To my uncles Mario and David

Shaolin monastery, Henan Province, China. 2004.

Steve McCurry

AKNOWLEDGEMENTS

This is the end of my PhD, a long journey and here I will take the opportunity to thank all the people that have been with me throughout my entire PhD.

First, I would like to thank my PhD supervisor Julien Bouvier. I joined his lab four years ago without really knowing what it meant to be a scientist, and he taught me the rigor and the scientific method. He trained me, technically and intellectually, to be a scientist, he dedicated time to my formation as a researcher and if I am here, now, willing to continue this career, it is because of his mentorship and guidance.

I am so grateful also to all the members of the lab. Aurélie, we almost started at the same time four years ago and she helped me a lot, not only with experiments, but also whenever I had problems or doubts, from dealing with French burocracy to finding microwaves. Edwin, with his computational skills allowed me to make order and sense and of all the experiments I performed. Andrea, who joined the lab in the last few months but it seems he is always been here. Séverine and Coralie, now they moved in other labs and other cities, but we shared the lab life for years, when I arrived they were the experts in the lab and they represented a point of reference for me, especially at the beginning. Alexis, for all the experiments we did together and the willpower he put into doing experiments. Indeed, he did not give up even when experiments did not work for several months. I would also like to thank Maria Giovanna, Diego, Alexandra and Emmanuel: colleagues, carpool mates and, most importantly, friends.

I would like to deeply thank Martina, especially for all the support and advices she gave me from the first time we met, without her everything would have been more problematic, I am simply glad of this. Gianguido, we shared the same city for two years during the master and other two years during my PhD, I was lucky to share with him these two experiences. Filippo, a very long friendship, he arrived with the perfect timing to help me to face the pandemic together with his "risotto alla zucca". Luca, which helped me in the moment of real need by hosting me for days at his home almost without notice. In addition, Matteo, Ivan and Anna that joined my journey in the last year, but immediately became part of this adventure.

Furthermore, I would like to thank all my family: my mother, my father, my brother, Desirée and the newcomer Pietro Enea that altogether encouraged and supported me over the years and I knew they were always on my side during my journey. I would like to thank also my relatives, cousins, uncles and aunts. I always felt their encouragement, in particular, my beloved uncles Mario and David that constantly showed me total support on the career I choose. I would like to thank also Sabrina that helped me from the very first moment I arrived in Paris, and I immediately knew that I could have always counted on her.

Furthermore, I want to thank all the people I did not mention explicitly here, that now are still in France or abroad, but also all the Italian friends that, with a visit or just a call, Beatrice also with her painting skills, made me feel better when I needed.

INTRODUCTION 1.1 The general neural basis of motor control

The picture I chose to introduce my PhD project shows, in an amazing way, a human performing an extremely complex motor behavior that requires the coordination of multiple motor actions simultaneously. In order to avoid falling down, indeed, the subject must control every muscle of his own body with high precision and timing. Performing such a composite motor behavior requires the involvement of many regions of the nervous system.

The motor task executed by the shaolin monk is difficult, and clearly not all the humans are able to perform it, however, to do the motor behaviors that we, and all vertebrates, accomplish every day to survive, represents one of the major functions of our nervous system. Most of the motor behaviors indeed require the execution of multiple motor actions simultaneously, for example hunting, escaping from predators, interacting with other animal, playing music. These motor behaviors are vital for animals and the ability of the brain to execute and modulate multiple motor actions simultaneously to perform such behaviors with the higher probability of success represents a topic of long-standing interest for neuroscientists.

In particular, for me, understanding how the nervous system recruits multiple motor actions in order to perform a complex/composite motor behavior represents one of the most interesting question to elucidate. Indeed, after a Master's degree in neuroscience, I decided to start a PhD investigation focused on the large-scale neuronal circuits that control composite motor behaviors.

Large-scale neuronal circuits involving multiple brain regions with different functions, from the encephalon to the spinal cord, are necessary to execute the desired motor actions. Indeed, our brain must consider information from the external environment and the inner body, compute and process such information in concert with our intentions. These brain areas will be referred to as "integrative/cognitive centers". The neural circuits that are appointed to produce the movements with specific timing and intensity are located along the brainstem and the spinal cord in a somatotopic manner (Figure 1). Such neural circuits will be termed "executory centers" throughout this manuscript. These executory centers comprise the motoneurons contacting the muscles recruited for a given movement and the interneurons that control and tune the motoneural activity (Figure 1C). However, how the motor signal is transmitted from the integrative/cognitive centers to the executory circuits is far from being completely resolved, and it still represents a big challenge in the field of neural motor control. In the following chapters, I will explain concepts and state-of the art notions that are necessary to understand the starting point and the aim of my PhD project. In particular, since I study the execution of multiple motor actions including orofacial, head, eye movements and locomotion, I will explain in the next chapters the actual notions related to specific motor actions.

Composite motor behaviors -example of orientation

Orientation is a complex motor behavior that requires the execution of multiple actions, for example regulating the speed, changing the locomotor direction, moving the head and the sensory apparatus towards an external cue. Animals can perform these motor actions either individually or simultaneously and can modulate their intensity in response to their needs at any moment. The main structure known to control orienting responses is the superior colliculus (SC, Figure 2) (Basso et al., 2021;Basso and May, 2017;Isa et al., 2021;May, 2006). It is well studied in monkeys, cats and rodents. The homologous structure in the non-mammalian vertebrates is called the optic tectum (Isa et al., 2021).

The SC is located in the dorsal part of the midbrain, and is organized in two divisions: the dorsal visuosensory division, and the ventral motor division (Figure 2). Moreover, the divisions are organized in layers. The stratum zonale (SZ), the startum griseum superficiale (SGS) and the stratum opticum (SO) compose the visuosensory division. The stratum griseum intermediale (SGI), the stratum album intermediale (SAI), the stratum griseum profondum (SGP), the stratum album profondum (SAP) compose the motor division (Basso and May, 2017;May, 2006).

The visuosensory layers of the superior colliculus receive inputs directly from a vast number of different regions of the brains including the retina, striatum, extrastiatum cortex, lateral geniculate nucleus, parabigeminal nucleus, pretectum and locus coeruleus (Basso et al., 2021;Basso and May, 2017). Notably, the inputs from the retina are segregated into the visuosensory division. Indeed, the retinal inputs from the temporal retina project to the rostral part of the visuosensory layers, while the nasal retinal inputs to the caudal superior colliculus, and inputs from the dorsal and ventral retina project to the lateral and ventral part (Isa et al., 2021). Therefore, the inputs from the retina are distributed in a retinotopic manner throughout the visuosensory layers. The outputs of the visuosensory division project to the lateral geniculate nucleus, pulvinar, tectopulvinar, parabigeminal nucleus and cortical areas of V3 and middle temporal area of the extrastriate [START_REF] Albano | Laminar origin of projections from the superficial layers of the superior colliculus in the tree shrew, Tupaia glis[END_REF]; together comprise the visuosensory layers, and the stratum griseum intermediale (SGI) together with the deeper layers comprise the motor layers. The schematic on the right shows the known neuronal types within the colliculus and their projection patterns. Narrow-field vertical cells (blue) project to the lateral geniculate nucleus (LGN), and the wide-field vertical cells (green) project to the pulvinar. Output neurons of the motor layers (brown) project to the rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF) to control vertical eye movements and to the paramedian pontine reticular formation (PPRF) to control horizontal eye movement. The motor layers also project upstream to the medial dorsal nucleus of the thalamus (MD). Abbreviations: SAI, stratum album intermediale; SAP, stratum album profundum; SGP, stratum griseum profundum; SZ, stratum zonale. (Basso and May, 2017). [START_REF] Bickford | Retinal and Tectal "Driver-Like" Inputs Converge in the Shell of the Mouse Dorsal Lateral Geniculate Nucleus[END_REF][START_REF] Clower | The inferior parietal lobule is the target of output from the superior colliculus, hippocampus, and cerebellum[END_REF][START_REF] Gale | Distinct representation and distribution of visual information by specific cell types in mouse superficial superior colliculus[END_REF][START_REF] Graham | Subcortical projections of six visual cortical areas in the owl monkey, Aotus trivirgatus[END_REF][START_REF] Harting | Projection of the mammalian superior colliculus upon the dorsal lateral geniculate nucleus: organization of tectogeniculate pathways in nineteen species[END_REF][START_REF] Lyon | A disynaptic relay from superior colliculus to dorsal stream visual cortex in macaque monkey[END_REF]. The outputs and the inputs of the motor layers link the SC to almost the entire neuraxis. Notably, extensive projections were found within the medullary reticular formation (Basso et al., 2021;Basso and May, 2017;Isa et al., 2021;May, 2006).

