Didier Le Ray

Laurent Juvin

Tanguy Boutin

François Auclair

Dr Réjean Dubuc
email: dubuc.rejean@uqam.ca

A neuronal substrate for a state-dependent modulation of sensory inputs in the brainstem

Keywords: MLR, Muscarinic, Sensory integration, Locomotion, Lamprey

Central networks modulate sensory transmission during motor behavior. Sensory inputs may thus have distinct impacts according to the state of activity of the central networks. Using an in vitro isolated lamprey brainstem preparation, we investigated whether a brainstem locomotor center, the mesencephalic locomotor region (MLR), modulates sensory transmission. The synaptic responses of brainstem reticulospinal (RS) cells to electrical stimulation of the sensory trigeminal nerve were recorded before and after electrical stimulation of the MLR. The RS cell responses were significantly reduced by MLR stimulation. Moreover, the depression of sensory transmission increased with the stimulation intensity of the MLR. Bath perfusion of atropine prevented the depression of sensory transmission, indicating that muscarinic receptor activation is involved. Previous studies have shown that, upon stimulation of the MLR, behavioral activity switches from a resting state to an active-locomotor state.

Therefore, our results suggest that a state-dependent modulation of sensory transmission to RS cells occurs in the behavioral context of locomotion and that muscarinic inputs from the MLR are involved.

Introduction

The neural control of movements is based on interactions between centrally generated motor commands and incoming sensory inputs. For instance, central neural networks require sensory inputs to adapt motor behavior to environmental conditions. Reciprocally, the central neural networks gate sensory transmission in a "state"-dependent fashion. Transmission of sensory signals can be modulated according to whether the animal is resting, standing or in movement [START_REF] Brown | Studies in the physiology of the nervous system. VlII:Neural balance and reflex reversal with a note on progression in the decerebrate guinea pig[END_REF][START_REF] Grillner | Locomotion in the spinal cat[END_REF][START_REF] Grillner | Contralateral reflex reversal controlled by limb position in the acute spinal cat injected with clonidine i[END_REF][START_REF] Rossignol | An analysis of mechanisms controlling the reversal of crossed spinal reflexes[END_REF]. Similarly, pain sensitivity is modulated in sleep vs. wake conditions [START_REF] Foo | Brainstem modulation of pain during sleep and waking[END_REF]. There are multiple examples of state-dependent modulation of sensory transmission in the mammalian spinal cord [START_REF] Rossignol | State-dependent responses during locomotion[END_REF][START_REF] Hultborn | State-dependent modulation of sensory feedback[END_REF][START_REF] Krawitz | State-dependent hyperpolarization of voltage threshold enhances motoneurone excitability during fictive locomotion in the cat[END_REF]. However, the underlying cellular mechanisms have not been identified yet.

Even less is known about modulation of sensory transmission in supraspinal networks.

Activity of brainstem cholinergic neurons can be adapted during the sleep-wake cycle [START_REF] El Mansari | Unitary characteristics of presumptive cholinergic tegmental neurons during sleep-waking cycle in freely behaving cats[END_REF][START_REF] Kayama | Firing of 'possibly' cholinergic neurons in the rat laterodorsal tegmental nucleus during sleep and wakefulness[END_REF]. Moreover, cholinergic neurons have been reported to play an important role in modulation of sensory transmission in the brainstem of mammals [START_REF] Fernández De Sevilla | Cholinergic modulation of synaptic transmission and postsynaptic excitability in the rat gracilis dorsal column nucleus[END_REF]. The cellular mechanisms of the muscarinic modulation have been described with little information relative to the specific pathways involved and to the behavioural circumstances during which the modulation was exerted. Thus, we began to examine state-dependent modulation of sensory transmission in the brainstem of lampreys, an animal preparation that has allowed us to examine cellular mechanisms as well as the detailed connectivity in relation to locomotor behavior. We have recently reported a powerful muscarinic modulation of sensory transmission or trigeminal inputs to the reticulospinal cells in the lamprey brainstem [START_REF] Ray | Muscarinic modulation of the trigemino-reticular pathway in lampreys[END_REF]. In this study, we showed that the local perfusion of a muscarinic agonist either on the reticulospinal or on the trigeminal relay cells induced a depression of synaptic transmission. However despite the characterisation of the cellular mechanisms involved, the origin and the functional relevance of this cholinergic modulation remained to be established.

We now show that trigeminal sensory transmission is depressed following the activation of the MLR. This effect was prevented by adding a muscarinic receptor antagonist to the perfusing Ringer's. We propose that the MLR is the origin of the previously described muscarinic modulation of sensory transmission from the trigeminal inputs to reticulospinal cells. The role of this modulation could be to adapt the responsiveness to the environment depending of the behavioural state of the animal (locomotion vs. rest). We now describe a neural pathway involved in modulating sensory transmission to brainstem neurons in relation to locomotion.

Methods

Electrophysiological experiments were performed in isolated in vitro brainstem preparations from larval lampreys, Petromyzon marinus (n=45), whereas immunohistochemistry was performed in adult animals (n=11). The larval lampreys were collected from the Morpion Stream near Lake Champlain in southern Québec, whereas the reproductive adult lampreys were collected from the Great Chazy River near Lake Champlain (NY, USA) and from the St-Mary's River between Lake Superior and Lake Huron (ON, Canada). All procedures conformed to the guidelines of the Canadian Council on Animal Care and were approved by the University Animal Care and Use Committees at the Université de Montréal and the Université du Québec à Montréal. Special care was taken to minimize suffering as well as the number of animals used in the experiments.

Electrophysiology

Under tricaine methanesulphonate anesthesia (MS 222, 100 mg/L, Sigma, Oakville, ON, Canada), the animals were incised along the ventral midline and eviscerated. The dissection was performed as previously described (Le [START_REF] Ray | Muscarinic modulation of the trigemino-reticular pathway in lampreys[END_REF] in cold Ringer's with the following composition (in mM): 130 NaCl, 2.1 KCl, 2.6 CaCl 2 , 1.8 MgCl 2 , 4 HEPES, 4 Dextrose, 1 NaHCO 3 . The preparation was pinned down to the Sylgard bottom of an experimental chamber perfused with oxygenated cold Ringer's (9ºC; pH 7.4). A complete transection was made above the mesencephalon for decerebration purposes and a lesion was made at the obex to separate the brainstem from the spinal cord (Fig. 1A). During the course of the experiments, the recording chamber was constantly perfused with Ringer's (2 ml/min).

Lamprey reticulospinal (RS) neurons (n=85) in the middle rhombencephalic reticular nuclei (MRRN) were impaled under visual guidance with sharp glass microelectrodes (4M K-acetate, ~100 M; Fig. 1A). The intracellular signals were recorded from the largest reticular neurons in the MRRN, including the Müller cells (see [START_REF] Rovainen | The Biology of Lampreys[END_REF] with an Axoclamp 2A (Axon Instruments Inc., Foster City, CA) and directed to a computer (sampling rate: 10 kHz) through a TL-1 DMA interface (Axon Instruments Inc.). In order to reduce the number of animals used in these experiments, one to five RS neurons were recorded per preparation. A long period of time (90 to 120 min) was allocated between successive RS neuron impalements to permit full recovery of the preparation.

The effects of drug application and trigeminal nerve stimulation were all tested at resting membrane potential (-76.8±0.7 mV; n=74; from -66mV to -90mV). Electrical stimulation of either the ipsi-or contralateral trigeminal sensory nerve (single shocks of 1.5 ms duration at 0.1 Hz) and of the MLR (1.5 ms shocks repeated at 3-10 Hz during 15-40 s) were performed with homemade glass-coated tungsten microelectrodes (4-5 M) using a Grass S88 stimulator (Grass Instrument Co., Quincy, MA). The trigeminal-evoked PSPs were first monitored for several minutes under control condition. They were then measured after MLR stimulation when the MLR-induced depolarization of the RS neuron had ceased and the membrane potential of the recorded cell had returned to control levels. Therefore, changes in trigeminal PSPs did not result from an indirect effect of the RS neuron being depolarized, such as a shunt due to a conductance opening. Atropine (10 M; Sigma) freshly dissolved in the Ringer's solution was bath applied and tested on 7 RS neurons (from 6 different preparations). In 5 RS cells, the input resistance (R in ) and the area of a 20-sec MLR stimulation-induced plateau were measured in normal Ringer's and during atropine perfusion.

Immunohistochemistry

The presence of muscarinic receptors was examined using immunohistochemistry in spawning-phase adult lampreys (n=11) ranging from 45 to 55 cm in total body length. Adult lampreys were chosen to carry out these experiments because they show more fully differentiated morphological characteristics (i.e. dendritic arbors) than larval animals. The dissection was similar to that for electrophysiological experiments in larvae. The preparations were fixed by immersion in a solution mixed with 2% paraformaldehyde/0.2% picric acid in 0.1 M phosphate buffer saline (PBS) at 4°C, pH 7.4 for 6 hours. After spending one night in a 30% sucrose solution at 4°C, the brains were frozen in 2-methylbutane. The rhombencephalon was then cut in 25µm-thick transverse sections using a cryostat (American Optical, Buffalo, NY) and mounted on gelatin-coated slides. Sections were dried on a warming plate at 37°C overnight. All subsequent steps were carried out at room temperature unless specified otherwise. The next day, the sections were first rinsed 3 times with PBS and endogenous peroxidase activity was blocked by incubation in a solution of 0.3% peroxide and 0.1% sodium azide for 10 min. The sections were rinsed 3 times in PBS and then incubated in a solution of PBS containing 5% normal rabbit serum (NRS) and 0.4% Triton X-100 for 1 hour, followed by a solution of PBS containing 1% NRS, 0.1% Triton X-100 and the primary antibody M35 (Argene Inc., N. Massapequa, NY) diluted 1:200 for 20 hours at 4°C. The next day, the sections were rinsed 3 times in PBS and then incubated in a solution of PBS containing 1% NRS, 0.1% Triton X-100, and a biotinylated rabbit anti-mouse antibody diluted 1:100 (Chemicon, Temecula, CA) for 1 hour. The sections were rinsed 3 times in PBS, incubated for 1 hour in a solution of the standard Vectastain Elite ABC kit (Vector, Burlingame, CA,) as prescribed by the manufacturer, and then rinsed again 3 times in PBS. The immunohistochemical staining was revealed by incubating the sections in a solution of 0.05% diaminobenzidine tetrahydrochloride in PBS for 5 min. The sections were then rinsed 3 times in PBS, dehydrated in alcohol, cleared with xylene and mounted with Entellan (VWR International, Montréal, QC, Canada).

