus and magnus [START_REF] Rathner | Cold-activated raphe-spinal neurons in rats[END_REF]. Such neurons could be involved in the results presented here. However, there are other control loops that regulate the central temperature through the vessels in the hairy skin, the interscapular brown adipose tissue (BAT) and the fusimotor fibers to limb muscles, all being silenced by neuronal inhibition of the raphe pallidus and/or magnus [START_REF] Mcallen | Multiple thermoregulatory effectors with independent central controls[END_REF]. Although they all should have a sympathetic premotor relay in these nuclei, these four sympathetic thermo-effector outflows differ in terms of thermal thresholds, relative responsiveness to core temperature, patterns and driving by neural pathways, and thus probably do not "crosstalk" at this level [START_REF] Mcallen | Multiple thermoregulatory effectors with independent central controls[END_REF][START_REF] Nagashima | Neuronal circuitries involved in thermoregulation[END_REF][START_REF] Ootsuka | Comparison between two rat sympathetic pathways activated in cold defense[END_REF][START_REF] Romanovsky | Thermoregulation: some concepts have changed. Functional architecture of the thermoregulatory system[END_REF][START_REF] Tanaka | Independent vasomotor control of rat tail and proximal hairy skin[END_REF]). The relative contribution of single individual neurons to these functions remains an open question.

Rostro-caudal localization of effective sites

Overall, the injection sites which elicited the most widespread and early latency vasodilation were located in the coronal planes posterior to the interaural line by 1.3-2.6 mm. 85% of experiments where the vasodilatation of all areas either on the tail or the paws occurred in less than 15 min were found in these regions. Again our observations agree with the report by [START_REF] Korsak | Rostral ventromedial medulla and the control of cutaneous vasoconstrictor activity following i.c.v. prostaglandin E 1[END_REF] showing a similar rostrocaudal extension of their effective sites for blocking by GABA the sympathetic vasoconstrictor drive in the tail. Similarly, brainstem neurons both antidromically activated from the lumbar dorso-lateral funiculus and activated by mild cold were found in the raphe nuclei between the coronal planes posterior to the interaural line by 1.8-2.3 mm [START_REF] Rathner | Cold-activated raphe-spinal neurons in rats[END_REF].

Overall, the literature related to thermoregulation points to coronal planes immediately rostral to the rostral pole of the inferior olivary complex at the level of the caudal half of the facial nucleus. This brainstem region is often referred as to the rostral raphe pallidus, although it generally does include the nucleus raphe magnus (e.g. Madden andMorrison 2003, 2005;[START_REF] Morrison | RVLM and raphe differentially regulate sympathetic outflows to splanchnic and brown adipose tissue[END_REF][START_REF] Morrison | Raphe pallidus neurons mediate prostaglandin E2-evoked increases in brown adipose tissue thermogenesis[END_REF][START_REF] Morrison | GABA-mediated inhibition of raphe pallidus neurons regulates sympathetic outflow to brown adipose tissue[END_REF][START_REF] Morrison | Reduced rearing temperature augments responses in sympathetic outflow to brown adipose tissue[END_REF][START_REF] Rathner | Central pathway for spontaneous and prostaglandin E 2 -evoked cutaneous vasoconstriction[END_REF]. In fact, very few studies were devoted to a systematic mapping.

As pointed out by Nason and [START_REF] Nason Mwjr | Modulation of sympathetic and somatomotor function by the ventromedial medulla[END_REF], it is unlikely that a microinjection into any single nucleus in RVM/rMR will affect neurons confined to that nucleus because these neurons have large dendritic arbors that cross cytoarchitectonic boundaries [START_REF] Gao | Somatodendritic and axonal anatomy of intracellularly labeled serotonergic neurons in the rat medulla[END_REF][START_REF] Mason | Somatodendritic morphology of on-and off-cells in the rostral ventromedial medulla[END_REF][START_REF] Newman | Distinguishing rat brainstem reticulospinal nuclei by their neuronal morphology. I. Medullary nuclei[END_REF][START_REF] Potrebic | Three-dimensional analysis of the dendritic domains of on-and off-cells in the rostral ventromedial medulla[END_REF]. [START_REF] Blessing | Lower brainstem pathways regulating sympathetically mediated changes in cutaneous blood flow[END_REF] discussed the conventional anatomical demarcation into RMg and RPa by raising the following points. The delimitation of boundaries is based particularly on the work of [START_REF] Taber | The raphe nuclei of the brain stem in the cat. I. Normal topography and cytoarchitecture and general discussion[END_REF] that follows the atlas of [START_REF] Meessen | A Cytoarchitectonic Atlas of the Rhombencephalon of the Rabbit[END_REF] for rabbits and the atlas of [START_REF] Olszewski | Cytoarchitecture of the Human Brain Stem[END_REF] for humans. However, the rabbit atlas combines all the ventral midline neurons in the rostral medulla and caudal pons as "RMg" and does not use the term "RPa." In contrast, the human atlas uses "RPa" and not "RMg". However, by convention, RMg in the rat is more rostral and dorsal, and

RPa is more caudal and ventral [START_REF] Paxinos | The Rat Brain in Stereotaxic Coordinates[END_REF].