First electrophysiological experiments on SC showed its coupling with saccadic eye movements (rapid eye movement toward an object of interest) [START_REF] Apter | Eye movements following strychninization of the superior colliculus of cats[END_REF][START_REF] Fuchs | A method for measuring horizontal and vertical eye movement chronically in the monkey[END_REF][START_REF] Schiller | Single-unit recording and stimulation in superior colliculus of the alert rhesus monkey[END_REF][START_REF] Wurtz | Activity of superior colliculus in behaving monkey. IV. Effects of lesions on eye movements[END_REF]. Furthermore, the site of electrical stimulation within the retinotopic map (receptive field of the SC) determines the amplitude and the direction of the eye movements, while the frequency of the stimulation controls the speed of the eye movements (Edelman andGoldberg, 2001, 2003;[START_REF] Gnadt | Sensorimotor transformation during eye movements to remembered visual targets[END_REF][START_REF] Hikosaka | Modification of saccadic eye movements by GABA-related substances. II. Effects of muscimol in monkey substantia nigra pars reticulata[END_REF][START_REF] Klier | The superior colliculus encodes gaze commands in retinal coordinates[END_REF][START_REF] Sparks | Signal transformations required for the generation of saccadic eye movements[END_REF][START_REF] Stanford | Site and parameters of microstimulation: evidence for independent effects on the properties of saccades evoked from the primate superior colliculus[END_REF][START_REF] Van Opstal | Comparison of saccades evoked by visual stimulation and collicular electrical stimulation in the alert monkey[END_REF].

The huge network that the superior colliculus forms with almost every region of the brain, with ascending and descending inputs and outputs, suggests a role in processing and transmitting sensory information to a vast number of areas [START_REF] Goldberg | Activity of superior colliculus in behaving monkey. II. Effect of attention on neuronal responses[END_REF][START_REF] Hall | Express saccades and superior colliculus responses are sensitive to shortwavelength cone contrast[END_REF][START_REF] White | Color-related signals in the primate superior colliculus[END_REF]. Saccadic eye movements is the typical motor action associated to the SC. However, the SC has also been shown to control other typical orienting movements. Indeed, multiple studied demonstrated that SC neurons control fin, limb, head and whisker movements [START_REF] Alstermark | Vestibular effects in long C3-C5 propriospinal neurones[END_REF][START_REF] Apter | Eye movements following strychninization of the superior colliculus of cats[END_REF][START_REF] Corneil | Neck muscle responses to stimulation of monkey superior colliculus. II. Gaze shift initiation and volitional head movements[END_REF][START_REF] Courjon | Direct evidence for the contribution of the superior colliculus in the control of visually guided reaching movements in the cat[END_REF][START_REF] Courjon | Electrical stimulation of the superior colliculus induces non-topographically organized perturbation of reaching movements in cats[END_REF][START_REF] Grantyn | Reticulo-spinal neurons participating in the control of synergic eye and head movements during orienting in the cat. II. Morphological properties as revealed by intra-axonal injections of horseradish peroxidase[END_REF][START_REF] Hemelt | Superior colliculus control of vibrissa movements[END_REF]Isa and Sasaki, 2002;[START_REF] Iwamoto | Disynaptic tectal and pyramidal excitation of hindlimb motoneurons mediated by pontine reticulospinal neurons in the cat[END_REF]Masullo et al., 2019;[START_REF] Sahibzada | Movements resembling orientation or avoidance elicited by electrical stimulation of the superior colliculus in rats[END_REF][START_REF] Suzuki | The role of the optic tectum for visually evoked orienting and evasive movements[END_REF].

Altogether, these observations suggest that the SC contributes to the control of multiple orienting motor components. However, how the signal from the SC is converted into a composite orienting behavior remains to be identified.

Composite motor behaviors -example of respiration and locomotion

Another example of combination of multiple motor actions to produce a complex/composite behavior is the coordination of breathing and locomotion. Indeed, when animals, including humans, start running, the breathing frequency immediately increases to match the increased metabolic needs, often coupled with an increase of respiratory amplitude [START_REF] Dejours | Control of respiration by certain neurogenic stimuli during muscular exercise in human][END_REF][START_REF] Flandrois | Control of breathing in the exercising dog[END_REF][START_REF] Haouzi | Frequency response of the input reaching the respiratory centres during moderate intensity exercise[END_REF][START_REF] Krogh | The regulation of respiration and circulation during the initial stages of muscular work[END_REF]Mateika andDuffin, 1992, 1995;[START_REF] Pearce | Dynamic and steady-state respiratory responses to bicycle exercise[END_REF][START_REF] Robinson | Respiratory adaptations to exercise[END_REF][START_REF] Tsuchiya | Dopaminergic modulation of exercise hyperpnoea via D(2) receptors in mice[END_REF] Here, I will explain the general basis of breathing, while that of locomotion will be covered in section 1.1.3 of this manuscript.

Breathing is a fundamental behavior that ensures the constant supply of O2 and the elimination of CO2. It is composed by two different phases, inspiration and expiration.

Inspiration is a systematically active mechanism that involves the concerted muscular contraction of dedicated inspiratory pump muscle groups, of which the diaphragm is the main muscle. Instead, expiration is a passive mechanism at rest due mainly to the relaxation of the inspiratory muscles and lung recoil. However, under certain physiological conditions where the respiratory drive is increased, like during high-level exercise, forced or active expiration takes place [START_REF] Iscoe | Control of abdominal muscles[END_REF][START_REF] Jenkin | Chapter 8 -Expiration: Breathing's other face[END_REF]. Breathing is a rhythmic behavior that is controlled by dedicated neurons of the brainstem that form a circuit called respiratory central pattern generator (respiratory CPG) or respiratory rhythm generator (which according to my terminology will form, with the phrenic motorneurons, the respiratory executory center). There are two main structures forming the respiratory CPG (Figure 3A): the preBotzinger Complex (preBötC) and the parafacial respiratory group (pRFG) [START_REF] Del Negro | Breathing matters[END_REF][START_REF] Feldman | Understanding the rhythm of breathing: so near, yet so far[END_REF].

The preBötC is the core rythmogenic microcircuit for generating inspiration [START_REF] Del Negro | Breathing matters[END_REF][START_REF] Feldman | Looking for inspiration: new perspectives on respiratory rhythm[END_REF][START_REF] Feldman | Breathing: rhythmicity, plasticity, chemosensitivity[END_REF][START_REF] Smith | Pre-Botzinger complex: a brainstem region that may generate respiratory rhythm in mammals[END_REF].

Multiple studies demonstrated the existence of the preBötC in different species, including humans [START_REF] Mutolo | Opioid-induced depression in the lamprey respiratory network[END_REF][START_REF] Pantaleo | Respiratory responses to somatostatin microinjections into the Botzinger complex and the pre-Botzinger complex of the rabbit[END_REF][START_REF] Ramirez | The human pre-Botzinger complex identified[END_REF][START_REF] Ramirez | Postnatal changes in the mammalian respiratory network as revealed by the transverse brainstem slice of mice[END_REF][START_REF] Schwarzacher | Neuroanatomical characteristics of the human pre-Botzinger complex and its involvement in neurodegenerative brainstem diseases[END_REF][START_REF] Smith | Pre-Botzinger complex: a brainstem region that may generate respiratory rhythm in mammals[END_REF][START_REF] Tupal | Testing the role of preBotzinger Complex somatostatin neurons in respiratory and vocal behaviors[END_REF][START_REF] Wenninger | Small reduction of neurokinin-1 receptor-expressing neurons in the pre-Botzinger complex area induces abnormal breathing periods in awake goats[END_REF]. The preBötC is located ventrally to the nucleus ambiguous within the ventral medulla and its constitutive neurons express the neurokinin 1 receptor (NK1R) and the neuropeptide somatostatin SST [START_REF] Feldman | Breathing: rhythmicity, plasticity, chemosensitivity[END_REF][START_REF] Gray | Modulation of respiratory frequency by peptidergic input to rhythmogenic neurons in the preBotzinger complex[END_REF][START_REF] Stornetta | A group of glutamatergic interneurons expressing high levels of both neurokinin-1 receptors and somatostatin identifies the region of the pre-Botzinger complex[END_REF].