Photomicrographs were taken with a 20X Plan Fluor objective (numerical aperture = 0.50) fitted on a Nikon Eclipse E600 microscope equipped with a DXM1200 digital camera. The contrast and brightness of photomicrographs were adjusted, and the figures designed using CorelDraw 12 (Corel Corp., Ottawa, ON, Canada) software.

The ability of the M35 pan-muscarinic monoclonal antibody to bind specifically to muscarinic cholinergic receptors of lampreys was previously confirmed using Western Blot analysis [START_REF] Smetana | Muscarinic receptor activation elicits sustained, recurring depolarizations in reticulospinal neurons[END_REF]. In the present experiments, omitting the M35 primary antibody from the immunohistochemical procedure resulted in a complete absence of labeling.

Statistical Analysis

The area of the PSPs evoked by the stimulation of the trigeminal nerve in RS cells was compared before (control condition) and after MLR stimulation (post-stimulation condition). All measurements were made when the RS cell membrane potential was stable.

The mean area of the trigeminal PSPs (in mV.ms) was calculated from the area of 10 PSPs recorded in each RS cell. Statistical comparisons were then made after averaging the mean areas of the different RS cells (mean area ±SEM). A total of 74 neurons were recorded in 34 animals. To test for sample independence when more than one cell were recorded per animal, the analyses on 74 neurons were compared to those when only one cell was taken per animal (34 neurons). There were no statistical differences between the analysis of 34 neurons and that of the entire population of 74 neurons. Therefore, all 74 neurons were included in the analysis.

The statistical significance of possible changes in the area of the PSPs was tested using a paired t-test when two groups were compared; a one-way ANOVA for repeated measures was used when more than two groups were compared. Some of the data were normalized for illustration (see histograms in Fig. 1).

Normalization was made with respect to control values after averaging the absolute control

values in order to obtain a SEM value for control values as well.

Results

MLR activation transiently depressed trigeminal-evoked EPSPs

Trains of stimuli applied for 15-40 s in the frequency range that was previously shown to elicit locomotion (2-10 Hz; [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF][START_REF] Ray | Nicotinic activation of reticulospinal cells involved in the control of swimming in lampreys[END_REF] markedly depressed sensory transmission to RS neurons in the MRRN. In the example illustrated in Figure 1B, the trigeminal sensory nerve on one side was stimulated at 0.1 Hz before and after applying a tonic 15 s, 3 Hz stimulation to the MLR. Stimulation of the MLR elicited a sustained depolarization in the recorded RS neuron (not shown, but see Le [START_REF] Ray | Nicotinic activation of reticulospinal cells involved in the control of swimming in lampreys[END_REF]. Once the stimulation of the trigeminal nerve was resumed after the MLR-induced RS neuron depolarization had stopped, the area of the trigeminal EPSPs was reduced by about 50%, from 162.6±11.9 to 79.5±3.8 mV•ms (Fig. 1B The muscarinic antagonist atropine (10 µM) was tested on the MLR ability to induce a depression of trigemino-reticular inputs in 7 RS neurons (out of the 74 RS neurons analyzed above). Under atropine, the synaptic responses were not depressed after MLR stimulation (n=7; 100±6.4% vs. 95.1±12.1 %, t 6 =0.717, P=0.51; Fig. 1C,right;Fig 1B bottom). In the same 5 cells into which the R in was measured before and after MLR stimulation, it was also measured after adding atropine. We found that R in did not change consecutive to atropine perfusion (n=5; 12.3±1.8 MΩ vs. 12.9±0.4 MΩ; F 3,16 =0.7921, P=0.52; Fig. 1D). Moreover the area of the MLRinduced sustained depolarization remained unchanged after atropine perfusion (n = 5; 100±9.5 % vs. 105.1±10.2 %; t 4 =0.5847, P=0.59; Fig. 1E). Taken together, these results indicate that the MLR-induced depression of the RS neuron response to trigeminal nerve stimulation is largely mediated by muscarinic acetylcholine receptors. In 3 experiments, the caudal half of the rhombencephalon (below the MRRN) was removed to eliminate most rhombencephalic cholinergic neurons located in the tegmentum. In this reduced preparation, stimulation of the MLR still consistently depressed (100±4.9% vs. 73.4±1.3 %; not illustrated) the EPSPs elicited by trigeminal stimulation.

In lampreys as in other vertebrates, stimulation of the MLR induces locomotor activity and the speed of swimming is directly proportional to the level of activation of the MLR (intensity or frequency of stimulation; [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF]. We then tested whether the depression exerted by activation of the MLR on sensory-evoked EPSPs also depended on the level of MLR activation.

Different MLR stimulation frequencies were then sequentially applied and the level of depression quantified in 6 RS neurons (from 6 different preparations). We observed that the stimulation of the MLR had significant effect over the PSPs area (F 5,30 =6.443, P=0.009). The Tukey's post hoc analysis revealed that after MLR stimulation at 3 Hz, the amplitude of EPSPs tended to decrease, but not significantly (87.9±6.7%, P>0.05). However, when MLR stimulation was increased to higher frequencies (5-6 Hz; 7-8 Hz; 9-10 Hz), the EPSPs amplitude decreased significally in comparison to control values (100± 6.2%) (respectively: 69.2±6.6% at 5-6 Hz, P<0.05; 63.8±4.3% at 7-8 Hz, P<0.01; 72.8±6.4% at 9-10 Hz, P<0.05; Fig. 1F-G). Within the stimulation frequency range used in this study, a maximal effect was reached at 7-8 Hz (63.8±4.3%) and higher stimulation frequency at 9-10 Hz did not produce further depression (72.8±6.4%) of the EPSP area (P=0.93).

Immunohistochemical identification of muscarinic receptors in rhombencephalic neurons

In order to identify the possible location where the muscarinic modulation could be exerted, an immunohistochemical detection of muscarinic receptors with the M35 pan-muscarinic monoclonal antibody was performed in the brainstem. Staining was seen in the MRRN as well as more laterally in the trigeminal relay area (Fig. 2).

Giant RS Müller cells of the MRRN, showed clear cell body and proximal dendrite staining, whereas the distal dendrites located in the more ventral and lateral areas of the tegmentum seemed to lack muscarinic receptors (Fig. 2A,B). The same pattern of labeling was seen in giant RS Müller cells in the mesencephalic reticular, and in the anterior and posterior rhombencephalic reticular nuclei (not illustrated). In addition, many other immunoreactive neurons of smaller size were found in all reticular nuclei, including a periventricular population lateral to and intermingled with the giant RS Müller cells of the MRRN (Fig. 2B, arrows), and another population ventral to the motoneurons of the seventh nerve (VII; Fig. 2D, large arrow).

Many neurons were also labeled in the nucleus of the trigeminal descending tract (dVn; [START_REF] Northcutt | Experimental determination of the primary trigeminal projections in lampreys[END_REF], which contains the second-order trigeminal sensory neurons that project directly to RS neurons [START_REF] Di Prisco | The trigeminal sensory relay to reticulospinal neurons in lampreys[END_REF]. The labeled cells were present at all rostrocaudal levels of the dVn. More particularly, many bipolar and multipolar neurons (small arrows) were present at the level of the motor nuclei of the fifth (V; Fig. 2C) and seventh (Fig. 2D) nerves.

Discussion

In both invertebrates and vertebrates, sensory information shapes motor commands at every level of the CNS in order for movements to be adapted to external and internal conditions. In turn, the central networks modulate sensory input transmission via both pre-and post-synaptic mechanisms (see [START_REF] Clarac | Central control components of a 'simple' stretch reflex[END_REF][START_REF] Hultborn | Spinal control of locomotionfrom cat to man[END_REF]. Many studies have highlighted phase-and/or state-dependent modulation of sensory transmission in the spinal cord (for a review, see [START_REF] Rossignol | Dynamic sensorimotor interactions in locomotion[END_REF]. In the brainstem as well, some control mechanisms of sensory inflow have been described related to locomotor activity [START_REF] Bussières | Phasic modulation of transmission from vestibular inputs to reticulospinal neurons during fictive locomotion in lampreys[END_REF][START_REF] Alford | Glutamate metabotropic receptor mediated depression of synaptic inputs to lampreys reticulospinal neurones[END_REF]. The detailed connectivity and neuronal elements involved are yet to be identified.

We now show in lampreys that activating the MLR induces a long lasting state-dependent depression of trigeminal sensory inputs to RS neurons and that muscarinic receptors are involved.

Long lasting depression of sensory inputs has previously been described either at the spinal cord level in decerebrate cats during fictive locomotion [START_REF] Gosgnach | Depression of group Ia monosynaptic EPSPs in cat hindlimb motoneurones during fictive locomotion[END_REF] or in man during walking [START_REF] Voigt | The human soleus Hreflex modulation during the transition from relaxed sitting to walking[END_REF]. In this work, we did not test the ability of MLR stimulation to induce a swimming behavior, but the same stimulation parameters have been used in previous studies to generate locomotor bouts in a semi-intact preparation [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF][START_REF] Ray | Nicotinic activation of reticulospinal cells involved in the control of swimming in lampreys[END_REF]. Because of this, we can assume that the depression observed in sensory transmission in response to stimulation of the MLR is related to locomotion.

The MLR of lampreys contains cholinergic neurons (Le [START_REF] Ray | Nicotinic activation of reticulospinal cells involved in the control of swimming in lampreys[END_REF]. Because the depression of sensory transmission to RS cells was shown to be significantly reduced by the muscarinic antagonist atropine, it is likely that activation of cholinergic neurons in the MLR will play a critical role in modulating sensory transmission. The simplest hypothesis (Fig. 3) would be cholinergic neurons in the MLR projecting to both RS and trigeminal relay cell areas. The dual projection is supported by the present observation that muscarinic receptors are located on both the RS neurons and the relay cells, and our previous observation that muscarinic agonists induce a sensory depression when injected either in the dVn or in the MRRN (Le [START_REF] Ray | Muscarinic modulation of the trigemino-reticular pathway in lampreys[END_REF]. The MLR-evoked muscarinic depression may thus occur at RS and relay cell areas.