We noted that the Heat loss Indexes following muscimol microinjection were 20-25% lower than the HLI seen during the maximal physiological vasodilatation achieved in rats maintained in thermo-neutral conditions (El Bitar et al. 2014a), suggesting that the ongoing hind-paws and tail fiber sympathetic activities were not completly silenced following the microinjections, in spite of a clear ceiling effect when microinjections were effective. The most parsimonious explanation for this observation is the oblong geometry of the involved brainstem structures, ~12 times longer than the diameter of the injected volume, preventing the drug to reach all structures potentially involved in controling vasomotor tone.

In summary, there are morphological and functional reasons to consider the RVM and rMR nuclei as a single RVM/rMR entity, as already proposed by [START_REF] Mason | Contributions of the medullary raphe and ventromedial reticular region to pain modulation and other homeostatic functions[END_REF]Mason et al. ( , 2005aMason et al. ( , 2005b[START_REF] Mason | Descending pain modulation as a component of homeostasis[END_REF][START_REF] Mason | From descending pain modulation to obesity via the medullary raphe[END_REF].

Is the RVM really specific for pain/nociception?

Our study shows, in the rat, a mutual covering of the regions involved in thermoregulation (at least, regarding the vasomotor tone of the tail and hind-paws) with those reported as being specifically involved in pain/nociception.

In spite of several warnings initiated by Thelma Lovick and Peggy Mason (Le [START_REF] Bars | Animal models of nociception[END_REF][START_REF] Lefler | Bulbospinal neurons of the rat rostromedial medulla are highly collateralized[END_REF][START_REF] Lovick | The medullary raphe nuclei: a system for integration and gain control in autonomic and somatomotor responsiveness?[END_REF][START_REF] Mason | Contributions of the medullary raphe and ventromedial reticular region to pain modulation and other homeostatic functions[END_REF]2005a;2005b;[START_REF] Mason | Descending pain modulation as a component of homeostasis[END_REF]2011;[START_REF] Mason | Medullary circuits for nociceptive modulation[END_REF], the specificity of the RVM regarding the control of pain/nociception mechanisms is repeatedly put forward or assumed (e.g. [START_REF] Fields | Central nervous system mechanisms of pain modulation[END_REF][START_REF] Heinricher | The brainstem and nociceptive modulation[END_REF][START_REF] Basbaum | The endogenous neuromodulation system[END_REF][START_REF] Heinricher | Descending control of nociception: Specificity, recruitment and plasticity[END_REF]). RVM neurons do not only project towards the dorsal horn of the spinal cord [START_REF] Fields | Brainstem control of spinal pain-transmission neurons[END_REF]1999), but also to the intermediolateral cell column, with a high degree of collateralization in both the rostro-caudal and dorsoventral axes [START_REF] Bacon | Electron microscopic evidence of a monosynaptic pathway between cells in the caudal raphe nuclei and sympathetic preganglionic neurons in the rat spinal cord[END_REF][START_REF] Basbaum | Three bulbospinal pathways from the rostral medulla of the cat: an autoradiographic study of pain modulating systems[END_REF][START_REF] Hossaini | Distribution of glycine/GABA neurons in the ventromedial medulla with descending spinal projections and evidence for an ascending glycine/GABA projection[END_REF][START_REF] Lefler | Bulbospinal neurons of the rat rostromedial medulla are highly collateralized[END_REF][START_REF] Loewy | Raphe pallidus and raphe obscurus projections to the intermediolateral cell column in the rat[END_REF][START_REF] Morrison | Axonal branching patterns and funicular trajectories of raphespinal sympathoinhibitory neurons[END_REF].

Numerous neurons in RVM/rMR were identified as sympathetic premotor neurons by early retrograde trans-synaptic labeling with the pseudo-rabies virus [START_REF] Smith | CNS cell groups projecting to sympathetic outflow of tail artery: neural circuits involved in heat loss in the rat[END_REF][START_REF] Strack | A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections[END_REF].

Quantitative data, obtained from 6 days survival experiments, revealed ~5 times more labeled neurons in raphe magnus and gigantocellular reticular pars alpha nuclei than within the raphe palidus nucleus [START_REF] Smith | CNS cell groups projecting to sympathetic outflow of tail artery: neural circuits involved in heat loss in the rat[END_REF]. [START_REF] Smith | CNS cell groups projecting to sympathetic outflow of tail artery: neural circuits involved in heat loss in the rat[END_REF] found the highest density at the level of the facial nucleus but the coronal planes facing the rostral part of the facial nucleus were not represented. Therefore, the neurons labeled in that study could have been mainly located in the RVM/rMR at the level of facial nucleus, although we cannot infer their rostral extent. However, it was stated more recently that transneuronally labeled neurons are distributed through the parapyramidal region, including all RVM/rMR elements [START_REF] Tóth | Serotonin-synthesizing neurons in the rostral medullary raphé/parapyramidal region transneuronally labelled after injection of pseudorabies virus into the rat tail[END_REF], at least within the coronal planes from 1.3 to 3.3 mm posterior to the interaural line [START_REF] Nakamura | Identification of sympathetic premotor neurons in medullary raphe regions mediating fever and other thermoregulatory functions[END_REF].