The pRFG is located ventrally of the facial motor nucleus (7N). It has been shown that the functional coupling between the pRFG and preBötC is necessary for the generation of the inspiratory rhythm at birth [START_REF] Mellen | Opioid-induced quantal slowing reveals dual networks for respiratory rhythm generation[END_REF][START_REF] Onimaru | Localization of respiratory rhythm-generating neurons in the medulla of brainstem-spinal cord preparations from newborn rats[END_REF][START_REF] Onimaru | A novel functional neuron group for respiratory rhythm generation in the ventral medulla[END_REF]. pRFG neurons express the transcription factor Phox2b [START_REF] Onimaru | CO2-sensitive preinspiratory neurons of the parafacial respiratory group express Phox2b in the neonatal rat[END_REF].

Notably, the pRFG overlaps with a region called retrotrapezoid nucleus (RTN) known for its chemogenetic role in crucial in detecting pCO2 changing and the pH variation and regulate the respiratory rhythm [START_REF] Amiel | Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome[END_REF][START_REF] Guyenet | Regulation of ventral surface chemoreceptors by the central respiratory pattern generator[END_REF][START_REF] Mitchell | Respiratory responses mediated through superficial chemosensitive areas on the medulla[END_REF][START_REF] Mulkey | Respiratory control by ventral surface chemoreceptor neurons in rats[END_REF][START_REF] Ramanantsoa | Breathing without CO(2) chemosensitivity in conditional Phox2b mutants[END_REF][START_REF] Ruffault | The retrotrapezoid nucleus neurons expressing Atoh1 and Phox2b are essential for the respiratory response to CO(2)[END_REF][START_REF] Smith | Brainstem projections to the major respiratory neuron populations in the medulla of the cat[END_REF][START_REF] Stornetta | Expression of Phox2b by brainstem neurons involved in chemosensory integration in the adult rat[END_REF].

First studies on breathing and locomotion were performed by stimulating the supraspinal regions diencephalic locomotor region (DLR) and mesencephalic locomotor region (MLR) in cats and lamprey (that are regions known to promote locomotion, I will discuss the MLR later in the manuscript, in section 1.2.1). The stimulation of these areas not only induces an increase of locomotion but also an increase in frequency and amplitude of breathing [START_REF] Dimarco | Immediate changes in ventilation and respiratory pattern associated with onset and cessation of locomotion in the cat[END_REF][START_REF] Eldridge | Stimulation by central command of locomotion, respiration and circulation during exercise[END_REF][START_REF] Eldridge | Exercise hyperpnea and locomotion: parallel activation from the hypothalamus[END_REF][START_REF] Gariepy | Specific neural substrate linking respiration to locomotion[END_REF][START_REF] Shik | Control of walking and running by means of electrical stimulation of the mesencephalon[END_REF].

Furthermore, studies on spinal circuits for locomotion (locomotor CPG, it will be discussed later in the manuscript) demonstrated, on neonatal spinal cord preparation, that the activation of such circuits induced an increase of respiratory frequencies (Le [START_REF] Gal | Modulation of respiratory network activity by forelimb and hindlimb locomotor generators[END_REF][START_REF] Gal | Remote control of respiratory neural network by spinal locomotor generators[END_REF][START_REF] Morin | Coordinations of locomotor and respiratory rhythms in vitro are critically dependent on hindlimb sensory inputs[END_REF].

A recent study led in my laboratory by another PhD student demonstrated that, during running exercise in mice, there is no lock between breaths and strides, meaning that the respiration phase is independent from the locomotor cycle (Figure 3B) [START_REF] Herent | Absent phasing of respiratory and locomotor rhythms in running mice[END_REF]. This work suggests that the adaptation to exercise may include different behaviordependent solutions. Indeed, whilst the transition from rest to moderate exercise could engage a default exercise breathing at fixed frequency, the engagement into faster displacement speeds appears to operate through a further increase of respiratory frequency. However, whether there is a neural substrate that links locomotor controlling circuits and the breathing CPG to increase the respiratory rate while the locomotor activity increases

is not yet determined. Nevertheless, humans asked to imagine performing running exercise showed an increase of breathing rate, even in the absence of actual exercise [START_REF] Decety | Analysis of actual and mental movement times in graphic tasks[END_REF][START_REF] Jahn | Imaging human supraspinal locomotor centers in brainstem and cerebellum[END_REF][START_REF] Karachi | Cholinergic mesencephalic neurons are involved in gait and postural disorders in Parkinson disease[END_REF][START_REF] Thornton | Identification of higher brain centres that may encode the cardiorespiratory response to exercise in humans[END_REF]. Furthermore, as I mentioned above, stimulating the MLR and DLR increase the respiratory rate in cats and lamprey.

Interestingly, it has been shown that the MLR projects, in the lamprey, to the paratrigeminal group, considered the homologous structure of the preBötC [START_REF] Gariepy | Specific neural substrate linking respiration to locomotion[END_REF][START_REF] Mutolo | Opioid-induced depression in the lamprey respiratory network[END_REF]. Therefore, the preBötC is a prime candidate for integrating locomotor signals and mediating respiratory changes to meet the energetic demand associated with exercise.

However, the existence of such mechanism as not been documented yet. [START_REF] Herent | Absent phasing of respiratory and locomotor rhythms in running mice[END_REF].

The "executory" centers: where the final drive to the muscles is elaborated

As I already mentioned in the first paragraph, the executory centers (composed by interneurons and motoneurons) are distributed throughout the brainstem and the spinal cord in a somatotopic manner. From the upper spinal cord to the lower spinal cord, these circuits control neck, forelimb, back and hindlimb movements, respectively (Figure 1B). The [START_REF] Kiehn | Decoding the organization of spinal circuits that control locomotion[END_REF]. most studied executory center in the spinal cord is the center for controlling locomotion.

Locomotion is a fundamental behavior that encompasses all the animal kingdom, and it can occur in many ways depending on the anatomical characteristics of the animals.

Swimming, flying, quadrupedal or bipedal walking are different ways to locomote.

The first hypothesis of the existence of a central spinal program sufficient to produce locomotion was proposed early in the XXth century [START_REF] Brown | Two Cases of Death from Post-Anaesthetic Acid Intoxication[END_REF]. Years later, was suggested the term locomotor Central Pattern Generator (CPG) [START_REF] Delcomyn | Neural basis of rhythmic behavior in animals[END_REF]. Initially the model proposed by Graham was the so-called "Half-centered model", consisting in two small neuronal networks connected through inhibitory neurons. When one was active, the other one was inhibited. Such model was used to explain the alternation between extensor and flexor muscles during locomotion [START_REF] Brown | On the nature of the fundamental activity of the nervous centres; together with an analysis of the conditioning of rhythmic activity in progression, and a theory of the evolution of function in the nervous system[END_REF]. This model was expanded with the idea of smaller network, called "Unit Burst Generator" (UBG), able to produce intrinsic rhythm activity [START_REF] Grillner | Entrainment of the spinal pattern generators for swimming by mechano-sensitive elements in the lamprey spinal cord in vitro[END_REF]. Pioneering electrophysiological experiments on ex vivo spinal cord preparations allowed to study the locomotor CPG in order to record the activity of the ventral roots of the spinal cord, where are located the fibers of the motoneurons contacting specific muscles involved in locomotor behaviors [START_REF] Kiehn | Spatiotemporal characteristics of 5-HT and dopamine-induced rhythmic hindlimb activity in the in vitro neonatal rat[END_REF].

Independently from the specie and the type of locomotion, the locomotor CPGs in the spinal cord are evolutionary conserved across vertebrates (Grillner and El Manira, 2020;[START_REF] Kiehn | Locomotor circuits in the mammalian spinal cord[END_REF][START_REF] Kiehn | Decoding the organization of spinal circuits that control locomotion[END_REF]. These networks contain the sufficient information to activate different muscles and coordinate the cycle and the rhythm of locomotion (Figure 4).

In the brainstem, reside the executory centers for breathing (already explained in section 1.1.2), ocular, and orofacial behaviors. The term orofacial behavior refers to any activity involving muscles of the face, the vibrissae, the jaw, the tongue or the airways, therefore including sniffing, chewing, licking, swallowing and vocalizing [START_REF] Moore | Hierarchy of orofacial rhythms revealed through whisking and breathing[END_REF].