Other lamprey cholinergic cells have been described in the lateral periventricular reticular formation of the caudal rhombencephalon, below the level of the MRRN down to the obex and just medial to the motoneurons of the vagal and glossopharyngeal nuclei [START_REF] Pombal | Distribution of choline acetyltransferaseimmunoreactive structures in the lamprey brain[END_REF]. These cells are unlikely to play a significant role here because the depression of sensory transmission to MRRN RS cells induced by stimulating the MLR was elicited equally well after a transection of the rhombencephalon just caudal to the MRRN. Similarly, muscarine-sensitive neurons were recently described in the caudal rhombencephalon [START_REF] Smetana | Muscarinic receptor activation elicits sustained, recurring depolarizations in reticulospinal neurons[END_REF] that were responsible for generating recurring sustained depolarization in RS cells of the MRRN. These neurons are not part of the trigemino-reticular sensory pathway and their contribution to the depressive effects described in the present study is unlikely. As indicated above, removal of the caudal half of the rhombencephalon (including these muscarinosensitive cells) did not prevent the MLR-induced depression of sensory transmission.. A muscarinic modulation of sensory transmission has also been described in mammals [START_REF] Fernández De Sevilla | Cholinergic modulation of synaptic transmission and postsynaptic excitability in the rat gracilis dorsal column nucleus[END_REF] where dorsal column sensory inputs to neurons in the gracilis nucleus were found to be modulated by the muscarinic agonist carbachol, via both pre-and postsynaptic effects. Muscarinic modulation of glutamatergic synapses has been reported in the past, and presynaptic mechanisms have been implicated [START_REF] Jiang | Presynaptic suppression of excitatory postsynaptic potentials in rat ventral horn neurons by muscarinic agonists[END_REF][START_REF] Scanziani | Presynaptic inhibition of excitatory synaptic transmission by muscarinic and metabotropic glutamate receptor activation in the hippocampus: are Ca2+ channels involved?[END_REF][START_REF] Bellingham | Presynaptic depression of excitatory synaptic inputs to rat hypoglossal motoneurons by muscarinic M2 receptors[END_REF][START_REF] Smolders | Muscarinic modulation of striatal dopamine, glutamate, and GABA release, as measured with in vivo microdialysis[END_REF][START_REF] Li | Role of presynaptic muscarinic and GABAB receptors in spinal glutamate release and cholinergic analgesia in rats[END_REF]. However, the source of these modulations has yet to be identified. At this stage, we cannot establish whether the effects occur pre-or postsynaptically in our system.

In lampreys, the MLR command is also largely mediated by glutamate in addition to acetylcholine [START_REF] Brocard | Differential contribution of reticulospinal cells to the control of locomotion induced by the mesencephalic locomotor region[END_REF][START_REF] Ray | Nicotinic activation of reticulospinal cells involved in the control of swimming in lampreys[END_REF]. Moreover, glutamate receptor agonists were shown to inhibit presynaptically the vestibular inputs to lamprey RS neurons [START_REF] Bussières | Phasic modulation of transmission from vestibular inputs to reticulospinal neurons during fictive locomotion in lampreys[END_REF] via the activation of metabotropic receptors [START_REF] Alford | Glutamate metabotropic receptor mediated depression of synaptic inputs to lampreys reticulospinal neurones[END_REF]. Whether such a glutamatergic control exists in the presently investigated trigeminoreticular pathway is not established yet. Conversely, whether the muscarinic modulation is specific to the trigeminal inputs or constitutes a more general control mechanism on sensory transmission to RS neurons, as well as the involvement of the MLR in such a global modulation remains to be established. Nevertheless, the present study provides the first demonstration that the MLR, traditionally given a role as a command center for locomotion (for reviews see [START_REF] Whelan | Control of locomotion in the decerebrate cat[END_REF][START_REF] Jordan | Initiation of locomotion in mammals[END_REF][START_REF] Dubuc | Locomotor regions in the midbrain (MLR) and diencephalon (DLR) Encyclopedia of Neuroscience[END_REF], can directly affect the transmission in a brainstem sensorymotor pathway that has been clearly implicated in eliciting escape swimming in lampreys [START_REF] Di Prisco | A cellular mechanism for the transformation of a sensory input into a motor command[END_REF][START_REF] Dubuc | Initiation of locomotion in lampreys[END_REF]. The MLR has also been shown to modulate sensory transmission in higher vertebrates. For insance, startle reflexes induced by acoustic, visual or tactile stimuli are depressed by the MLR [START_REF] Fendt | Brain stem circuits mediating prepulse inhibition of the startle reflex[END_REF]. In addition, our present study shows that the level of the trigemino-reticular depression increases with the level of MLR activation, suggesting that the more intense is the locomotion induced by the MLR, the more the latter structure depresses sensory transmission. Therefore, locomotion induced in a "goal-directed" manner could be associated with a state-dependent reduction of potentially disturbing sensory inputs, thereby facilitating the control exerted by central structures on the RS system. Pre-and/or post-synaptic effects could be involved.

  The ANOVA was followed by a Tukey's post hoc test when significance was achieved. The sphericity assumption was verified using the Mauchly criterion. If rejected, a Greenhouse-Geisser correction was performed. The same statistical analyses were used when comparing the R in and the area of the depolarization plateaus. For all the statistics, (n) refers to the number of neurons. Data are expressed as mean ± SEM. Differences were considered as statistically significant when P value was less than 0.05. All the statistics were obtained using both Prism software (GraphPad software, inc., La Jolla, CA, USA) and SPSS Statistics 17.0 software (SPSS inc., Chicago, IL, USA).

  top). On average (n=74 cells), the area of the synaptic responses was significantly decreased from 100±14.5% (before) to 70.1±11.1% after MLR stimulation (t 73 =5.689, P=0.002; Fig.1C, left). The EPSPs progressively recovered after 20 min. The R in of 5 RS cells were compared before (12.3±1.8 MΩ) and after MLR stimulation (13.7±1.4 MΩ), and no significant changes were found (F 3,16 =0.7921, P=0.52; Fig.1D).

Figure 1 :

 1 Figure 1: MLR activation depresses trigeminal sensory inputs to RS neurons. A: Experimental setup for stimulation of a trigeminal sensory nerve and the MLR as well as recording RS cells intracellularly. B: In normal Ringer's (top), a 3 Hz stimulation of the MLR for 15 s (arrow) induced a long-lasting reduction in the size of the EPSP evoked in an intracellularly recorded RS neuron by electrical stimulation (repeated every 10s) of a trigeminal sensory nerve. The perfusion of atropine (10 µM, bottom) prevented the EPSP depression induced by MLR activation. Insets: Superimposition of control (1) and post-MLR activation (2) trigeminal EPSPs. Scale bars: 2 mV, 10 ms. C: Histograms illustrating the mean reduction of the trigeminal EPSP area, expressed as the percentage of control values, after MLR activation in control Ringer's (left; n=74 cells) and under 10 µM atropine perfusion (right; n=7 cells). ***: p<0.001. D: Histograms illustrating the lack of input resistance (R in ) changes in five tested RS neurons after MLR stimulation both in normal Ringer's (two right bars) and under atropine perfusion (two left bars). E: Histograms illustrating the lack of effect of atropine perfusion on the MLR-evoked plateau potential in five tested RS neurons. F: Averages of five trigeminal EPSPs (black traces) recorded after the MLR was stimulated for 15 s at four different frequencies. The traces are superimposed with the averaged control EPSP (grey traces). Scale bars: 5 mV, 50 ms. G: Histograms illustrating the mean area of the EPSPs for six neurons as in F. The values are expressed as percentage of control values. The significance of the differences from control values are indicated by * (p<0.05) and ** (p<0.01), whereas significance of the differences between the stimulation groups are indicated by § § (p<0.01). Complete recovery was always observed several tens of minutes after MLR stimulation (right bar; light grey).

Figure 2 :

 2 Figure 2: Muscarinic receptor immunoreactivity in the MRRN and the dVn. A,B: Photomicrographs of transverse sections through the MRRN showing large immunoreactive RS neurons and their proximal dendrites. The bottom drawings indicate the localization of the sections in the brainstem. C,D: Photomicrographs of the lateral portion of transverse sections passing through two different rostro-caudal levels of the MRRN (see bottom drawings) and showing immunoreactive neurons (arrows) of different shapes in the dVn at the level of the motor nuclei of the fifth (V in C) and seventh (VII in D) nerves.

Figure 3 :

 3 Figure 3: Schematic illustration of a putative pathway by which the MLR could control 5 trigeminal sensory inputs to RS neurons. Left: Trigeminal projections to reticulospinal cells in the MRRN network and the localization of the MLR. Right (enlargement of the boxed area in the left schematic):The simplest hypothesis would be that the MLR exerts a muscarinic control on the trigemino-reticular pathway at both the RS neuron and the relay cell levels.

Acknowledgements:

This study was supported by the following grants: individual and group grants from the Canadian Institutes of Health Research to R.D. (CIHR, grant numbers: 15129 and 15176); individual grant from the Natural Sciences and Engineering Research Council of Canada to R.D. (NSERC, grant number: 217435-01); group grant from the Fonds de la Recherche en Santé du Québec (FRSQ) to R.D. (Groupe de Recherche sur le Système Nerveux Central, grant number: 5249). L.J. received a fellowship from the Fondation pour la Recherche Médicale, from the Groupe de Recherche sur le Système Nerveux Central of the FRSQ, and a Jasper fellowship from the Université de Montréal. The authors wish to express their gratitude to D. Veilleux for her excellent technical assistance, to C. Valiquette for his skilful programming of data analysis software and to F. Bernard for his help with figure making. We than W. D. Swink, M. K. Jones and R. Bergstedt from the Lake Huron Biological Station (MI, USA), R. McDonald and E. Achtemichuk of the Sea Lamprey Control Centre in Sault Ste. Marie (ON, Canada), and B.A. Young and W. Bouffard of the U.S. Fish and Wildlife Service of Vermont (VT, USA) for their kind supply of lampreys.

Abbreviations:

CNS, Central nervous system; dVn, nucleus of the trigeminal descending tract; EPSPs, Excitatory postsynaptic potentials; MLR, mesencephalic locomotor region; MRRN, middle rhombencephalic reticular nuclei; PSPs, Postsynaptic potentials; RS, Reticulospinal.


Didier Le Ray

Laurent Juvin

Dimitri Ryczko

Dr Réjean Dubuc
email: dubuc.rejean@uqam.ca

Juvin, Ryczko Le Ray

Supraspinal control of locomotion: The mesencephalic locomotor region

Keywords: locomotion, supraspinal control, mesencephalic locomotor region, sensory modulation, cholinergic transmission, lamprey

Locomotion is a basic motor function generated and controlled by genetically defined neuronal networks. The pattern of muscle synergies is generated in the spinal cord, whereas neural centers located above the spinal cord in the brainstem and the forebrain are essential for initiating and controlling locomotor movements. One such locomotor control center in the brainstem is the Mesencephalic Locomotor Region (MLR), first discovered in cats and later found in all vertebrate species tested to date.