Finally, cold exposure increases the expression of Fos immunoreactivity not only in RPa and PPy [START_REF] Bonaz | Induction of Fos immunoreactivity in the rat brain after cold-restraint induced gastric lesions and fecal excretion[END_REF][START_REF] Cano | Anatomical substrates for the central control of sympathetic outflow to interscapular adipose tissue during cold exposure[END_REF]Martinez et al. 2001;[START_REF] Morrison | GABA-mediated inhibition of raphe pallidus neurons regulates sympathetic outflow to brown adipose tissue[END_REF]) but also in RMg, between the coronal planes posterior to the interaural line by 1.3-3.3 mm [START_REF] Nakamura | Identification of sympathetic premotor neurons in medullary raphe regions mediating fever and other thermoregulatory functions[END_REF].

If one moves back to the literature related to nociception, namely the so-called "on-" and "off-" cells recorded within the medial brainstem, one is struck by the similar rostro-caudal distribution of the sites, all pointing out the coronal planes including the facial nucleus. Interestingly, [START_REF] Vanegas | Tail-flick related activity in medullospinal neurons[END_REF] attempted to map "on-" and "off-" neurons that project to the spinal cord, and found that these were located in brain regions 1.9-2.7 mm posterior to the interaural line. In summary, the most effective sites for blocking by muscimol the cutaneous vasoconstrictor fibers matches exactly the brainstem sites where "on-" and "off-" cells were recorded over the last years, notably those projecting to the spinal cord.

Pharmacological manipulations of RVM/rMR in pain studies

Administration of muscimol exclusively within RVM/rMR results in a significant decrease in TFL while administration of the competitive GABA A receptor antagonist bicuculline (but not the glycine receptor antagonist strychnine) induces an increase of the TFL [START_REF] Drower | GABAergic modulation of nociceptive threshold: effects of THIP and bicuculline microinjected in the ventral medulla of the rat[END_REF][START_REF] Gilbert | GABAergic modulation of descending inhibitory systems from the rostral ventromedial medulla (RVM). Dose-response analysis of nociception and neurological deficits[END_REF][START_REF] Heinricher | GABA-mediated inhibition in rostral ventromedial medulla: role in nociceptive modulation in the lightly anesthetized rat[END_REF][START_REF] Heinricher | Interference with GABA transmission in the rostral ventromedial medulla: disinhibition of off-cells as a central mechanism in nociceptive modulation[END_REF][START_REF] Nason Mwjr | Modulation of sympathetic and somatomotor function by the ventromedial medulla[END_REF]). Similar results have been reported with paw withdrawal and hot-plate tests [START_REF] Gilbert | GABAergic modulation of descending inhibitory systems from the rostral ventromedial medulla (RVM). Dose-response analysis of nociception and neurological deficits[END_REF][START_REF] Martenson | A possible neural basis for stress-induced hyperalgesia[END_REF]).

These increases or decreases of TFL are generally interpreted in terms of hypo-or hyper-algesia, respectively. However, other interpretations are conceivable, available and advisable. The tail-flick test does not achieve the criterion of construct validity because it does not effectively measure the targeted construct, i.e. a quantitative nociceptive response, presumed to reflect an animal's perception of pain (Le [START_REF] Bars | Animal models of nociception[END_REF][START_REF] Bars | Current animal test and models of pain[END_REF]. Indeed, the reaction time measured in the conventional tail-flick test (and all other tests using conventional progressive heating) is the sum of (1) the time to achieve the threshold for the behavioral reaction and (2) the behavioral latency [START_REF] Benoist | Peripheral and central determinants of a nociceptive reaction: an approach to psychophysics in the rat[END_REF][START_REF] Pincedé | Psychophysics of a nociceptive test in the mouse[END_REF]. If the reaction time is the only measured end-point, there is no way of knowing whether the variation was produced by changes of either the basal skin temperature or the threshold temperature, or both. Using a simple model for computing the TFL in the rat, taking into account the power of the radiant heat source, the initial skin temperature, the core temperature and the site of stimulation on the tail [START_REF] Benoist | Peripheral and central determinants of a nociceptive reaction: an approach to psychophysics in the rat[END_REF], we predicted that the change in tail temperature 15 minutes after effective muscimol injection would lead to a 30-35% reduction of the TFL.

Interestingly, [START_REF] Heinricher | GABA-mediated inhibition in rostral ventromedial medulla: role in nociceptive modulation in the lightly anesthetized rat[END_REF] described a decrease of the TFL of approximately 30% at 12 min post-injection of muscimol (50 ng; 0.5 μl), maintained throughout the half an hour observation period, a finding reproduced by [START_REF] Meng | An analgesia circuit activated by cannabinoids[END_REF]. Both observations match remarkably the predicted variations of TFL related to changes in baseline skin temperature. Interestingly, [START_REF] Tjølsen | Animal models of analgesia[END_REF] attributed the reduction of TFL following lesions of the raphe-spinal serotoninergic system to the increase in skin temperature.