Notably, such behaviors must be performed without fatal blockages of the airflow. Furthermore, orofacial movements can be performed in synchrony with eye movements, for example during exploration the nose, wiskers and eyes can point towards the same direction in response to an external stimulus. Therefore, these motor actions, if needed in synergy, must be well-coordinated and require the control of multiple muscles. Indeed, it has been demonstrated that in rats the coordinated activity of 26 pairs of muscles and five cranial nerves are needed to correctly perform eating, drinking and swallowing, that altogether compose the essential complex behavior known as feeding (Barlow, 2009a, b). The motoneurons of the brainstem are organized in nuclei controlling specific muscles (Figure 5).

The main nuclei known to control voluntary movements are the oculomotor (III nerve), trochlear (V), abducens (VI) for eye movements, while the trigeminal (V), facial (VII), ambiguous (X) and hypoglossal (XII) for orofacial movements [START_REF] Cordes | Molecular genetics of cranial nerve development in mouse[END_REF]. Examples of motor actions controlled by these motoneurons and executory centers in which they are embedded are the eye and neck movements, whisking, chewing, sniffing and breathing (Guthrie, 2007;Ruder and Arber, 2019).

An orofacial behavior that has been well-studied over the last decades is whisking, that consist in rapid movements of the mystacial vibrissae used for touch, in order to explore the external environment. Notably, the whisker moments are associated and coordinated with sniffing, and therefore, breathing [START_REF] Moore | How the brainstem controls orofacial behaviors comprised of rhythmic actions[END_REF]. The central oscillator for whisking is located in the vibrissa intermediate reticular nucleus (vIRt), a ventral region of the caudal brainstem [START_REF] Moore | Hierarchy of orofacial rhythms revealed through whisking and breathing[END_REF]. The neurons of the vIRt indeed fire in coordination with the retraction of the vibrissae. It has been proposed that rhythmic whisking is controlled by an oscillatory signal from the inhibitory vIRt neurons to the facial motor nucleus (7N) during the retraction phase [START_REF] Moore | Hierarchy of orofacial rhythms revealed through whisking and breathing[END_REF][START_REF] Moore | How the brainstem controls orofacial behaviors comprised of rhythmic actions[END_REF]. Such inhibition might be counterbalanced by an excitatory drive to the motoneurons of the 7N, causing the oscillatory back-and-forth whisker movements [START_REF] Deschenes | Inhibition, Not Excitation, Drives Rhythmic Whisking[END_REF]. Furthermore, the activity of the vIRt might be controlled by the respiratory CPG (preBotzinger Complex) in order to tightly coordinate whisking and breathing [START_REF] Deschenes | Inhibition, Not Excitation, Drives Rhythmic Whisking[END_REF][START_REF] Kleinfeld | More than a rhythm of life: breathing as a binder of orofacial sensation[END_REF][START_REF] Smith | Pre-Botzinger complex: a brainstem region that may generate respiratory rhythm in mammals[END_REF]. Recently, it has been showed that vIRt neurons comprises Parvalbumin-expressing inhibitory neurons and that their silencing impairs regular whisking [START_REF] Takatoh | Constructing an adult orofacial premotor atlas in Allen mouse CCF[END_REF].

Importantly, as I explained before, these executory centers are controlled by integrative/cognitive centers upstream. In the next paragraph, I will describe the classic organization of descending tracts contacting the executory centers. I will depict the main tracts, and, as I will explain later in the manuscript, my work focuses in particular on the reticulospinal tract.

The supraspinal control of executory centers

While the spinal cord and the brainstem executory centers contain all the neural components to produce the motor act itself, their activity is under obligatory control of supraspinal-spinal structures. Over the past decades, scientists dedicated their work in investigating how the nervous system plans and controls motor actions and how it coordinates them in order to perform composite motor behaviors. Efforts were done for identifying the descending pathways that relay a motor signal from the integrative/cognitive centers to the executory centers [START_REF] Kuypers | The Descending Pathways to the Spinal Cord, Their Anatomy and Function[END_REF][START_REF] Kuypers | Anatomy of the descending pathways[END_REF]. The classical organization of the mammal brain, groups the descending motor pathways in corticospinal, vestibulospinal, rubrospinal, cerebellospinal and reticulospinal tract is shown in Figure 6.

The corticospinal tract (i.e., pyramidal tract) originates within the motor cortex and sends axons to the spinal cord with most of the fibers (the percentage varies within different species; about 90% of the fibers in primates, including humans) decussating contralaterally at the level of the pyramids of the brainstem. The corticospinal tract is mostly known for the control for distal muscles. Its main role is to control fine digital movements (Lemon, 2008), Note that the corticospinal tract sends collateral fibers to the reticular formation, the classical nomenclature referred to this pathway as corticobulbar tract. Realized using BioRender templates. (B) Relationship between the development of the corticospinal tract and the emergence of fine motor control abilities. In rodents, there are no direct connections between corticospinal neurons and the cervical motoneurons which innervate forelimb muscles, but brainstem pathways and spinal interneurons relay cortical input to motor neurons. Most of the corticospinal fibers in rodents travel in the dorsal columns. In non-human primates and humans, direct corticospinal connections with motoneurons have evolved, together with an increase in the size and number of the corticospinal fibers. This is reflected in an increase in the size of the excitatory postsynaptic potential (EPSP) elicited by cortical neurons in hand motoneurons. The primate CST is located mostly in the lateral columns, and a significant proportion of CS fibers (∼10%) descend ipsilaterally. The development of the CST correlates with the improvement in the index of dexterity, particularly in the ability to perform fingerthumb precision grip (Lemon, 2008). Figure 5B. Furthermore, it has been demonstrated that the development of the corticospinal tract presents crucial differences between species [START_REF] Kuypers | Anatomy of the descending pathways[END_REF]Lemon, 2008). In particular, it has been demonstrated that in humans, the corticospinal tract forms a direct monosynaptic connection with motoneurons in the cervical spinal cord controlling digital muscles, whilst, in rodents, such connectivity is absent [START_REF] Alstermark | Lack of monosynaptic corticomotoneuronal EPSPs in rats: disynaptic EPSPs mediated via reticulospinal neurons and polysynaptic EPSPs via segmental interneurons[END_REF]. This might explain why in primates the digital movements are highly skilled. However, in mice, digital movements for behaviors including grasping and grooming are essential. As we will see later in the manuscript, evidences suggest that such behaviors in mice might be controlled by brainstem neurons. Furthermore, in mice, it has been studied that the corticospinal tract does not impact locomotor activity and, in the lumbar spinal cord its main role is to modulate sensory inputs [START_REF] Digiovanna | Engagement of the Rat Hindlimb Motor Cortex across Natural Locomotor Behaviors[END_REF][START_REF] Moreno-Lopez | The corticospinal tract primarily modulates sensory inputs in the mouse lumbar cord[END_REF].

The vestibulospinal tract originates in the vestibular nuclei and projects the axons ipsilaterally along the spinal cord and controls primarily the head and eyes movements for body balance [START_REF] Mccall | Descending Influences on Vestibulospinal and Vestibulosympathetic Reflexes[END_REF][START_REF] Sugiuchi | Functional synergies among neck muscles revealed by branching patterns of single long descending motor-tract axons[END_REF]. The vestibular nucleus receives inputs from contralateral vestibular nucleus, somatosensory inputs from the spinal cord, from the cerebellum, from multiple areas of the cerebral cortex [START_REF] Bibliography Akbarian | Corticofugal projections to the vestibular nuclei in squirrel monkeys: further evidence of multiple cortical vestibular fields[END_REF][START_REF] Fukushima | Corticovestibular interactions: anatomy, electrophysiology, and functional considerations[END_REF][START_REF] Guldin | Connections from the neocortex to the vestibular brain stem nuclei in the common marmoset[END_REF][START_REF] Pogossian | Organization of afferent projections to the ventral and dorsal regions of the cat lateral vestibular nucleus: an HRP study[END_REF][START_REF] Wilson | Cortical influences on the vestibular nuclei of the cat[END_REF].

Notably, extensive vestibular projections were observed within the brainstem reticular formation, where originates the reticulospinal tract [START_REF] Bolton | Response of pontomedullary reticulospinal neurons to vestibular stimuli in vertical planes. Role in vertical vestibulospinal reflexes of the decerebrate cat[END_REF][START_REF] Peterson | Properties of projections from vestibular nuclei to medial reticular formation in the cat[END_REF][START_REF] Schor | Horizontal rotation responses of medullary reticular neurons in the decerebrate cat[END_REF].

The rubrospinal tract originates within the magnocellular red nucleus of the brainstem. Rubrospinal projections are more abundant contralaterally. Studies suggest that the rubrospinal tract is involved in controlling the velocity of the execution of the voluntary movements of the limbs [START_REF] Kuypers | Anatomy of the descending pathways[END_REF]Lemon, 2008). Interestingly, In humans, the rubrospinal tract seems to be less developed [START_REF] Yamamoto | Descending pathways to the spinal cord in teleosts in comparison with mammals, with special attention to rubrospinal pathways[END_REF].