Over the last years, we have investigated the cellular mechanisms by which this locomotor region operates in lampreys. The lamprey MLR is a well circumscribed region located at the junction between the midbrain and hindbrain. Stimulation of the MLR induces locomotion with an intensity that increases with the stimulation strength.

Glutamatergic and cholinergic monosynaptic inputs from the MLR are responsible for excitation of reticulospinal (RS) cells that in turn activate the spinal locomotor networks.

The inputs are larger in the rostral than in the caudal hindbrain RS cells. MLR stimulation on one side elicits symmetrical excitatory inputs in RS cells on both sides and this is linked to bilateral projections of the MLR to RS cells. In addition to its inputs to RS cells, the MLR activates a well-defined group of muscarinoceptive cells in the brainstem that feedback strong excitation to RS cells in order to amplify the locomotor output. Finally, the MLR gates sensory inputs to the brainstem through a muscarinic mechanism. It appears therefore that the MLR not only controls locomotor activity, but it also filters sensory influx during locomotion.

Introduction

The neural organization underlying locomotion -one of the most basic motor actsis remarkably similar in different species of vertebrates. The muscle synergies responsible for propulsion are generated by neural networks in the spinal cord interacting with sensory signals [START_REF] Grillner | Control of locomotion in bipeds, terapods, and fish[END_REF]Grillner, 1985;[START_REF] Rossignol | Neural control of stereotypic limb movements[END_REF][START_REF] Rossignol | Dynamic sensorimotor interactions in locomotion[END_REF]. These spinal networks, known as Central Pattern Generators (CPG) for locomotion, are activated and controlled by specific supraspinal structures, which also receive sensory inputs [START_REF] Armstrong | Supraspinal contributions to the initiation and control of locomotion[END_REF][START_REF] Orlovsky | Neural Control of Locomotion, From Mollusc to Man[END_REF][START_REF] Rossignol | Neural control of stereotypic limb movements[END_REF][START_REF] Rossignol | Dynamic sensorimotor interactions in locomotion[END_REF][START_REF] Shik | Neurophysiology of locomotor automatism[END_REF]. The supraspinal control of locomotion includes forebrain structures, specific locomotor centers in the forebrain and brainstem, and command cells in the lower brainstem that activate the spinal CPGs (Fig. 1). The detailed contribution of forebrain structures to locomotion has not been resolved yet. However, the role of motor cortex is better known; it contributes to precision walking requiring an exact foot placement, such as on an uneven terrain (Beloozerova et al., 1993a;Beloozerova et al., 1993b;[START_REF] Bretzner | Contribution of the motor cortex to the structure and the timing of hindlimb locomotion in the cat: a microstimulation study[END_REF]reviewed in Drew et al., 2008). The basal ganglia are believed to play a role in the selection of locomotor behaviors [START_REF] Grillner | Selection and initiation of motor behavior[END_REF][START_REF] Grillner | Neural bases of goaldirected locomotion in vertebrates--an overview[END_REF]. A striking feature relative to the supraspinal control of locomotion is the presence of forebrain and brainstem locomotor centers specifically dedicated to initiating and controlling locomotion (for reviews see: [START_REF] Armstrong | Supraspinal contributions to the initiation and control of locomotion[END_REF][START_REF] Dubuc | Locomotor regions in the midbrain (MLR) and diencephalon (DLR). Encyclopedia of Neuroscience[END_REF][START_REF] Dubuc | Locomotor regions in the midbrain (MLR) and diencephalon (DLR). Encyclopedia of Neuroscience[END_REF]Grillner, 1988;Grillner, 1997;[START_REF] Jordan | Initiation of locomotion in mammals[END_REF][START_REF] Jordan | Brain stem control of locomotion[END_REF][START_REF] Orlovsky | Neural Control of Locomotion, From Mollusc to Man[END_REF][START_REF] Whelan | Control of locomotion in the decerebrate cat[END_REF]. One such region is located in the diencephalon and another in the mesencephalon. These two locomotor control centers are respectively referred to as the Diencephalic Locomotor Region (DLR;[START_REF] El Manira | Diencephalic projection to reticulospinal neurons involved in the initiation of locomotion in adult lampreys Lampetra fluviatilis[END_REF][START_REF] Grillner | Neural bases of goaldirected locomotion in vertebrates--an overview[END_REF] and the Mesencephalic Locomotor Region (MLR; [START_REF] Shik | Control of walking and running by means of electric stimulation of the midbrain[END_REF]. This review article will focus primarily on the MLR and on recent findings obtained from one species of vertebrates, the lamprey.

The mesencephalic locomotor region (MLR) in vertebrates

The MLR has been identified in the 1960s by a research group in Moscow [START_REF] Shik | Control of walking and running by means of electric stimulation of the midbrain[END_REF]. This region receives inputs from the basal ganglia, the lateral hypothalamus and the medial hypothalamus through the periaqueductal gray matter [START_REF] Jordan | Initiation of locomotion in mammals[END_REF]. The mammalian MLR consists of two nuclei, the nucleus cuneiformis (CN) and the pedunculopontine nucleus (PPN). Electrical or chemical stimulation of the MLR induces bouts of locomotion [START_REF] Shik | Control of walking and running by means of electric stimulation of the midbrain[END_REF][START_REF] Garcia-Rill | Chemical activation of the mesencephalic locomotor region[END_REF] via the activation of the reticulospinal (RS) pathways (Gracia-Rill and Skinner 1987;[START_REF] Orlovsky | Work of the reticulo-spinal neurones during locomotion[END_REF][START_REF] Steeves | Autoradiographic demonstration of the projections from the mesencephalic locomotor region[END_REF]. The mechanisms that underlie the activation of the MLR when the basal ganglia are activated are not fully understood. It appears that the selection of a relevant motor program by the basal ganglia would rely on disinhibition [START_REF] Hikosaka | Role of the forebrain in oculomotor function[END_REF]. It was proposed that the ventral pallidum and the SNr could act similarly and inhibit the MLR [START_REF] Grillner | Intrinsic function of a neuronal network -a vertebrate central pattern generator[END_REF][START_REF] Takakusaki | Forebrain control of locomotor behaviors[END_REF]. As such, locomotion would result from a disinhibition of the MLR by the ventral pallidum or the SNr. Experimental findings support this hypothesis. For instance, electrical stimulation of the SNr prevents MLRinducted locomotion [START_REF] Takakusaki | Function of cortical basal nuclei: Pathophysiology of Parkinson's disease][END_REF], suggesting that the SNr inhibits locomotor activity. In addition, injections of the GABA A antagonist bicuculline in the MLR induce bouts of locomotion [START_REF] Garcia-Rill | Posterior midbrain-induced locomotion[END_REF].

Classically, electrical or chemical stimulation of the MLR in decerebrate cats elicits motor output that has been subdivided in two phases. First, there is an increase in muscle tone allowing the animal to fully support its weight; this is followed by the locomotor phase [START_REF] Shik | Control of walking and running by means of electric stimulation of the midbrain[END_REF]. In the first initial experiments describing the MLR of cats, it was found that the frequency of locomotion was graded in relation to the stimulation intensity. At low MLR stimulation intensities, the animals walked; as the intensity increased, they trotted and then galloped. This initial stunning observation provided the basis for qualifying this particular brainstem region as "dedicated to control a locomotor output". The exact anatomical substrate of the MLR has been on the other hand subjected to debate. The most effective site to induce locomotion was a region comprising the CN and possibly a part of the PPN [START_REF] Mori | Site-specific postural and locomotor changes evoked in awake, freely moving intact cats by stimulating the brainstem[END_REF]. Activation of the CN in a walking cat increased the speed of locomotion [START_REF] Sterman | Modification of locomotor performance by reticular formation and basal forebrain stimulation in the cat: evidence for reciprocal systems[END_REF].

Similarly in rats, chemical activation of the PPN elicited only brief episodes of locomotion in comparison to those elicited by activation of the CN (Garcia-Rill, 1985;Garcia-Rill, 1990). The CN was thus proposed as the most effective site eliciting locomotion (for review see Grillner 1997). Whether a part or the entire PPN participated in the initiation of locomotion was not as clear; because of the proximity of these two structures, experimental results have been difficult to interpret. Several studies have tried to dissect the specific role of the CN and the PPN in the control of locomotion.

Because locomotion is also strongly modulated by its behavioural context, it was suggested that the MLR could be subdivided in different modules that would be activated in a context dependent manner [START_REF] Sinnamon | Preoptic and hypothalamic neurons and the initiation of locomotion in the anesthetized rat[END_REF]. For instance, it was proposed the MLR elicited locomotion in three different contexts and could therefore be subdivided into three main functional areas: "an exploratory system", "an appetitive system" and "a defensive system" [START_REF] Sinnamon | Preoptic and hypothalamic neurons and the initiation of locomotion in the anesthetized rat[END_REF]. This concept of organization was supported by experimental findings. For example, injection of glutamate in the CN induced a sequence of freezing, darting and fast running (Mitchell, 1988a(Mitchell, , 1988b). An "escape" locomotor behavior was observed both in cats and rats, when the CN was stimulated (Depoortere, 1990a(Depoortere, , 1990b;;[START_REF] Mori | Site-specific postural and locomotor changes evoked in awake, freely moving intact cats by stimulating the brainstem[END_REF][START_REF] Sirota | Locomotion of the cat on stimulation of the mesencephalon (Article in Russian) Fiziol[END_REF]. On the other hand, injections of GABA antagonist in the PPN induced locomotion that was apparently more related to startle [START_REF] Ebert | The mesencephalic locomotor region is activated during the auditory startle response of the unrestrained rat[END_REF][START_REF] Garcia-Rill | Posterior midbrain-induced locomotion[END_REF]. It was proposed that the PPN would itself be divided into two specific regions, a ventral and a dorsal component [START_REF] Milner | Electrical and chemical activation of the mesencephalic and subthalamic locomotor regions in freely moving rats[END_REF]. The dorsal part of the PPN would be part of the MLR as a locomotor controlling region, whereas the ventral part would consist of a muscle tone inhibitory system [START_REF] Takakusaki | Function of cortical basal nuclei: Pathophysiology of Parkinson's disease][END_REF]. Chemical activation of the dorsal part of the PPN was shown to increase locomotion in intact rats; an opposite effect was observed when ventral part of the PPN was activated [START_REF] Milner | Electrical and chemical activation of the mesencephalic and subthalamic locomotor regions in freely moving rats[END_REF]. Functional MRI has recently revealed an increased BOLD signal in the MLR during mental imagery of walking and running in healthy volunteers [START_REF] Jahn | Imaging human supraspinal locomotor centers in brainstem and cerebellum[END_REF]. In addition, recent clinical trials have shown improvement in posture and gait after stimulating the pedunculopontine nucleus (a part of the MLR [START_REF] Jordan | Initiation of locomotion in mammals[END_REF]

in

Parkinson's patients [START_REF] Lozano | Deep brain stimulation for parkinsonian gait disorders[END_REF][START_REF] Mazzone | Implantation of human pedunculopontine nucleus: a safe and clinically relevant target in Parkinson's disease[END_REF][START_REF] Stefani | Bilateral deep brain stimulation of the pedunculopontine and subthalamic nuclei in severe Parkinson's disease[END_REF]. There is considerable new evidence that cholinergic mesencephalic neurons would be involved in gait and postural disorders in Parkinson's disease [START_REF] Karachi | Cholinergic mesencephalic neurons are involved in gait and postural disorders in Parkinson disease[END_REF].