It should be noted that such considerations are not restricted to the tests based on the utilization of thermal stimuli. For example, the vasodilatation of the paws induced by high ambient temperatures exacerbates the second phase of the formalin test [START_REF] Rosland | The formalin test in mice: the influence of ambient temperature[END_REF][START_REF] Tjølsen | The formalin test: an evaluation of the method[END_REF]. It follows that the increased responsiveness to formalin injection following muscimol administration in RVM/rMR [START_REF] Gilbert | GABAergic modulation of descending inhibitory systems from the rostral ventromedial medulla (RVM). Dose-response analysis of nociception and neurological deficits[END_REF] could also be largely due to the unavoidable vasodilatation.

Conclusion

The functional blockade of the RVM/rMR by muscimol elicits an important increase of skin temperature of the paws and tail, eliciting a reduction of the core temperature. The effective zones match very exactly the brain regions defined as specifically devoted to the control of nociception. Because changes in skin temperature can have a strong effect on the usual tests of pain using radiant heat such as the TFL, our results indicate that the evidence that pharmacological manipulations of RVM/rMT modulate pain-specific descending modulatory pathways could, actually, be explained by unaccounted changes in vasomotor tone. (T min = 24.2°C), the adjusted maximal skin temperature at the end of the process (T max = 32.4°C), the amplitude of skin temperature variation (ΔT skin = T max -T min = 8.2°C), the inflection point (t X , T x = T min + ΔT skin /2) [(8.8 min, 28.3°C)] of the sigmoid represented by a red point. The beginning (t min ) and end (t max ) of the ascending phase of vasodilatation were then calculated by considering the 10 and 90% percentiles of ΔT skin : t min = t x -Ln(1/0.1-1) * s -1 = 4.9 min and t max = t x -Ln(1/0.9-1) * s -1 = 13.9 min, represented by white Boltzmann sigmoid (see Fig. 1) from the ipsi-(T paw-ipsi ) and contralateral (T paw-contra ) hind-paw skin temperature, respectively. The experiments were divided in three groups on the basis of the distribution of the beginning (t min ) of the ascending phase of vasodilatation from the ipsilateral hind-paw. A. Group 1: t min < 7.5 min). B. Group 2: 7.5 < t min < 15 min. C. Group 3: t min > 15 min. See text and table 1A. Boltzmann sigmoid (see Fig. 1) from the skin temperature of the proximal (T tail-prox ) and distal (T tail-dist ) part of the tail, respectively. The experiments were divided in three groups on the basis of the distribution of the beginning (t min ) of the ascending phase of vasodilatation on the distal part of the tail. A. Group 1: t min < 7.5 min). B. Group 2: 7.5 < t min < 15 min. C. Group 3: t min > 15 min. See text and table 1B. from the proximal part of the tail. F. Variation of core temperature (Δ°C). In the 1 st group, T core declined linearly as early as the first minute post-injection); for the 2 nd group, T core decline is parallel to the 1 st 's but with 15-min delay and the 3 rd group's T core remained stable. G. Variation of mean arterial blood pressure (ΔmmHg). Note a slight transitory increase of MAP in all groups, shortly after the microinjection followed by a sustained drop. This was followed by a slow decline in the first group, stabilization in the 3 rd group blood, while the 2 nd group spread out in-between. H. Corresponding variations of heart rate (Δbpm). I.

Variation of end-tidal CO 2 (Δ%). The corresponding numerical data are provided in table 3.

Figure 10. We proposed and verified experimentally a simple model for computing the TFL in the rat, taking into account the power of the radiant heat source, the initial temperature of the skin, the core temperature or the site of stimulation on the tail [START_REF] Benoist | Peripheral and central determinants of a nociceptive reaction: an approach to psychophysics in the rat[END_REF]). This model is used here to compute the predictable variations of the TFL introduced by muscimol in the positive cases of the present experiments.

Since the decisional and motor latencies were estimated 134 and 4 ms, respectively [START_REF] Benoist | Peripheral and central determinants of a nociceptive reaction: an approach to psychophysics in the rat[END_REF] and the middle of the tail (T mid-tail , blue line). In the control situation, the model provides tail-flick latencies (red lines) in the 3-4 seconds range for α in the 0.08-0.2°C 2 /s range: the model foretells a 30-35% decrease of the TFL following muscimol microinjections in the sites indicated by a black circle in Fig. 9A.