The cerebellospinal tract has been mostly investigated for its role in motor learning, postural adjustments and forelimb movements [START_REF] Asanuma | Nucleus interpositus projection to spinal interneurons in monkeys[END_REF][START_REF] Carrea | The anatomy of the primate brachium conjunctivum and associated structures[END_REF][START_REF] Fukushima | Direct fastigiospinal fibers in the cat[END_REF][START_REF] Liang | Projections from the brain to the spinal cord in the mouse[END_REF][START_REF] Matsushita | The location of spinal projection neurons in the cerebellar nuclei (cerebellospinal tract neurons) of the cat. A study with the horseradish peroxidase technique[END_REF][START_REF] Nudo | Descending pathways to the spinal cord: a comparative study of 22 mammals[END_REF][START_REF] Sathyamurthy | Cerebellospinal Neurons Regulate Motor Performance and Motor Learning[END_REF][START_REF] Wang | Global Connectivity and Function of Descending Spinal Input Revealed by 3D Microscopy and Retrograde Transduction[END_REF].

The reticulospinal tract originates in the brainstem reticular formation and its projections were observed along the entire spinal cord. In particular, ipsilateral reticulospinal projections are more abundant than contralateral projections. The reticulospinal tract controls multiple voluntary movements including forelimb and orofacial movements, breathing and locomotion. Notably, fibers from other descending tracts have been shown to project and collateralize to the brainstem, as it is demonstrated for the corticospinal and cerebellospinal tracts [START_REF] Batton | Fastigial efferent projections in the monkey: an autoradiographic study[END_REF][START_REF] Brodal | The termination of the brachium conjunctivum descendens in the nucleus reticularis tegmenti pontis. An experimental anatomical study in the cat[END_REF]Lemon, 2008;[START_REF] Tolbert | A demonstration of the dentato-reticulospinal projection in the cat[END_REF]. These observations suggest that the reticulospinal tract might relay motor signals from the other descending tracts. Indeed, the reticulospinal tract is considered a crucial descending motor pathway that relays the motor command to the executory centers within the spinal cord and the brainstem allowing the animals to perform vital behaviors and survive in the external environment (Brownstone and Chopek, 2018;[START_REF] Humphries | Is there a brainstem substrate for action selection?[END_REF]. Such role of the reticular neurons represents the starting point of my investigations.

The aim of the following chapter is therefore to accompany the reader to the investigations that were done in the last decades on the study of reticulospinal neurons, and the reticular formation in general, and its involvement in controlling motor actions.

The Brainstem Reticular Formation

The brainstem reticular formation is a region of the brain that controls many vital, and highly conserved motor behaviors, including orofacial, respiratory, forelimb and locomotor movements but also head and trunk movements as well as uro-genito-sexual functions. The reticular formation is key structure that relays motor commands from the higher brain areas (integrative/cognitive centers) to the executory centers within the spinal cord.

The reticular formation is a vast ventral region with non-discrete boundaries that spans throughout the brainstem (midbrain, pons and medulla oblungata). It receives inputs from multiple brain regions with different functions including the cerebellum, the thalamus, the cortex, visual, vestibular, and auditory pathways. It contacts an incredible number of brain regions with ascending and descending projections: thalamus, cerebellum, red nucleus, basal ganglia, hypothalamus and spinal cord. With such heterogeneous efferences and afferences connectivity, the reticular formation is a major candidate for relaying information from and to different regions of the brains and for controlling multiple motor functions.

However, investigating the functions and the anatomy of the reticular formation has been challenging for decades due to the difficult accessibility of this brain region. Indeed, it is composed most likely by multiple cell types, differing by neurotransmitters, morphology, genetic identity and projections profile, and it is formed by borderless nuclei, but the cells are highly intermingled (Drew et al., 1986;Valverde, 1961). It is also located deep in the brainstem, making access to recording and imaging tools very tedious (Figure 7). However, the development of new methods of investigation, including optogenetics, chemogenetics and cutting-edge viral tracing, nowadays allows us to thoroughly investigate neural circuits that before were inaccessible and elucidate their anatomy and functions [START_REF] Deisseroth | Optogenetics: 10 years of microbial opsins in neuroscience[END_REF]Fenno et al., 2014;[START_REF] Roth | DREADDs for Neuroscientists[END_REF]Stepien et al., 2010;Wickersham et al., 2007). For my study, I took advantage from such new techniques to investigate, anatomically and functionally, the reticular formation.

Furthermore, the reticular formation has been proposed to have a role not only in relaying the motor signal between upstream and downstream circuits. Instead, it might be also a region with a certain degree of action selection. Indeed, it has been hypothesized that the reticular formation forms a dense network that might provide the ability to extract formation in an adult albino rat, using a pyridine-silver method. B, horizontal section through the facial and ambiguus nuclei. Rat, 21 days old. Cox method. Abbreviations: A.tr., triangular area containing the fibers to the nucleus dorsomedialis of Astrom. Br.c., superior N.tr.sp.V, spinal nucleus of the trigeminal nerve. N., tr.sp.V, spinal tract and nucleus of the trigeminal nerve. Ol.ac., accessory superior olive. Ol.i., inferior olive. Ol.ped.c., olivary peduncle, crossed part. Ol.s., superior olive. P.g., periaqueductal gray. P1.b.f.v-l., bulbar prolongation of the ventrolateral funiculus of the spinal cord. Pyr., pyramidal tract. R., raphe. R.d.m.o., subnucleus reticularis dorsalis medullae oblongatae of Meessen and Olszewski. R.pc., nucleus reticularis parvicellularis. R.v., nucleus reticularis ventralis. S.g., substantia gelatinosa of dorsal horn and nucleus caudalis of the trigeminal nerve. Str. ac .d., stria acustica dorsalis. T.r-s., rubrospinal tract. Tr.s., tractus solitarius. Tr.sp.V, spinal tract of trigeminal nerve. V.d., descending vestibular nucleus. V.1., lateral vestibular nucleus. V.m., medial vestibular nucleus. V,VI,VII,VIII,IX-X,XIII, rootlets of cranial nerves. N.V,N.VI,N.VII,N.XII, motor nuclei of craial nerves. N.X, nucleus ambiguous. IX-Xef, efferent rootlets of the vago-glosso pharyngeal nerve. G.VII, Genu of the facial nerve. (Valverde, 1961). correlated sensory information, the internal connectivity might guarantee the substrate for the coordination of distinct behavioral components, and the individual cells might drive the appropriate motor system [START_REF] Humphries | The brainstem reticular formation is a small-world, not scale-free, network[END_REF][START_REF] Humphries | Is there a brainstem substrate for action selection?[END_REF].

The Mesencephalic Locomotor Region

As I already mentioned before, one of the most conserved motor behaviors that is performed by all animals is the locomotion. It can be performed in multiple ways: quadrupedal, bipedal, swimming, flying. In non-limbed animals, the execution of locomotion requires the activity of axial muscles that control the body, while in limbed animals, it requires in addition the coordinated activity of limb muscles. Locomotion is composed by three distinct phases: initiation, locomotor episode and termination. These three phases can be performed in multiple ways, based on the speed, gait, limb coordination, direction, depending on the behavioral need at any time.

The neural circuits necessary for the execution of locomotion reside in the spinal cord (locomotor CPG, this concept has been already described previously in the manuscript, section 1.1.3). However, the activity of these executory centers depends from the motor signal coming from supraspinal integrative/cognitive centers. While the executory centers within the spinal cord are well-studied [START_REF] Kiehn | Locomotor circuits in the mammalian spinal cord[END_REF][START_REF] Kiehn | Decoding the organization of spinal circuits that control locomotion[END_REF], as I explained above in the manuscript the mechanism by which integrative/cognitive centers of the brain contact the executory centers is poorly understood.

One of the most important findings in the field of motor control is the discovery of a region of the brainstem located in the dorsal mesopontine border of the cats that, when stimulated electrically, was inducing locomotion. It was termed the Mesencephalic Locomotor Region (MLR) [START_REF] Shik | Control of walking and running by means of electric stimulation of the midbrain[END_REF][START_REF] Shik | Control of walking and running by means of electrical stimulation of the mesencephalon[END_REF]. Furthermore, as the stimulation strength increased, higher speed locomotion is produced, from slow walk to trot and to gallop. This region was found evolutionary conserved in many different species (lamprey, mice, and primates) [START_REF] Mori | Site-specific postural and locomotor changes evoked in awake, freely moving intact cats by stimulating the brainstem[END_REF][START_REF] Ryczko | The multifunctional mesencephalic locomotor region[END_REF][START_REF] Shik | Neurophysiology of locomotor automatism[END_REF].