The MLR does not project directly to the spinal cord, but it activates hindbrain RS cells that in turn activate spinal cord locomotor networks (Garcia-Rill and Skinner, 1987a, 1987b[START_REF] Garcia-Rill | The pedunculopontine nucleus[END_REF][START_REF] Grillner | Control of locomotion in bipeds, terapods, and fish[END_REF] for review see [START_REF] Rossignol | Neural control of stereotypic limb movements[END_REF]. After its first discovery in cats [START_REF] Shik | Control of walking and running by means of electric stimulation of the midbrain[END_REF], the MLR was also identified in many other species of vertebrates including rats [START_REF] Skinner | The mesencephalic locomotor region (MLR) in the rat[END_REF], stingrays (Bernaud, 1991), guinea pigs (Marlkinsky, 1991), lampreys [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF], salamanders [START_REF] Cabelguen | Bimodal locomotion elicited by electrical stimulation of the midbrain in the salamander Notophthalmus viridescens[END_REF] and rabbits [START_REF] Musienko | Postural performance in decerebrated rabbit[END_REF]. The most remarkable feature of the MLR was the locomotor activity that increased in speed as the stimulation of the MLR was increased. This was seen in all the animal species investigated. Moreover, it was shown in salamanders that MLR stimulation elicited the two modes of locomotor activity displayed by these animals, walking and swimming. At low MLR stimulation, the motor output elicited was characterized by limb movements associated with stepping.

As the MLR stimulation was increased, the stepping movements increased in frequency.

With further increases in stimulation strength, the limbs moved under the animal's belly and swimming movements were elicited. Altogether, these observations indicate that the MLR is responsible for the initiation of swimming, walking, trotting or galloping in different species of vertebrates. Moreover, observations made in salamanders indicate that this brainstem region can control two different modes of locomotion in the same animal.

The supraspinal control of locomotion in lampreys

The lamprey model has provided first-hand information on the cellular mechanisms of vertebrate locomotion. The general organization of the lamprey nervous system is strikingly similar to that of mammals, but the presence of considerably fewer neurons results in a reduced complexity that has been very useful to examine the cellular mechanisms underlying motor behavior. The lamprey model has paved the way for several important discoveries. One of these is the detailed characterization of a vertebrate CPG for locomotion [START_REF] Buchanan | Newly identified 'glutamate interneurons' and their role in locomotion in the lamprey spinal cord[END_REF]. The brainstem mechanisms responsible for initiating and controlling locomotion have also been successfully uncovered in lampreys with an array of in vitro techniques, with the added benefit of including all relevant brain structures needed for locomotor control, and the ability to monitor the active locomotor behavior.

One common feature of all vertebrate species is the crucial role played by RS cells in relaying MLR inputs to the spinal CPGs for locomotion. The RS cells receive peripheral and central inputs, integrate these signals, and generate a coherent descending motor command. In lampreys, RS cells have been described anatomically and physiologically.

They constitute about 90% of the neurons projecting to the spinal cord [START_REF] Bussières | Les systèmes descendants chez la lamproie. Étude anatomique et fonctionnelle[END_REF]Davis andMcClellan, 1994a, 1994b;[START_REF] Stefani | Bilateral deep brain stimulation of the pedunculopontine and subthalamic nuclei in severe Parkinson's disease[END_REF]. The lamprey RS cells are located in one mesencephalic reticular nucleus (MRN) and in three rhombencephalic reticular nuclei, the anterior (ARRN), the middle (MRRN) and the posterior (PRRN; [START_REF] Brodin | Reticulospinal neurons in lamprey: Transmitters, synaptic interactions and their role during locomotion[END_REF]Davis andMcClellan, 1994a, 1994b;[START_REF] Nieuwenhuys | Topological analysis of the Brain Stem of the lamprey Lampetra fluviatilis[END_REF][START_REF] Nieuwenhuys | The brain of the lamprey in a comparative perspective[END_REF][START_REF] Swain | Cytoarchitecture of spinalprojecting neurons in the brain of the larval sea lamprey[END_REF]. There are around 2500 RS cells and ≈ 85% of them reside in the PRRN and MRRN [START_REF] Bussières | Les systèmes descendants chez la lamproie. Étude anatomique et fonctionnelle[END_REF]. There are clear homologies with reticular nuclei in other vertebrate species, including mammals [START_REF] Cruce | Evolution of motor systems: the reticulospinal pathways[END_REF]. The ARRN and MRRN are located in the "lamprey pons" and contain large RS cells (Müller cells; [START_REF] Rovainen | Physiological and anatomical studies on large neurons of central nervous system of the sea lamprey (Petromyzon marinus) I. Müller and Mauthner cells[END_REF] that send their axons medially in the spinal cord. These two nuclei are similar to the superior and middle reticular nuclei in fish, which are respectively homologous to nuclei pontis oralis and caudalis of mammals [START_REF] Cruce | Evolution of motor systems: the reticulospinal pathways[END_REF]. The PRRN, located in the "lamprey medulla oblongata", contains RS cells that send axons laterally, similarly to the nucleus gigantocellularis in mammals (also discussed in [START_REF] Brocard | Differential contribution of reticulospinal cells to the control of locomotion induced by the mesencephalic locomotor region[END_REF]. The axons of large RS neurons make synaptic contacts with several classes of spinal neurons and some of these are part of the locomotor CPG [START_REF] Buchanan | Identification of interneurons with colateral, caudal axons in the lamprey spinal cord: synaptic interactions and morphology[END_REF][START_REF] Ohta | Monosynaptic excitatory amino acid transmission from the posterior rhombencephalic reticular nucleus to spinal neurons involved in the control of locomotion in lamprey[END_REF][START_REF] Rovainen | Synaptic interactions of reticulospinal neurons and nerve cells in the spinal cord of the sea lamprey[END_REF]. The prevalent neurotransmitter is glutamate, although 5-HT and neuropeptides are also present in some RS cells (reviewed in [START_REF] Brodin | Reticulospinal neurons in lamprey: Transmitters, synaptic interactions and their role during locomotion[END_REF].

As in other species, the MLR was first characterized functionally in the lamprey by its ability to initiate and control locomotion when electrically stimulated [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF]. The neuroanatomical substrate of the MLR has remained more elusive, but recent studies have provided new insights relative to this. Stimulation experiments demonstrated that the brainstem area responsible for the control of locomotion (i.e., the MLR) is located at the mesopontine border, close to the wall of the mesencephalic ventricle [START_REF] Brocard | Differential contribution of reticulospinal cells to the control of locomotion induced by the mesencephalic locomotor region[END_REF][START_REF] Ray | Muscarinic modulation of the trigeminoreticular pathway in lampreys[END_REF][START_REF] Brocard | The transformation of a unilateral locomotor command into a symmetrical bilateral activation in the brainstem[END_REF][START_REF] Ray | Nicotinic activation of reticulospinal cells involved in the control of swimming in lampreys[END_REF][START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF][START_REF] Smetana | A parallel cholinergic brainstem pathway for enhancing locomotor drive[END_REF]. The most efficient area for eliciting locomotion is a region containing a group of cholinergic cells close to a large RS cell, I1. This area corresponds to the caudal pole of the laterodorsal tegmental (LTD) nucleus. Increasing the MLR stimulation intensity elicited faster and faster swimming movements of greater amplitude [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF]Fig. 2).

The forebrain projections to the lamprey MLR have not been as extensively studied as in mammals. There are GABAergic inputs from the caudal portion of the medial pallium [START_REF] Ménard | GABAergic projections to the mesencephalic locomotor region in the lamprey Petromyzon marinus[END_REF], a region that could correspond to the amygdala of mammals. As shown in mammals, the lamprey MLR is under a tonic inhibition.

Locomotion induced by glutamate ejection in the MLR is suppressed by co-ejection of GABA receptor agonist. Moreover, the GABA receptor antagonist, gabazine elicits bouts of swimming when injected in the MLR of a semi-intact lamprey [START_REF] Ménard | GABAergic projections to the mesencephalic locomotor region in the lamprey Petromyzon marinus[END_REF].

The forebrain connections to the MLR as well as their neurochemical identity are still not fully identified.

Downstream effects of the MLR

Inputs to the MLR in lampreys are just starting to be explored, but the output projections from the MLR have been defined more extensively. The MLR projections are relayed within the hindbrain reticular formation. Several lines of evidence indicate that the MLR projections to RS neurons are monosynaptic. First, electrical stimulation of the MLR evokes short-latency EPSPs that are maintained during repetitive MLR stimulation at high frequency. Second, the synaptic responses in RS cells, in a Ringer's solution enriched in divalent cations, still followed a 25-Hz MLR stimulation [START_REF] Brocard | Differential contribution of reticulospinal cells to the control of locomotion induced by the mesencephalic locomotor region[END_REF][START_REF] Ray | Nicotinic activation of reticulospinal cells involved in the control of swimming in lampreys[END_REF].