A: paws (see Fig. 3) 3 and6). Results (expressed as means ± confident interval 95 %) obtained following Boltzmann sigmoid regression of individual curves of skin temperature following microinjections of muscimol (see Fig. 1). For both the paws (A) and the tail (B), the experiments were divided in three groups on the basis of the beginning time t min of the ascending phase of vasodilatation. Group 1: t min < 15 min for all skin areas; Group 2: t min < 15 min for at least one skin area; Group 3 (not shown): t min > 15 min for all skin areas (see Fig. 3 and6). Keys: t X = abscissa of the inflection point of the sigmoid, ∆t = duration of the ascending phase of vasodilatation, T min = adjusted skin temperature during the control period, ΔT skin = amplitude of skin temperature variation. ΔT skin /∆t = speed of warming. The regression coefficients R² of the adjusted curves were always highly significant (> 0.98). [START_REF] Gordon | Rat tail skin temperature monitored noninvasively by radiotelemetry: characterization by examination of vasomotor responses to thermomodulatory agents[END_REF][START_REF] Romanovsky | Selected contribution: ambient temperature for experiments in rats: a new method for determining the zone of thermal neutrality[END_REF][START_REF] Székely | Skin temperature-skin blood flow: assessment of thermoregulatory changes[END_REF], represented as means and their 95% confidence interval. Results obtained from current study (a) are compared to a previous study (b) in the extreme vasomotor tone status, namely vasoconstriction (A) and vasodilation (B). During vasoconstriction, there were no significant statistical differences between HLI observed in the two studies. By contrast, the HLI for both hind-paws and following muscimol microinjections were significantly lower than the HLI seen during the physiological vasodilation elicited during thermo-neutrality (El Bitar et al. 2014a).

Table 3 (see Fig. 9). Results (means ± confident interval 95%) expressed in terms of difference to mean value recorded during the control period. (1) The data represented in the five left hand columns were obtained following Boltzmann sigmoid regression of individual curves of skin temperature following microinjections of muscimol (see Fig. 1). The experiments were divided in three groups on the basis of the beginning time t min of the ascending phase of vasodilatation. Group 1: t min < 15 min for all skin areas; Group 2: t min < 15 min for at least one skin area; Group 3 (not shown): t min > 15 min for all skin areas (see Fig. 9B-9E). Keys: T min = adjusted skin temperature during the control period, ΔT skin = amplitude of skin temperature variation, t X = abscissa of the inflection point of the sigmoid, ∆t = duration of the ascending phase of vasodilatation. The regression coefficients R² of the adjusted curves were always highly significant (> 0.98). (2) The data represented in the four right hand columns are the corresponding data related to the vital signs (T core , MAP, HR and ETCO 2 ) recorded 30 minutes following the microinjection of muscimol (see Fig. 9F-9I). The mean values for T core , MAP, HR and ETCO 2 during the control period were [83][84][85][86][87][88][89][90][320][321][322][323][324][325][326][327][328][329][330][331][332])%, respectively. There was no statistical difference among the three groups concerning T core , MAP, HR and ETCO 2 during the control period (P = 0.78, 0.25, 0.77 and 0.63, respectively).

  temperature at the end of the process of vasodilatation (°C) T min Adjusted skin temperature during the control period (°C) T paw-contra Temperature of the plantar aspect of the hind-paw, contralateral to the injection site (°C) T paw-ipsi Temperature of the plantar aspect of the hind-paw, ipsilateral to the injection site (°C) T skin Temperature of the skin (°C) T tail-dist Temperature of the distal part of the tail (°C) T tail-mid Temperature of the mid part of the tail (°C) T tail-prox Temperature of the proximal part of the tail (°C) T x Ordinate of the inflection point of Boltzmann sigmoid (°C) tail-dist Distal area of the tail, located at 3 cm from the tip tail-mid Intermediate area of the tail, located at mid-tail tail-prox Proximal area of the tail, located at 3 cm from the root of the tail TFL Tail-flick latency (s) T tail-mid Temperature of the mid part of the tail (°C) T tail-prox Temperature of the proximal part of the tail (°C) T warm Warming temperature (°C) x latero-lateral coordinate with reference to the interaural line y ventro-dorsal coordinate with reference to the interaural line z rostro-caudal coordinate with reference to the interaural line

  134 and 4 ms, respectively[START_REF] Benoist | Peripheral and central determinants of a nociceptive reaction: an approach to psychophysics in the rat[END_REF]. Considering a site of stimulation on the mid-tail, the distance to the dorsal horn entry zone is approximately 200 mm. The model provides the following equation for the expected tail-flick latency: TFL (s) = [(36.8 -0.73 * T mid-tail ) 2 /α + 90/(0.041 * T core -0.47) + 110/(0.041 * T mid-tail -0.47) + 138]/1000 where α is the slope of the squared temperature variation (in °C2 /s) generated by the power of the radiant heat source.Let us recall at this point that, when the skin is exposed to a constant power source of infrared radiation, the temperature increases with the square root of time, according to the law of radiant heat transfer T = T mid-tail + a.t 0.5 or, expressed in terms of temperature variation T -T mid-tail = a.t 0.5 . This quadratic relationship becomes linear in t by squaring the two terms of the equation: (T -T mid-tail ) 2 = a 2 .t = α.t where α is the slope of the squared temperature variation generated by the power of the radiant heat source [see Fig.

Figure 1 .