Moreover, a recent work on MLR, still not peer-reviewed at the time of the writing of this manuscript, has identify the homologous structure of the MLR in zebrafish [START_REF] Carbo-Tano | Functional coupling of the mesencephalic locomotor region and V2a reticulospinal neurons driving forward locomotion[END_REF]. The MLR is composed of two main subnuclei, the Pedunclopontine nucleus (PPN) and the Cuneiform nucleus (CNF) [START_REF] Ryczko | Dopamine and the Brainstem Locomotor Networks: From Lamprey to Human[END_REF]. For years, which region of the MLR was involved in promoting movements or whether they were concurring together in inducing locomotion remained unknown. Recent works studied, using novel methods of investigation, the contribution of the two brain structures to the locomotor behavior.

Researchers investigated the glutamatergic cells expressing the type 2 vesicular glutamate transporter (VGlut2 neurons), located in both the PPN and CnF, and the cholinergic cells located in the PPN. Using optogenetics, investigators were able to selectively activate these cells and found that the stimulation of VGlut2 neurons in the PPN trigger slow locomotion, whereas the VGlut2 neurons in CnF induce high speed locomotion (Caggiano et al., 2018;Josset et al., 2018). These results suggested that the CnF elicits straight and fast (escapelike) locomotion while the PPN triggers slow, exploratory-like locomotion.

Moreover, studies suggest that MLR might mediate exercise hyperpnea [START_REF] Dimarco | Immediate changes in ventilation and respiratory pattern associated with onset and cessation of locomotion in the cat[END_REF][START_REF] Eldridge | Stimulation by central command of locomotion, respiration and circulation during exercise[END_REF][START_REF] Gariepy | The interactions between locomotion and respiration[END_REF][START_REF] Kawahara | Coupling between respiratory and locomotor rhythms during fictive locomotion in decerebrate cats[END_REF]. In lamprey, it has been shown that stimulation of MLR increases respiratory output and that the MLR projects to the homologous structure of the PreBotc [START_REF] Gariepy | Specific neural substrate linking respiration to locomotion[END_REF]. MLR stimulation in cats induces the respiratory and cardiovascular increases that could occur during locomotor episodes and prior to the movement [START_REF] Eldridge | Stimulation by central command of locomotion, respiration and circulation during exercise[END_REF]. These results suggest that the MLR might control in parallel locomotor and autonomic responses, including breathing. Indeed, that is the main topic of the project of onother PhD student within the laboratory, in which I participated.

Interestingly, projections from excitatory neurons of the MLR were found in the reticular formation, suggesting that in the medullary reticular formation there might be neurons relaying the locomotor-promoting signal from the MLR to the spinal cord [START_REF] Shefchyk | Reversible cooling of the brainstem reveals areas required for mesencephalic locomotor region evoked treadmill locomotion[END_REF]. Therefore, it has been proposed that the glutamatergic excitatory neurons in the medullary reticular formation might perform such function. Identifying these neurons might represents a key discovery in investigating the contribution of the reticular formation in the control of movements.

The medullary reticular formation

Within the last century, many scientists focused their investigations in describing the organization of the medullary reticular formation in regions and nuclei. In particular, most of research depicted the anatomy of the rodent reticular formation. The edges of the reticular formation are still not clearly defined and, as well as the name of the structures, they can vary between studies. Therefore, to help the reader, in the following chapter I explain the nomenclature of the medullary reticular formation that I will follow throughout the manuscript.

The classical organization of the reticular formation divides this structure in three regions (median, medial and lateral) (Chopek et al., 2021). The medial medullary reticular formation comprises rostrally the caudal part of the nucleus reticularis pontis caudalis (PnC), continuing with the Gigantocellular nucleus (Gi). The Gi is one of the bigger nuclei of the medullary reticular formation and spans across the ventral pons and the medulla oblongata, from the facial nucleus to the obex (from -5.5 to -7.20 from bregma) (Franklin and Paxinos, 2007;Valverde, 1961). Ventral to the Gi there are the alpha part of the Gi (GiA), more rostrally, and the ventral part of the Gi (GiV), more caudally. Other nuclei of the medullary reticular formation are the dorsal paragigantocellular nucleus (DPGi), the parvocellular reticular nucleus (PCRt), intermediate reticular zone (IRt) and the lateral paragigantocellular nucleus (LPGi). At the most caudal part of the medulla (caudal to -7.20 from bregma), there are the ventral part (MdV) and the dorsal part (MdD) of the medullary reticular formation (Chopek et al., 2021;Franklin and Paxinos, 2007).

One of the main contributions in investigating the organization and the neurons of the medullary reticular formation was done by Valverde in 1961. His work described with an incredible accuracy (especially considering the techniques available at the time) the anatomy of the reticular neurons highlighting the huge variety of the morphology of the cells within the various nuclei of the reticular formation.

"The nucleus reticularis gigantocellularis […] make up the principal site of origin of the long distance efferent projections of the Reticular Formation. […] The characteristic tendency of the axons of their cells to form a longitudinal tract […]. The tract reaches the spinal cord".

With these words, Valverde described the neurons of the reticular formation projecting at different levels of the spinal cord. These neurons, called reticulospinal (RS) neurons, are commonly considered the fundamental cells carrying the motor signal from the integrative/cognitive to the executory centers in order to switch them on or off and modulate their activity. In particular, within the RS, there might be the neurons controlling a fundamental behavior already introduced in this manuscript: locomotion. Indeed, RS neurons are thought to send, in response to MLR activity, the motor command to the locomotor CPG located in the spinal cord (Brownstone and Chopek, 2018;[START_REF] Dubuc | Initiation of locomotion in lampreys[END_REF][START_REF] Mcclellan | Activation of 'fictive swimming' by electrical microstimulation of brainstem locomotor regions in an in vitro preparation of the lamprey central nervous system[END_REF][START_REF] Noga | Mechanism for activation of locomotor centers in the spinal cord by stimulation of the mesencephalic locomotor region[END_REF]. Furthermore, axonal projections of the RS neurons have been found in multiple regions of the spinal cord, suggesting that RS neurons might control also other important motor function beside locomotion [START_REF] Riddle | Direct and indirect connections with upper limb motoneurons from the primate reticulospinal tract[END_REF][START_REF] Shapovalov | The evolution of neuronal systems for suprasegmentary motor control (review)[END_REF][START_REF] Sivertsen | Pontine reticulospinal projections in the neonatal mouse: Internal organization and axon trajectories[END_REF][START_REF] Szokol | Differential origin of reticulospinal drive to motoneurons innervating trunk and hindlimb muscles in the mouse revealed by optical recording[END_REF].

In parallel to the anatomical studies, for a functional investigation, first experiments on the brainstem were performed on decerebrated animals. After the lesion, it has been observed that the physiological functions remained intact or, at least, partially active. For decades, this strategy represented the main strategy to investigate brainstem functions.

Experiments on decerebrated animals (only the brainstem remained intact), showed in different works that certain motor functions, like locomotion, grooming and responding to acoustic stimuli, remained intact or only partially limited [START_REF] Bjursten | Behavioural repertory of cats without cerebral cortex from infancy[END_REF][START_REF] Roh | Modules in the brain stem and spinal cord underlying motor behaviors[END_REF][START_REF] Whelan | Control of locomotion in the decerebrate cat[END_REF].

Altogether, these works demonstrated that the brainstem is an essential region of the brain in controlling multiple motor behaviors. Furthermore, these results suggested that, at the level of the brainstem there might be a certain degree of action selection. Indeed, between the regions left intact in decerebrated animals, the reticular formation has been proposed as the main structure for selecting and performing different motor actions [START_REF] Humphries | Is there a brainstem substrate for action selection?[END_REF].

As I mentioned previously, the first obstacle in investigating the reticular formation is represented by the difficult in identifying and manipulate only one neuronal type. This is due to the high level of intermingling of the cells in the reticular formation. Moreover, the nuclei of the reticular formation are deeply located in the ventral part of the brainstem and there is a lack of molecular tools to manipulate them. This represented for decades a huge impediment to anatomically and functionally investigate reticular and reticulospinal neurons.