Glutamatergic and Cholinergic MLR outputs

The MLR of mammals contains populations of cholinergic neurons (see [START_REF] Jordan | Initiation of locomotion in mammals[END_REF]. It is also the case in lampreys where cholinergic neurons have been found within the isthmic region [START_REF] Pombal | Distribution of choline acetyltransferaseimmunoreactive structures in the lamprey brain[END_REF]. The possibility that the cholinergic neurons would be located within the MLR per se was addressed directly using immunohistochemical ChAT staining at the site of electrical stimulation that was found to initiate and control locomotion in lampreys (Le [START_REF] Ray | Nicotinic activation of reticulospinal cells involved in the control of swimming in lampreys[END_REF]. Two distinct groups of ChAT-immunoreactive neurons were observed in an area that included the isthmus and the caudal mesencephalon. One group consisted of densely clustered cells located medially close to the ventricular border. Another group of cholinergic cells was more loosely distributed further rostrally and laterally within the tegmentum. Comparison with other species (cats: [START_REF] Mitani | Cholinergic projections from the laterodorsal and pedunculopontine tegmental nuclei to the pontine gigantocellular tegmental field in the cat[END_REF]rats: Jones, 1990;and amphibians: Marin et al., 1997) suggested that the first group would correspond to the laterodorsal tegmental nucleus (LDT), whereas the second one to the PPN. In mammals (Garcia-Rill and Skinner, 1987a;[START_REF] Lai | Brainstem projections to the ventromedial medulla in cat: retrograde transport horseradish peroxidase and immunohistochemical studies[END_REF][START_REF] Mesulam | Cholinergic innervation of cortex by the basal forebrain: cytochemistry and cortical connections of the septal area, diagonal band nuclei, nucleus basalis (substantia innominata), and hypothalamus in the rhesus monkey[END_REF][START_REF] Skinner | Locomotor projections from the pedunculopontine nucleus to the medioventral medulla[END_REF], cholinergic neurons in these nuclei project to the reticular formation. This suggests a role for ACh in the MLR control of locomotion. However, numerous neurons projecting to RS cells are located in regions of the lamprey MLR that do not contain ChAT-positive neurons [START_REF] Brocard | The transformation of a unilateral locomotor command into a symmetrical bilateral activation in the brainstem[END_REF]. This implies that other neurotransmitter systems may also be involved (see below). Electrophysiological experiments are also in accord with a role of cholinergic transmission in mediating MLR effects onto RS cells. In mammals and birds, local injections of cholinergic agonists in the reticular formation elicit locomotion (Garcia-Rill & Skinner, 1987a;[START_REF] Sholomenko | Avian locomotion activated by brainstem infusion of neurotransmitter agonists and antagonists. I. Acetylcholine excitatory amino acids and substance P[END_REF]. In these animal species however, a direct link between a cholinergic command originating from the MLR and locomotion was not established. The role of cholinergic inputs was examined in semi-intact preparations of lampreys. The MLR was stimulated at 5 Hz, and the evoked EPSPs in RS cells summed up until the threshold for spiking was reached and swimming activity was elicited (see also [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF]. In the presence of the nicotinic antagonist D-tubocurarine (30-50 µM), applied selectively to the brainstem and not to the spinal cord using a partioned recording chamber, the membrane potential of RS neurons remained mostly below spiking threshold and swimming was prevented, even under MLR stimulation intensity that would normally induced swimming. In fact, swimming could only be induced by significantly increasing the MLR stimulation intensity. In addition, the application of the selective cholinesterase inhibitor physostigmine (100 µM) to the "brainstem" chamber partition largely enhanced the MLR-evoked compound EPSPs and facilitated the RS cell depolarization and the occurrence of swimming (Le [START_REF] Ray | Nicotinic activation of reticulospinal cells involved in the control of swimming in lampreys[END_REF]. The amplitude and slope of the MLR-evoked monosynaptic EPSP were largely, but not completely blocked by the nicotinic antagonists D-tubocurarine (30-100 µM) or α-bungarotoxin (0.1 µM).

Adding a mixture of NMDA and non-NMDA glutamate receptor antagonists (200 µM AP5 and 25 µM CNQX, respectively) further reduced the EPSPs. Taken together, these results suggested that MLR inputs to RS cells use cholinergic and glutamatergic transmission.

Additional support for this was the observation that a direct application of nicotinic receptor agonists in either the MRRN or the PRRN evoked swimming in a semi-intact preparation. Comparable results were obtained in an in vitro isolated brainstem-spinal cord preparation in which fictive locomotion was induced by nicotinic receptor agonists.

In addition, when applied on preparation already displaying a slow fictive locomotor activity under NMDA perfusion, cholinergic agonists speeded up the locomotor rhythm early after their application. This effect did not occur when the fictive locomotor rhythm had already stabilized to its faster rhythm under NMDA perfusion or after a previous ejection of the cholinergic agonist had already accelerated the fictive locomotor rhythm.

TTX-resistant depolarizing responses were generated in intracellularly recorded RS neurons by local application of nicotinic agonists onto the recorded cell. When repeated before the RS neuron membrane potential returned to resting value, the nicotinic responses showed summation properties allowing the neuron to reach spiking threshold and to generate a sustained firing of action potentials. Such temporal summation of responses may be an important for the slow buildup of RS cell depolarization and the delayed swimming onset that occur at low intensities of MLR stimulation. Indeed, in lampreys [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF] as in mammals (Garcia-Rill & Skinner, 1987b;[START_REF] Iwakiri | Stimulus effects of the medial pontine reticular formation and the mesencephalic locomotor region upon medullary reticulospinal neurons in acute decerebrate cats[END_REF] several seconds of repeated MLR stimulation are required to induce locomotion. Furthermore, the onset delay of swimming decreases as the intensity or frequency of stimulation increases, probably due to the enhancement of RS depolarization by the nicotinic response summation. According to this, the buildup of the response to a 5 Hz stimulation of the MLR was dramatically reduced in the presence of a nicotinic antagonist and largely increased in the presence of physostigmine, which resulted in the blockade or the facilitation of the initiation of swimming activity, respectively (Le [START_REF] Ray | Nicotinic activation of reticulospinal cells involved in the control of swimming in lampreys[END_REF].

Differential MLR inputs to RS cells in the MRRN and PRRN

Although it is well established that the MLR elicits locomotor behavior by activating RS cells in different species of vertebrates, the detailed connectivity between the MLR and RS cells has remained unresolved. Such connectivity was examined in lampreys by comparing the recruitment of large MRRN and PRRN RS cells using paired intracellular recordings and increasing stimulation strength of the MLR [START_REF] Brocard | Differential contribution of reticulospinal cells to the control of locomotion induced by the mesencephalic locomotor region[END_REF]; such an approach was not used in any other vertebrate species previously. MRRN cells displayed spiking activity at low MLR stimulation strength, whereas PRRN cells begin to discharge at higher intensities, when MRRN cells have already reached their maximal spiking frequency. The respective contribution of the MRRN and PRRN in locomotor control was also investigated by selectively injecting a mixture of ionotropic glutamate receptor antagonists in each of the two reticular nuclei [START_REF] Brocard | Differential contribution of reticulospinal cells to the control of locomotion induced by the mesencephalic locomotor region[END_REF]. Injections over the entire MRRN prevented locomotion, even during MLR stimulation at high intensities. Injections over the PRRN only decreased locomotion intensity. According to these observations, RS cells receive differential inputs from the MLR, such that RS cells in the rostral hindbrain discharge more importantly at low swimming intensities and RS cells located more caudally begin spiking at higher swimming speeds.

Bilateral MLR inputs to RS cells

Another interesting aspect of the MLR is that it will elicit bilaterally symmetrical locomotion even when it is stimulated only on one side [START_REF] Shik | Control of walking and running by means of electric stimulation of the midbrain[END_REF][START_REF] Shik | Neurophysiology of locomotor automatism[END_REF][START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF]. This has been a feature observed in the different animal species investigated to date. A unilateral electrical (E) or chemical (C) activation of the MLR produces symmetrical locomotion in the guinea pig (C: [START_REF] Marlinsky | The effect of procaine injection into the medullary reticular formation on forelimb muscle activity evoked by mesencephalic locomotor region and vestibular stimulation in the decerebrated guinea-pig[END_REF], lamprey (E and C: [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF], rabbit (E: [START_REF] Musienko | Postural performance in decerebrated rabbit[END_REF], rat (E: [START_REF] Skinner | The mesencephalic locomotor region (MLR) in the rat[END_REF], salamander (E: [START_REF] Cabelguen | Bimodal locomotion elicited by electrical stimulation of the midbrain in the salamander Notophthalmus viridescens[END_REF], and stingray (E: [START_REF] Bernau | Identification of the midbrain locomotor region and its relation to descending locomotor pathways in the Atlantic stingray, Dasyatis sabina[END_REF] for review see [START_REF] Orlovsky | Neural Control of Locomotion, From Mollusc to Man[END_REF]. One question relates to how a unilateral stimulation of the MLR is converted into a bilateral symmetrical locomotor bout? At which level (or levels) does the symmetry appear?

We recently addressed these questions in a study combining anatomical, electrophysiological, Ca 2+ imaging, and kinematic analysis in lampreys. We found that MLR inputs to RS cells are at least partly responsible for the transformation of a unilateral MLR stimulation into bilaterally symmetrical locomotor output [START_REF] Brocard | The transformation of a unilateral locomotor command into a symmetrical bilateral activation in the brainstem[END_REF]. Direct evidence for the symmetry of MLR inputs to RS cells was provided by simultaneously recording the intracellular responses of bilateral pairs of identifiable homologous RS cells from the MRRN and the PRRN to stimulation of the MLR on one side [START_REF] Brocard | The transformation of a unilateral locomotor command into a symmetrical bilateral activation in the brainstem[END_REF]. The synaptic responses on both sides were very similar in shape, amplitude, and threshold intensity. Increasing the intensity of MLR stimulation produced a strikingly similar increase in the magnitude of the responses on both sides (Fig. 3). Because the technique of intracellular recordings limits our conclusions to a small number of large-size paired RS cells, Ca 2+ imaging experiments were performed on brainstem-isolated preparations. In accord to what been found in the large cells, a bilaterally symmetrical activation of smaller-sized RS cells of the MRRN and PRRN was seen when unilaterally stimulating the MLR.

Monosynaptic inputs from the MLR to RS neurons were known to be present in lampreys [START_REF] Brocard | Differential contribution of reticulospinal cells to the control of locomotion induced by the mesencephalic locomotor region[END_REF]. In [START_REF] Brocard | The transformation of a unilateral locomotor command into a symmetrical bilateral activation in the brainstem[END_REF], it was shown that MLR projects monosynaptically to RS cells not only on the ipsilateral, but also on the contralateral side. In a high-divalent cation solution, the synaptic responses of simultaneously-recorded homologous RS cells persisted and exhibited a constant latency during high-frequency stimulation. Moreover, during gradual replacement of normal Ringer's solution with a Ca 2+ -free solution, the magnitude of responses showed a gradual reduction with a similar time course in the homologous RS cells. These results provided strong evidence for monosynaptic inputs from the MLR to RS cells on both sides.