 1 Figure 1. Procedure to analyze the muscimol-induced variations of skin temperature. A & B. Abscissa: time in min with reference to the end of the microinjection. Ordinate: temperature in °C. The grey area corresponds to the time intervals < 7.5 min and 7.5-15 min post-injection, delimited by vertical dotted lines. A. Analysis of temperature variations observed from an ipsilateral hind-paw (T paw-ipsi ). The thin yellow line is the temporal evolution of the temperature recorded during the 5-min control period and the following 30-min post-injection. The thick yellow line is the corresponding adjusted sigmoid Boltzmann curve T adj = T min + ΔT skin /{1 + exp[-(t -t x )]/s}, with R² = 0.99 (equation in the upper left corner of the graph). The adjusted parameters of the function are shown in red, namely the adjusted skin temperature during the control period

  points. B. Calculation of the beginning of the ascending phase of vasodilatation observed from the corresponding three other recorded zones (T paw-contra , T tail-prox , T tail-dist ). The thin and thick curves represent the genuine and adjusted curves, respectively. The green, dark blue and light blue curves correspond to the contralateral hind-paw, proximal and distal parts of the tail, respectively (corresponding equations provided in the upper left hand corner of the graph). Calculations of t min were made as in A, except for the proximal part of the tail for which a 15% percentile was used in place of the 10% (see text). C. Histogram showing the distribution of the beginning of the ascending phase of vasodilatation (t min ) for the ipsilateral hind-paws, allowing the categorization into three groups: group 1: t min < 7.5 min; group 2: 7.5 < t min < 15 min; group 3: t min > 15 min.

Figure 2 .

 2 Figure 2. Example of effects elicited by muscimol microinjection in the RVM. A. Example of pictures taken at 0, 5, 10, 15, 20, 25, 30 min post-injection of a thermographic movie recorded with a 320 * 240 pixels resolution (See the corresponding Supporting video S1). The false colors scale of temperature is shown in the right hand part of the picture. B. Temporal evolution of the temperatures recorded during the 5-min control and 30-min post-injection periods. Abscissa: time in min. Ordinate: temperature in °C. The eight analyzed zones (10 pixels each) are indicated on the drawing in the right hand part of the figure. T paw-ipsi(yellow) and T paw-contra (green) corresponds to the skin temperature of the ipsi-and contralateral hind-paw, respectively. T tail-prox (dark blue) and T tail-dist (light blue) corresponds to the skin temperatures of the proximal and distal part of the tail, respectively; there are three additional intermediate blue sites between these two sites. T amb (brown) corresponds to the ambient temperature measured from a small piece of wood. Note the stability of the control period in vasoconstriction with skin temperatures near T amb . Following the microinjection, the ipsilateral hind-paw skin temperature raised first, followed by the proximal and distal parts of the tail and at last the contralateral hind-paw. C. Temporal evolution of core temperature (T core ).Abscissa: time in min. Ordinate: temperature in °C. Note the decrease in core temperature, starting shortly after the rise of T paw-ipsi . D. Corresponding Heat loss Index [HLI = (T skin -T amb )/(T core -T amb )[START_REF] Romanovsky | Selected contribution: ambient temperature for experiments in rats: a new method for determining the zone of thermal neutrality[END_REF]] for the seven skin areas considered. E. Temporal evolution of MAP. Abscissa: time in min.Ordinate: MAP in mmHg. Note the slight transitory increase in MAP shortly after the microinjection, followed by a sustained drop and then a progressive stabilization after 8-min post-injection. F. Temporal evolution of HR. Abscissa: time in min. Ordinate: HR in beat per minute (bpm). Note the slight transitory increase in HR shortly after the microinjection, followed by a sustained drop and then a progressive stabilization after 9-min post-injection. G. Temporal evolution of ETCO 2 . Abscissa: time in min. Ordinate: ETCO 2 in %. Note the parallel changes in E, F and G. H. Localization of the site of injection drawn on a frontal section of the brain in plane -2.3 mm caudal to the inter-aural line. The center of the injection site is located 0.3 mm lateral to the midline and 0.4 mm below the inter-aural line. Abbreviations: 7: facial nucleus, GiA: gigantocellular reticular nucleus pars alpha; ml: medial lemniscus; PPy: parapyramidal nucleus; py: pyramidal tract; RMg: raphe magnus nucleus; RPa: raphe pallidus nucleus.

Figure 3 .

 3 Figure3. Temporal evolution of the skin temperature recorded from the hind-paws, following muscimol microinjections. Abscissa: time in min, Ordinate: temperature variation in °C (mean ± 95% confidence interval). As indicated in the left hand lower corner, yellow and green curves are data adjusted to a

Figure 4 .

 4 Figure 4. Overall effects elicited by muscimol microinjection on the skin temperature recorded from the hind-paws. A. contralateral hind-paw; B. ipsilateral hind-paw. Schema of frontal sections of the brain from inter-aural z = -0.8 (top) to z = -3.3 mm (bottom), adapted from[START_REF] Paxinos | The Rat Brain in Stereotaxic Coordinates[END_REF]. The circles indicate the center of the corresponding injection sites, with a diameter scaled to a 50 nl sphere. The black symbols represent the early latency group with an onset of vasodilatation in less than 7.5 min. The grey symbols correspond to the intermediate latency group with an onset of vasodilatation between 7.5 and 15 min. The white symbols stand for the group that included the experiments with an onset of vasodilatation in more than 15-min (or did not react at all). Abbreviations: 7: facial nucleus; GiA: gigantocellular reticular nucleus pars alpha; ml: medial lemniscus; PPy: parapyramidal nucleus; py: pyramidal tract; RMg: raphe magnus nucleus; RPa: raphe pallidus nucleus.