There are different parameters that we can consider to define the neurons belonging to a cell type: by their location, the neurotransmitter released by the cells, the size of the soma, the axonal projections, the function, the genetic profile. Along the years, using techniques as optogenetics, chemogenetics and viral tracing, different research tried to isolate and study the functions of the reticular neurons according to one or more of these parameters.

In the next paragraphs, I will describe the essential studies focused on the role of the medullary reticular formation in controlling forelimb and orofacial movements, breathing and locomotion.

The medullary reticular neurons and forelimbs movements

As I explained before RS might not control only locomotion, indeed reticular neurons had been studied in the past decades in the context of different motor functions. One of the sets of motor actions discovered to be controlled by the reticular neurons is represented by the forelimb movements. Indeed, in most animals forelimbs are used to reach and grab objects, allowing the animals to perform essential behaviors in exploration, manipulation and grooming. However, whether there are different cell types controlling individual forelimb movements or whether they are controlled by a unique population, is poorly known.

Studies suggested that the lateral regions of the reticular formation might control distal forelimb movements [START_REF] Kuypers | The Descending Pathways to the Spinal Cord, Their Anatomy and Function[END_REF]Lemon, 2008). Furthermore, recent studies showed that the glutamatergic cells of the ventral part of the reticular formation control grasping and the cells located in the lateral rostral medulla control reaching and food handling [START_REF] Esposito | Brainstem nucleus MdV mediates skilled forelimb motor tasks[END_REF][START_REF] Ruder | A functional map for diverse forelimb actions within brainstem circuitry[END_REF]. These studies highlight the complexity of controlling different skilled forelimb movements and rise an important concept that each component of these motor actions might be controlled by different reticular centers instead of a unitary control by extensive collateralization.

The medullary reticular neurons and the orofacial and respiratory movements

Numerous studies over the last decades focused on the reticular formation and its role in motor control. Importantly it has been showed that this complex structure is involved in controlling multiple motor actions including orofacial movements and respiration. As I already discussed above, with the term orofacial movements, we indicate a vast number of motor actions that are often coordinated with respiration like whisking and sniffing. Abbreviations: 5N, fifth motor nucleus; 7N, seventh motor nucleus; 12N, hypoglossal motor nucleus; BötC, Bötzinger complex; Dbx1, developing brain homeobox protein 1; IRt, intermediate reticular nucleus; MN, motor neuron; MRF, medullary reticular formation; PCRt, parvicellular reticular nucleus; preBötC, pre-Bötzinger complex; PRF, pontine reticular formation; rVRG, rostral ventral respiratory group; vGAT, vesicular GABA transporter; vGlut2, vesicular glutamate transporter 2; vIRt, vibrissa zone of the intermediate reticular nucleus (Ruder and Arber, 2019).

Such movements are controlled by dedicated pools of motoneurons located within the brainstem: the cranial motor nuclei (Guthrie, 2007). Understanding the premotor circuits controlling these motor nuclei represents a big challenge. Many studies focus on revealing the presynaptic connectome of the brainstem motor nuclei [START_REF] Deschenes | Inhibition, Not Excitation, Drives Rhythmic Whisking[END_REF][START_REF] Stanek | Monosynaptic premotor circuit tracing reveals neural substrates for oro-motor coordination[END_REF][START_REF] Takatoh | New modules are added to vibrissal premotor circuitry with the emergence of exploratory whisking[END_REF][START_REF] Takatoh | Constructing an adult orofacial premotor atlas in Allen mouse CCF[END_REF].

Other studies focus on investigating the brainstem circuits that control respiration, and it has been showed that the preBötC complex is a main structure in controlling breathing and it has access to the phrenic nerve via the rostral ventral respiratory group [START_REF] Del Negro | Breathing matters[END_REF][START_REF] Feldman | Understanding the rhythm of breathing: so near, yet so far[END_REF] (see section 1.1.2). It is extremely important to mention that different orofacial behaviors might be active synergistically but also can occur isolated from the others depending from the behavioral needs, suggesting that there might be separated circuits for each orofacial behavior but they might be also in communication when they are recruited in coordination [START_REF] Moore | Hierarchy of orofacial rhythms revealed through whisking and breathing[END_REF].

The medullary reticular neurons and locomotor movements

Different studies showed that the locomotor-promoting signal from the MLR does not reach directly the spinal cord, but is rather relayed by the medullary reticular formation (Brownstone and Chopek, 2018;[START_REF] Dubuc | Initiation of locomotion in lampreys[END_REF]Jordan et al., 2008;[START_REF] Noga | Mechanism for activation of locomotor centers in the spinal cord by stimulation of the mesencephalic locomotor region[END_REF][START_REF] Shefchyk | Reversible cooling of the brainstem reveals areas required for mesencephalic locomotor region evoked treadmill locomotion[END_REF]. Moreover, in many different species including the lamprey ducks, geese, guinea pig, cats [START_REF] Marlinskii | Participation of the medial reticular formation of the medulla oblongata in the supraspinal control of locomotor and postural activities in the guinea pig[END_REF][START_REF] Mcclellan | Activation of 'fictive swimming' by electrical microstimulation of brainstem locomotor regions in an in vitro preparation of the lamprey central nervous system[END_REF][START_REF] Steeves | Stimulation of the pontomedullary reticular formation initiates locomotion in decerebrate birds[END_REF] the medullary reticular formation is crucial in controlling locomotion. Recent work focused on investigating whether the medullary reticular formation neurons relay the motor command from the MLR to the spinal cord. Furthermore, the medullary reticular formation receives inputs from other regions of the brain that are known to control locomotion, from the cerebellar locomotor region [START_REF] Mori | Cerebellar-induced locomotion: reticulospinal control of spinal rhythm generating mechanism in cats[END_REF] and the subthalamic locomotor region [START_REF] Sinnamon | Locomotion elicited by lateral hypothalamic stimulation in the anesthetized rat does not require the dorsal midbrain[END_REF].

It has been shown that RS neurons are fast conducting [START_REF] Degtyarenko | Locomotor modulation of disynaptic EPSPs from the mesencephalic locomotor region in cat motoneurons[END_REF][START_REF] Noga | Mechanism for activation of locomotor centers in the spinal cord by stimulation of the mesencephalic locomotor region[END_REF]Orlovskii, 1970a) and that they are active during spontaneous locomotion (Drew et al., 1986;[START_REF] Matsuyama | Lumbar commissural interneurons with reticulospinal inputs in the cat: morphology and discharge patterns during fictive locomotion[END_REF][START_REF] Shimamura | Discharge patterns of reticulospinal neurons corresponding with quadrupedal leg movements in thalamic cats[END_REF][START_REF] Shimamura | Reticular neuron activities associated with locomotion in thalamic cats[END_REF]. Anatomically, RS projections were founded both in the ipsilateral and contralateral ventrolateral funiculus [START_REF] Steeves | Effect of noradrenaline and 5hydroxytryptamine depletion on locomotion in the cat[END_REF] and in different regions of the spinal cord, comprising the lumbar spinal cord, where is located the locomotor CPG [START_REF] Jankowska | Spinal interneuronal networks in the cat: elementary components[END_REF][START_REF] Matsuyama | Lumbar commissural interneurons with reticulospinal inputs in the cat: morphology and discharge patterns during fictive locomotion[END_REF]. However, while in non-mammalian animal models the stimulation of the medullary reticular formation clearly promotes locomotion, experiments on the mammalian medullary reticular formation showed controversial results. Mainly, it has been proposed that the divergent outcomes observed might be due to the cell heterogeneity of the RS neurons (Arber and Costa, 2018;Brownstone and Chopek, 2018;Jordan et al., 2008;[START_REF] Orlovskii | Work of reticulo-spinal neurons during locomotion[END_REF]Ruder and Arber, 2019). Indeed, RS neurons can differ based on their neurotransmitter and works focused on both excitatory, including glutamatergic and serotonergic, and inhibitory (both GABAergic and glycinergic) RS neurons with the idea that the first type would promote locomotion, while the latter would prevent it (Jordan et al., 2008). Certainly the most important source of signal that may favor locomotor, and motor actions in general, is thought to be glutamatergic RS neurons. Indeed, the bath application of glutamate is the most effective way to obtain locomotor-like activities on isolated spinal cord preparations from lamprey, rodents and cats [START_REF] Dale | Excitatory amino acid receptors in Xenopus embryo spinal cord and their role in the activation of swimming[END_REF][START_REF] Douglas | The effects of intrathecal administration of excitatory amino acid agonists and antagonists on the initiation of locomotion in the adult cat[END_REF]Grillner and El Manira, 2020;[START_REF] Grillner | Entrainment of the spinal pattern generators for swimming by mechano-sensitive elements in the lamprey spinal cord in vitro[END_REF][START_REF] Kudo | Morphological and physiological studies of development of the monosynaptic reflex pathway in the rat lumbar spinal cord[END_REF]. On the contrary, blocking NMDA receptors in the spinal preparation, and in intact animals, impairs locomotion [START_REF] Chau | Effects of intrathecal glutamatergic drugs on locomotion I. NMDA in short-term spinal cats[END_REF][START_REF] Giroux | Effects of intrathecal glutamatergic drugs on locomotion. II. NMDA and AP-5 in intact and late spinal cats[END_REF], including that evoked by stimulation of the MLR. Furthermore, it has been showed that the optogenetic stimulation of glutamatergic brainstem neurons on isolated brainstem spinal cord preparation, can promote locomotor-like activities [START_REF] Hagglund | Activation of groups of excitatory neurons in the mammalian spinal cord or hindbrain evokes locomotion[END_REF]. Recently, the glutamatergic neurons in the lateral paragigantocellular nucleus (LPGi) of the brainstem were shown, when activated with optogenetics, to promote locomotion in freely-moving mice [START_REF] Capelli | Locomotor speed control circuits in the caudal brainstem[END_REF]. On the contrary, it was reported that the stimulation of the glutamatergic neurons in the adjacent Gi does not initiate locomotion per se, but help stabilize postural tone and tune the ongoing pattern [START_REF] Lemieux | Glutamatergic neurons of the gigantocellular reticular nucleus shape locomotor pattern and rhythm in the freely behaving mouse[END_REF]. Others nevertheless argue for a role of Gi glutamatergic neurons in favoring locomotion, notably following spinal cord injury [START_REF] Engmann | The Gigantocellular Reticular Nucleus Plays a Significant Role in Locomotor Recovery after Incomplete Spinal Cord Injury[END_REF]. Therefore, while there is accumulating evidence for a role of some glutamatergic RS neurons in promoting movements, including locomotion in response to MLR signals, the identity, nature and location of such locomotor-promoting neurons is still not fully resolved. Furthermore, I will illustrate in the next chapter that considering smaller subsets of neurotransmitter-defined RS neurons will reveal that even excitatory ones can mediate a functional inhibition. Therefore, the glutamatergic status is only a poor predictor of the function of RS neurons and may include a substantial diversity.