Simultaneous recordings of homologous RS cells of the MRRN on both sides also revealed a symmetrical output in frequency when bouts of symmetrical swimming (Fig. 4) are generated by unilateral stimulation of the MLR in a semi-intact preparation [START_REF] Brocard | The transformation of a unilateral locomotor command into a symmetrical bilateral activation in the brainstem[END_REF]. When increasing the MLR stimulation, the increase in discharge frequency was identical for the left and right RS cells, and the swimming frequency proportionally increased [START_REF] Brocard | The transformation of a unilateral locomotor command into a symmetrical bilateral activation in the brainstem[END_REF].

Anatomical experiments confirmed that, in the MLR on one side, neurons projecting to the left MRRN were intermingled with neurons projecting to the right MRRN: a unilateral injection of a retrograde tracer into the MRRN revealed labeled cells bilaterally in the MLR [START_REF] Brocard | The transformation of a unilateral locomotor command into a symmetrical bilateral activation in the brainstem[END_REF]; see also [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF]. Bilateral injections of two different tracers in the MRRN revealed that the same MLR cells very rarely projected to both MRRN at the same time [START_REF] Brocard | The transformation of a unilateral locomotor command into a symmetrical bilateral activation in the brainstem[END_REF]. The anatomical projections were bilaterally asymmetrical: retrograde markers injected in the MRRN on one side always revealed fewer labeled cells in the contralateral MLR, indicating that the descending projections from the MLR to RS neurons were slightly biased ipsilaterally [START_REF] Brocard | The transformation of a unilateral locomotor command into a symmetrical bilateral activation in the brainstem[END_REF]. Because the MLR inputs to RS cells are perfectly symmetrical as well as the swimming behavior elicited upon a unilateral stimulation of the MLR, the anatomical asymmetry must then be physiologically compensated [START_REF] Brocard | The transformation of a unilateral locomotor command into a symmetrical bilateral activation in the brainstem[END_REF].

Bilateral descending projections from the MLR to RS neurons have also been described anatomically in the cat [START_REF] Garcia-Rill | Connections of the mesencephalic locomotor region (MLR). II. Afferents and efferents[END_REF][START_REF] Steeves | Autoradiographic demonstration of the projections from the mesencephalic locomotor region[END_REF] and rat [START_REF] Garcia-Rill | Projections of the mesencephalic locomotor region in the rat[END_REF]. Electrophysiological experiments in cats revealed bilateral inputs (Garcia-Rill and Skinner, 1987;[START_REF] Orlovsky | Work of the reticulo-spinal neurones during locomotion[END_REF]. The bilateral anatomical projections seen cats were on the other hand asymmetrical [START_REF] Steeves | Autoradiographic demonstration of the projections from the mesencephalic locomotor region[END_REF].

Injections of [3H]proline and [3H]leucine into the MLR revealed descending neurons

from the MLR that were located mainly on the ipsilateral side. In line with this anatomical observation, Garcia-Rill and Skinner (1987b) provided electrophysiological evidence in the cat that the MLR projected mainly to ipsilateral RS cells, that in turn projected to the ipsilateral spinal cord. Noga and colleagues showed that the pattern of post synaptic responses measured intracellularly in alpha-motoneurons (innervating flexor, extensor, or bi-functional muscles) in the L6-L7 spinal cord segments was similar whether the ipsilateral or the contralateral MLR was stimulated. The segmental latency of the first locomotor EPSP detected in motoneuronal response was also similar on both sides [START_REF] Noga | Mechanism for activation of locomotor centers in the spinal cord by stimulation of the mesencephalic locomotor region[END_REF]. They proposed that the slight asymmetry of the descending signal generated by unilateral MLR stimulation could be compensated by descending RS neurons projecting contralaterally directly or indirectly through spinal commissural neurons, to end up with a symmetrical motor output [START_REF] Noga | Mechanism for activation of locomotor centers in the spinal cord by stimulation of the mesencephalic locomotor region[END_REF]. As indicated above, we found in lampreys an anatomical bilateral asymmetry in the projections between the MLR-RS cell projections. On the other hand, our physiological experiments revealed a striking symmetry in the MLR-RS inputs, suggesting that the anatomical asymmetry was fully compensated physiologically. Whether this is the case in mammals remains to be established. The physiological connections between the MLR in RS cells on both sides have not been examined directly. In an attempt to do so, Orlovsky used intracellular recordings of RS cells and stimulated the MLR on each side with two different stimulating electrodes [START_REF] Orlovsky | Work of the reticulo-spinal neurones during locomotion[END_REF]. Small differences in the positioning of the stimulating electrodes and/or differences in the impedance of the two stimulating electrodes could account for differences in the size of synaptic responses of RS cells on both sides.

It appears therefore that unilateral activation of the MLR produces a bilateral symmetrical locomotor output. The bilateral projections from the MLR to RS cells are likely to play a crucial role in the symmetrical locomotor activity. Further experiments are needed to establish the contribution of bilateral connections in the spinal cord to this symmetry as well as the possible contribution of sensory inputs. Moreover, it is not known whether crossing connections innervating the RS cells themselves in the hindbrain could also strengthen the bilateral symmetry observed during locomotion induced by a unilateral MLR stimulation. These issues should be examined further.

A locomotor boosting mechanism within the brainstem

The MLR has traditionally been described as a neural control area in the brainstem, which activates specific populations of reticulospinal neurons to initiate and control locomotion. As such, the MLR has been considered as part of a serial control system for locomotion (Forebrain structures → MLR → RS cells → spinal CPGs) Recently, we described in lampreys a parallel projection from the MLR to a group of hindbrain neurons that, in turn, provide additional excitation to reticulospinal cells to amplify the locomotor output. These interesting findings were made as we were examining the role of muscarinic receptor activation of RS cells [START_REF] Smetana | Muscarinic receptor activation elicits sustained, recurring depolarization in reticulospinal neurons[END_REF].

We found that lamprey RS cells were activated by bath application of a cholinergic muscarinic agonist. Muscarine elicited sustained membrane depolarizations (~ 5 s duration) in RS cells that recurred at periodicity of ~ 60s. Interestingly, the depolarizations occurred simultaneously in pairs of homologous RS cells recorded intracellularly and calcium imaging showed that entire populations of RS cells were activated synchronously. The sustained depolarizations were also associated with ventral discharges, suggesting they could somehow participate in locomotion.

The effects of muscarine disappeared when TTX was added to the perfusion Ringer's suggesting that muscarine was not acting directly onto RS cells, but most likely through neurons that were presynaptic to the RS cells. Indeed, it was found that the depolarizations resulted from the activation a group of hindbrain muscarinoceptive interneurons that projected to RS neurons. Lesion studies as well as local muscarine injections revealed that the population of muscarinoceptive cells was located between the caudal border of the MRRN and rostral border of the PRRN. Bilateral injections of atropine in this region prevented the depolarization of RS neurons when muscarine was bath applied.

There were cells in this region showing immunoreactivity for muscarinic receptors. In addition, calcium imaging experiments revealed that cells in this region displayed sustained rises in intracellular calcium to bath application of muscarine. These calcium responses persisted in the presence of TTX [START_REF] Smetana | A parallel cholinergic brainstem pathway for enhancing locomotor drive[END_REF]. It was also shown that the muscarinoceptive cells provided strong bilateral glutamatergic inputs to the RS cells. Paired recordings of RS neurons and muscarinoceptive interneurons were carried out; depolarization of the muscarinoceptive cell induced short latency EPSCs in the RS cell, suggesting that the connections were monosynaptic [START_REF] Smetana | A parallel cholinergic brainstem pathway for enhancing locomotor drive[END_REF].

The physiological significance of these connections was then determined. First, it was found that these muscarinoceptive cells received inputs from the MLR that were blocked by the muscarinic receptor antagonist, atropine. The direct connectivity between the MLR and the muscarinoceptive neurons was also confirmed anatomically.

Cells were labeled and the MLR and both sides after injecting biocytin into the muscarinoceptive cell region. These results confirmed that the MLR not only projected to RS neurons MLR, but it also projected to a group of muscarinoceptive cells that in turn sent by natural excitation to RS neurons. This parallel pathway amplifies significantly the synaptic input received by the RS cells during the activation of the MLR.

The specific role was confirmed using a semi-intact swimming preparation. Bilateral injections of atropine in the muscarinoceptive cell area considerably depressed the depolarization responses are the RS cells to MLR stimulation and modified the locomotor output (Fig. 5). The linear relation between swimming frequency and MLR stimulation intensity was dramatically depressed by the bilateral injection of atropine in the muscarinoceptive neurons area [START_REF] Smetana | A parallel cholinergic brainstem pathway for enhancing locomotor drive[END_REF].

There is a strikingly linear relationship between the MLR stimulation intensity and locomotion frequency in many animal species. We have now found in lampreys that when the MLR is intensively activated, a population of muscarinoceptive cells located in the hindbrain is recruited to literally "boost" the locomotor output. Whether this mechanism is present in other vertebrate species remains to be determined. In rat, the ejection of cholinergic agonists (carbachol, methacholine and arecoline) in the medioventral medulla induces locomotion that is prevented by atropine, thus suggesting a role for muscarinic receptors [START_REF] Kinjo | Medioventral medulla-induced locomotion[END_REF]. The neural substrate is however still unknown in mammals.