Figure 5 .

 5 Figure5. Three-dimensional mapping of the hind-paw response latencies t min as a function of injection sites.The responses obtained from the left and right paw were merged, by expressing the location of the injection site as ipsilateral (positive x-axis) vs. contralateral (negative x-axis) relative to the paw. On the left graphs, the interpolated response latencies are indicated by the false colors (scale shown in the lower right hand part of the figure). They are adjusted on frontal sections of the brain from inter-aural z = -0.8 (top) to z = -3.3 mm (bottom) with y being the ventro-dorsal coordinate, again with reference to the interaural line. The right drawings are adapted from[START_REF] Paxinos | The Rat Brain in Stereotaxic Coordinates[END_REF]. Abbreviations: 7: facial nucleus; GiA: gigantocellular reticular nucleus pars alpha; LPGi: lateral paragigantocellular nucleus; ml: medial lemniscus; PPy: parapyramidal nucleus; py: pyramidal tract; RMg: raphe magnus nucleus; RPa: raphe pallidus nucleus.

Figure 6 .

 6 Figure 6. Temporal evolution of the skin temperature recorded from the tail, following muscimol microinjections. Abscissa: time in min, Ordinate: temperature variation in °C (mean ± 95% confidence interval). As indicated in the left hand lower corner, dark blue and blue colors are data adjusted to a

Figure 7 .

 7 Figure 7. Overall effects elicited by muscimol microinjection on the skin temperature recorded from the tail. A. proximal part of the tail; B. distal part of the tail. Symbols and abbreviations as in figure 4.

Figure 8 .

 8 Figure 8. Three-dimensional mapping of the tail response latencies t min as a function of injection sites. A. proximal part of the tail. B. distal part of the tail. The interpolated response latencies are indicated by the false colors (scale shown in the bottom of the figure). They are adjusted on frontal sections of the brain from inter-aural z = -0.8 (top) to z = -3.3 mm (bottom) with x and y being the lateral and ventro-dorsal coordinate, respectively, again with reference to the interaural line. Symbols and abbreviations as in figure 5.

Figure 9 .

 9 Figure9. A. Schema of frontal sections of the brain from inter-aural -0.8 (top) to -3.3 mm (bottom), adapted from[START_REF] Paxinos | The Rat Brain in Stereotaxic Coordinates[END_REF]. Symbols and abbreviations as in figure4. The data were synthesized by regrouping the experiments in three new groups, again on the basis of the beginning time t min of the ascending phase of vasodilatation, considering all skin areas either on the tail or the paws. The black symbols represent the early latency group with t min < 15 min for all considered skin areas either on the tail or the paws. The grey symbols correspond to the intermediate latency group with t min < 15 min for at least one of these areas. The white symbols correspond to the late latency group with t min > 15 min for all considered skin areas. B. Variation of skin temperature (Δ°C) recorded from the ipsilateral hind-paw. C.Variation of skin temperature (Δ°C) recorded from the contralateral hind-paw. D. Variation of skin temperature (Δ°C) recorded from the distal part of the tail. E. Variation of skin temperature (Δ°C) recorded

  and considering a site of stimulation on the mid-tail far-off the dorsal horn entry zone by 200 mm, the model provides the following equation giving the tail-flick latency: TFL (s) = [(36.8 -0.73 * T mid-tail ) 2 /α + 90/(0.041 * T core -0.47) + 110/(0.041 * T mid-tail -0.47) + 138]/1000 where α is the slope of the squared temperature variation, witness of the power of the radiant heat source. Experimental data are from the experiments where the beginning of the ascending phase of vasodilatation was < 15 min either on the tail or the paws (Black group in figure9); they include both the mean temperatures of the core (T core , brown line)

  24.2 + 8.2/{1 + exp[-0.57 * (t -8.8)]} T adj = 23.9 + 7.4/{1 + exp[-0.55 * (t -16.6)]} T adj = 25.2 + 6.4/{1 + exp[-0.15 * (t -18.4)]} T adj = 23.6 + 7.8/{1 + exp[-0.21 * (t -22.3all paws AND tail < 15 min (n = 23) one paw OR tail < 15 min (n = 41) all paws AND tail > 15 min (n = 96) D: skin temperature T tail-dist (Δ°C) arterial blood pressure MAP (ΔmmHg) H: heart rate (Δbpm) I: end tidal CO 2 (Δ%) B: skin temperature T paw-ipsi (Δ°C) C: skin temperature T paw-contra (Δ°C)

  Table 2 compares results of skin temperature, converted in terms of HLI, obtained in the present study (acute onset group 1, black symbols) to previous experiments performed on anesthetized rats maintained in thermo-neutral conditions (El Bitar et al. 2014a).

Table 1 (see Fig.

 1 P < 0.01 with respect to the corresponding value of the ipsilateral paw. P < 0.05 with respect to the corresponding value of the ipsilateral paw.