Gating of sensory inputs on RS neurons by the MLR

During locomotion, sensory inputs shape the activity of the central generating and controlling neural networks according to external and internal constraints. In turn, the central networks gate sensory influx [START_REF] Brown | Studies in the physiology of the nervous system. VIII: neural balance and reflex reversal with a note on progression in the decerebrate guinea pig[END_REF][START_REF] Grillner | Locomotion in the spinal cat[END_REF][START_REF] Grillner | Contralateral reflex reversal controlled by limb position in the acute spinal cat injected with clonidine i[END_REF][START_REF] Rossignol | An analysis of mechanisms controlling the reversal of crossed spinal reflexes[END_REF]. Gating has been described extensively in the mammalian spinal cord [START_REF] Hultborn | State-dependent modulation of sensory feedback[END_REF][START_REF] Krawitz | State-dependent hyperpolarization of voltage threshold enhances motoneurone excitability during fictive locomotion in the cat[END_REF][START_REF] Rossignol | Stimulus--response relationships during locomotion[END_REF], but the underlying cellular mechanisms have not been identified yet. In lampreys, we found that muscarinic receptor activation depressed sensory inputs to RS cells (Le [START_REF] Ray | Muscarinic modulation of the trigeminoreticular pathway in lampreys[END_REF]. The efficacy of the sensory-motor connection between trigeminal afferents and RS neurons was then tested in the context of a MLR-induced locomotor behavior (Le [START_REF] Ray | A neuronal substrate for a state-dependent modulation of sensory inputs in the brainstem[END_REF]. For this purpose, intracellular recordings were made from RS cells in isolated brainstem preparations while the trigeminal sensory root on one side was electrically stimulated at a low frequency (0.1 Hz). The trigeminal EPSP was monitored before and after a 3 Hz train of stimuli was applied to the MLR for 15 s. Consecutively to MLR tonic activation, a powerful depression (>60%) of the trigeminal EPSP was observed in RS cells (Fig. 6). On average, both the peak and amplitude of the synaptic responses were significantly decreased and showed a progressive recovery about 30 min in vitro (Le [START_REF] Ray | A neuronal substrate for a state-dependent modulation of sensory inputs in the brainstem[END_REF].

It was found that the depression of the trigeminal-evoked EPSPs also depended on the level of MLR activation (Le [START_REF] Ray | A neuronal substrate for a state-dependent modulation of sensory inputs in the brainstem[END_REF]. Different frequencies of stimulation were randomly applied to the MLR in order to reproduce different levels of locomotor activity in the isolated brainstem. We found that the level of depression was linearly correlated to the frequency of the MLR stimulation. The higher was the MLR stimulation frequency, the stronger was trigeminal EPSP depression until a maximal depression was obtained at 7-8 Hz. Increasing the stimulation frequency further did not produce more depression. Interestingly, the sensory depression was maximal for MLR stimulation frequencies that were sub-maximal for swimming speeds (see [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF].

The time course of the depressive effects produced by the activation of the MLR suggested the involvement of metabotropic mechanisms, and because the MLR contains cholinergic neurons, the implication of muscarinic receptors was tested. Activating the in the presence of the muscarinic receptor antagonists, atropine, MLR stimulation produced a far less depression of trigeminal EPSPs in RS cells (~15% EPSP reduction).

There was still a residual depression suggesting that other neurotransmitter systems can be involved. Altogether, these results indicate that the MLR-induced depression of the RS neuron response to trigeminal nerve stimulation is largely mediated by muscarinic acetylcholine receptors. This is supported by the immunohistochemical demonstration of the presence of muscarinic receptors on RS cells (Le [START_REF] Ray | A neuronal substrate for a state-dependent modulation of sensory inputs in the brainstem[END_REF].

Immunohistochemical labeling was also seen on cells located in the trigeminal descending tract [START_REF] Northcutt | Experimental determination of the primary trigeminal projections in lampreys[END_REF], which was fond to contain the second-order trigeminal sensory neurons that relay directly trigeminal inputs to RS neurons [START_REF] Di Prisco | The trigeminal sensory relay to reticulospinal neurones in lampreys[END_REF].

Local pressure application of ACh or its muscarinic agonist pilocarpine onto the recorded RS neuron or in the trigeminal relay area reproduced the depressive effects of the MLR activation onto the trigeminal EPSP, without affecting the membrane potential of the recorded RS cell (Le [START_REF] Ray | Muscarinic modulation of the trigeminoreticular pathway in lampreys[END_REF]. Conversely, EPSP enhancement was observed when atropine (or scopolamine, another muscarinic antagonist) was substituted for the muscarinic agonists in the ejection pipette or when it was bath-applied. This suggests the existence of a tonic inhibitory control exerted via muscarinic receptors onto the trigemino-reticular connection. In addition, pre-incubation with atropine totally prevented the depressive effects of the muscarinic agonists. Several lines of evidence also indicate that the muscarinic modulation is predominantly exerted on the NMDA receptor-mediated component of the glutamatergic EPSP elicited by trigeminal stimulation, without affecting the glycinergic one (see [START_REF] Di Prisco | Trigeminal inputs to reticulospinal neurones in lampreys are mediated by excitatory and inhibitory amino acids[END_REF]: the depolarizing responses to direct application of NMDA onto the recorded RS cells were enhanced by atropine, whereas the responses to AMPA application were not.

Moreover, blocking NMDA receptors with AP5 abolished the effects of muscarinic agonists and antagonists on the trigeminal-evoked EPSP; muscarinic drug applications usually had little effect on the early part of the synaptic responses. In contrast to most of the cases reported in the literature [START_REF] Jiang | Presynaptic suppression of excitatory postsynaptic potentials in rat ventral horn neurons by muscarinic agonists[END_REF][START_REF] Scanziani | Presynaptic inhibition of excitatory synaptic transmission by muscarinic and metabotropic glutamate receptor activation in the hippocampus: are Ca2+ channels involved?[END_REF][START_REF] Bellingham | Presynaptic depression of excitatory synaptic inputs to rat hypoglossal motoneurons by muscarinic M2 receptors[END_REF][START_REF] Smolders | Muscarinic modulation of striatal dopamine, glutamate, and GABA release, as measured with in vivo microdialysis[END_REF], the muscarinic modulation of the RS responses to glutamate relies mainly on postsynaptic mechanisms in lampreys (i.e., at the level of the RS neuron itself). Whether this is also the case within the trigeminal relay area where muscarinic modulation also clearly occurs will need to be examined.

As reported above, stronger trigeminal stimulation induces sustained depolarizations in RS neurons, which trigger swimming activity in the spinal cord [START_REF] Di Prisco | Sensory-evoked NMDA plateau potentials and their role in the initiation of locomotion[END_REF], 2000). There is evidence that a tonic muscarinic inhibition of this sustained activity occurs (Le [START_REF] Ray | Muscarinic modulation of the trigeminoreticular pathway in lampreys[END_REF]: the duration of the depolarizing plateaus displayed a 5-folds increase when atropine was perfused on the brainstem; the threshold for inducing a sustained depolarization was reduced by half; the firing rate during maximal responses was increased more than twice. Because the RS neuron input resistance, resting potential, and after-hyperpolarization were not affected by atropine perfusion, the enhancement of the sustained depolarizations likely resulted from a specific action on the synaptic response to trigeminal excitatory inputs. Interestingly, when depolarizations were evoked by local ejection of NMDA onto the recorded RS neuron, atropine unmasked membrane potential oscillations that occurred on top of the depolarizing plateau, and spiking occurred on top of each oscillation during the whole duration of the NMDA-evoked oscillatory behavior (Le [START_REF] Ray | Muscarinic modulation of the trigeminoreticular pathway in lampreys[END_REF]. The persistence of these oscillations under TTX suggested an intrinsic nature for the NMDA-induced activity. In the lamprey RS neurons, sustained depolarizations require the activation of NMDA receptor [START_REF] Di Prisco | A cellular mechanism for the transformation of a sensory input into a motor command[END_REF] by trigeminal inputs, and a 30-Hz electrical stimulation of a trigeminal nerve could also trigger membrane potential oscillations in RS cells in the presence of atropine (Le [START_REF] Ray | Muscarinic modulation of the trigeminoreticular pathway in lampreys[END_REF]. Experiments performed on the isolated hindbrain demonstrated that RS oscillations could also occur in the absence of atropine and were blocked by the local ejection of a muscarinic agonist onto the recorded RS neuron (Le [START_REF] Ray | Muscarinic modulation of the trigeminoreticular pathway in lampreys[END_REF]. Because the spinal cord was removed, the NMDA-induced oscillations could not result from the ascending spinal inputs [START_REF] Dubuc | The role of spinal cord inputs in modulating the activity of reticulospinal neurons during fictive locomotion in the lamprey[END_REF][START_REF] Vinay | The spino-reticulo-spinal loop can slow down the NMDAactivated spinal locomotor network in lamprey[END_REF]Vinay et al. 1998a, b).

Conclusions

The supraspinal control locomotion relies in large part on neural regions within the brainstem and forebrain specifically dedicated to locomotion. The MLR is one such region at the mesopontine border identified in all vertebrate species examined to date.

It is believed to channel brainstem and forebrain inputs to then activate populations of RS cells in order to initiate and control locomotion. Examining the detailed downstream effects of the MLR in lampreys has yielded new information relative to the role of this region (Fig. 7). Inputs from the MLR to RS cells are strikingly symmetrical on both sides and this will play a significant role in generating symmetrical locomotion. The MLR does not only project to RS cells, but it also sends powerful inputs to a population of muscarinoceptive cells that provide additional excitation of RS cells, considerably amplifying locomotor output. In addition to controlling locomotor output, the MLR has now been shown to gate sensory inputs in the brainstem. The detailed mechanisms by which this sensory modulation operates have not been fully identified, but muscarinic receptors are involved. Future research will be needed to elucidate the detailed mechanisms involved as well as the functional importance of such sensory modulation. (Adapted from [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF].

Figure 1 :

 1 Figure 1: The supraspinal control of locomotion. The general organization of the

Figure 2 :

 2 Figure 2: The velocity of swimming movements is correlated to the intensity of MLR

Figure 3 :

 3 Figure 3: Post synaptic potentials recorded in homologous RS cells on both sides in

Figure 4 :

 4 Figure 4: Homologous reticulospinal (RS) cells display a symmetrical output in frequency

Figure 5 :

 5 Figure 5: Inactivation of muscarinoceptive neurons slows down swimming movement

Figure 6 :

 6 Figure 6: Synaptic transmission of trigeminal sensory nerve inputs to RS neurons is

Figure 7 :

 7 Figure 7: Schematic representation of the proposed brainstem locomotor control

Figure 1 Le

 1 Figure 1

Figure 2 Le

 2 Figure2

Figure 3 Le

 3 Figure3

Figure 4 Le

 4 Figure4

Figure 5 Le

 5 Figure5

Figure 6

 6 Figure6

Acknowledgements

We are very grateful to Danielle Veilleux for her help with the manuscript, to Christian Valiquette for his skilful programming of data analysis software, and to Frédéric Bernard for his help with the figures. We would like to thank François Auclair for his comments on the manuscript. R.D. receives funding from the Canadian Institutes of Health Research (CIHR; individual and group grants), from the Natural Sciences and Engineering Research Council of Canada (NSERC), from the Fonds de la Recherche en Santé du Québec (FRSQ; group grant).