			T min (°C)	ΔT skin (°C)	t min (min)	t X (min)	Δt (min)	ΔT skin /∆t (°C/min)
	ipsilateral	group 1	25.4	7.4	3.6	6.6	6.6	1.2
			(25.2-25.7)	(7.2-7.7)	(3.4-3.8)	(7.4-5.8)	(7.4-5.8)	(1.0-1.6)
		group 2	25.4	7.7	11.3	15.3	7.9	7.9
	contralateral group 1	(25.2-25.7) 25.6	(7.4-8.0) 7.2	(11.2-11.5) 14.6 *	(16.0-14.6) 18.0 *	(6.7-9.0) 6.8	(6.7-9.0) 1.1
		group 2	(25.5-25.8) 25.4	(7.0-7.4) 7.9	(12.8-16.3) 23.5 *	(15.8-20.2) 27.8 *	(5.9-7.7) 8.5	(0.9-1.3) 0.9
			(25.3-25.6)	(7.6-8.2)	(22.0-25.0)	(29.6-25.9)	(7.8-9.2)	(0.8-1.0)
	B: tail (see Fig. 6)
			T min (°C)	ΔT skin (°C)	t min (min)	t X (min)	Δt (min)	ΔT skin /∆t (°C/min)
	proximal	group 1	27.7	4.4 *	2.4 *	8.1	14.2 *	0.3 *
		group 2	(27.4-28.0) 27.7	(4.1-4.7) 4.4 *	(2.4-2.5) 8.2 *	(8.9-7.2) 16.1	(11.9-16.5) 15.7 *	(0.2-0.4) 0.3 *
	distal	group 1 group 2	(27.3-28.2) 25.2 (25.0-25.5) 25.4	(4.0-4.9) 5.6 * (5.2-6.0) 6.2 *	(8.1-8.4) 2.4 * (2.0-2.8) 11.2	(16.9-15.3) 8.6 * (7.1-10.2) 17.5 *	(13.7-17.7) 14.7 * (11.2-18.2) 12.5 *	(0.2-0.4) 0.4 * (0.3-0.5) 0.5 *
			(25.0-25.8)	(5.7-6.7)	(11.0-11.4)	(18.6-16.4)	(10.7-14.4)	(0.4-0.6)

* *

Table 2 .

 2 Summary of skin vasomotor tone expressed in terms of Heat Loss Index [HLI = (T skin -T amb )/(T core -T amb )]

		(A) HLI during vasoconstriction		(B) HLI during vasodilation
		( a )	(b)	P	(a)	(b)	P
		control period	vasoconstriction periods		30 min following	vasodilation periods
		(present study)	(El Bitar et al. 2014a)		muscimol	(El Bitar et al. 2014a)
	T paw-ipsi	(0.10-0.11) 0.10	(0.09-0.16) 0.13	0.12	0.65-0.74) 0.69	(0.85-0.98) 0.92	< 0.00001
	T tail-prox	(0.22-0.25) 0.24	(0.21-0.26) 0.23	0.64	(0.57-0.67) 0.62	(0.72-0.83) 0.77	< 0.0001
	T tail-dist	(0.04-0.04) 0.04	(0.01-0.05) 0.03	0.06	(0.46-0.63) 0.55	(0.69-0.80) 0.74	< 0.0001

ACKNOWLEDGMENTS

We thank Professors Pascal Carrive, François Cesselin and Léon Plaghki for advice in the preparation of the manuscript. Nabil El Bitar was supported by a grant from the Société Française d'Etude et de Traitement de la Douleur (SFETD) et l'Institut UPSA de la douleur (IUD).

AUTHOR CONTRIBUTIONS

Conceived and designed the experiments: NEB, BP, DLB. Performed the experiments: NEB, BP, DLB.

Analyzed the data: NEB, BP, DLB, AM, GH. Wrote the paper: NEB, BP, AM, DLB.

Supporting Video. Thermographic recording at 1 Hz with a 320x240 pixels resolution, compressed and presented in 14 seconds, made during the 5-min control and 30-min post-injection periods. The thermography is presented in false colors (scale in the left). The graph is the corresponding temporal evolution of the skin temperature recorded on 5 points distributed from the base to the tip of the tail (blue points) and the right (yellow point) and left (green point) hind-paws as indicated on the right hand picture.

The ambient temperature was measured on an inert piece of wood (brown point). The localization of the site of injection of muscimol (50 nmol, 50 nl) is drawn on a frontal section of the brain in plane -2.3 mm caudal to the inter-aural line. Abbreviations: Gi: gigantocellular reticular nucleus; GiA: gigantocellular reticular nucleus pars alpha; ml: medial lemniscus; RMg: raphe magnus nucleus; RPa: raphe pallidus nucleus; py: pyramidal tract. (20.1-27.7 39 (31-48) 0.5 (0.4-0.5) contralateral paw 25.9 (25.7-26.1) 6.9 (6.2-6.6) 9.6 (8.7-10.4) 13.0 (11.6-14.2) 6.7 (5.7-7.6) 1.0 (0.8-1.2) proximal tail 27.8 (27.4-28.1) 4.2 (3.9-4.5) 5.3 (5.0-5.6) 10.9 (9.8-11.9)