Investigations of inhibitory neurons in the medullary reticular formation indicate that they may form a general signal for motor inhibition. One best example that incriminates reticulospinal inhibitory neurons is the induction of muscular atonia of the whole body during REM sleep [START_REF] Vetrivelan | Medullary circuitry regulating rapid eye movement sleep and motor atonia[END_REF]. Regarding inhibition during wakefulness, it was shown in the xenopus tadpole that head contact with obstacles activates GABAergic descending pathways that immediately terminate swimming [START_REF] Perrins | Sensory activation and role of inhibitory reticulospinal neurons that stop swimming in hatchling frog tadpoles[END_REF].

Furthermore, the activation of inhibitory neurons located in the GiA, GiV and Gi, blocks ongoing locomotion [START_REF] Capelli | Locomotor speed control circuits in the caudal brainstem[END_REF]. A motor inhibition signal, including for locomotion, may also be conveyed by inhibitory reticular neurons projecting to subcortical structures, including the thalamus [START_REF] Giber | A subcortical inhibitory signal for behavioral arrest in the thalamus[END_REF]. Therefore, evidence so far implicate a general role of inhibitory neurons of the medullary reticular formation, being descending or not, in mediating motor arrest. Here again, the possibility for differences among cell-types and spatial location is not documented.

Aside the excitatory and inhibitory amino acids, serotonergic medullary neurons may play a significant role. Although they are not systematically classified as belonging to the reticular formation, one should stress that the raphe nucleus is not the only source of serotonin in the brainstem. In particular, several laboratories have investigated serotonergic RS neurons that are located in the Parapyramidal region (Ppy), largely overlapping with the lateral paragigantocellular reticular nucleus (LPGi). The idea that a descending serotonergic pathway might promote locomotion is prompted from several observations. First, on neonatal spinal cord preparation, the application of serotonin promotes locomotor-like activities [START_REF] Cazalets | Two types of motor rhythm induced by NMDA and amines in an in vitro spinal cord preparation of neonatal rat[END_REF][START_REF] Cazalets | Activation of the central pattern generators for locomotion by serotonin and excitatory amino acids in neonatal rat[END_REF]Cowley andSchmidt, 1994, 1997;[START_REF] Schmidt | The role of serotonin in reflex modulation and locomotor rhythm production in the mammalian spinal cord[END_REF]. Secondly, the transplantation of serotonergic cells within the thoracic cord activates locomotion [START_REF] Ribotta | Activation of locomotion in adult chronic spinal rats is achieved by transplantation of embryonic raphe cells reinnervating a precise lumbar level[END_REF]. A more recent research demonstrated that using electrical stimulation of the Ppy region in a brainstem-spinal cord rat preparation elicits fictive locomotion, an effect dependent on spinal serotonergic receptors (Liu and Jordan, 2005). Furthermore, serotonergic neurons, including in the Ppy, show increased expression of the immediate early gene c-fos after MLR-induced locomotion [START_REF] Opris | Activation of Brainstem Neurons During Mesencephalic Locomotor Region-Evoked Locomotion in the Cat[END_REF]. Overall, serotonergic descending tracts may play a role in evoking or stabilizing locomotion, although here again, without a clear identification of the incriminated cells nor of their connectivity to the locomotor CPG.

Finally, activation the noradrenergic and dopaminergic receptors elicits fictive locomotion in neonatal rat spinal cord preparation [START_REF] Gabbay | Alpha-1 adrenoceptor agonists generate a "fast" NMDA receptorindependent motor rhythm in the neonatal rat spinal cord[END_REF][START_REF] Kiehn | Effects of noradrenaline on locomotor rhythm-generating networks in the isolated neonatal rat spinal cord[END_REF]. However, few experiments were performed on noradrenergic and adrenergic RS medullary neurons. Indeed, noradrenergic and adrenergic RS neurons are found in limited number in two restricted areas of the brainstem: the A5 and A11 cells group of the brainstem Clark [START_REF] Bjorklund | Evidence for a major spinal cord projection from the diencephalic A11 dopamine cell group in the rat using transmitter-specific fluorescent retrograde tracing[END_REF][START_REF] Qu | Projections of diencephalic dopamine neurons into the spinal cord in mice[END_REF]. While the RS dopaminergic neurons of A11 cell group have not been tested, the depletion of noradrenergic RS cells of the brainstem A5 group does not alter the ongoing locomotion [START_REF] Jasmin | Long-term effects of decreased noradrenergic central nervous system innervation on pain behavior and opioid antinociception[END_REF][START_REF] Steeves | Effect of noradrenaline and 5hydroxytryptamine depletion on locomotion in the cat[END_REF].

Given the importance of the RS neurons in body movements and in locomotion, it is crucial to consider the RS system as a target for therapies aimed to the recovery of the motor circuits following trauma. In particular, Spinal cord injury (SCI) is an incurable condition which leads to a highly debilitating loss of motor and sensory functions below the injury often including the ability to walk and stand [START_REF] Sekhon | Epidemiology, demographics, and pathophysiology of acute spinal cord injury[END_REF][START_REF] Varma | Spinal cord injury: a review of current therapy, future treatments, and basic science frontiers[END_REF][START_REF] Wilson | Clinical predictors of neurological outcome, functional status, and survival after traumatic spinal cord injury: a systematic review[END_REF]. The interruption or weakening of the descending neuronal tracts, and in particular of the reticulospinal tract, is the major cause of loss of motor autonomy following SCI. Therefore, strategies aiming at enhancing the survival or regrowth of severed descending axons are highly promising for promoting recovery after SCI. It has been showed that the RS neurons collectively show strong plasticity following SCI [START_REF] Lu | Motor axonal regeneration after partial and complete spinal cord transection[END_REF][START_REF] Pearse | cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury[END_REF][START_REF] Zorner | Chasing central nervous system plasticity: the brainstem's contribution to locomotor recovery in rats with spinal cord injury[END_REF] and may actually be the tract the most disposed to plasticity [START_REF] Asboth | Cortico-reticulo-spinal circuit reorganization enables functional recovery after severe spinal cord contusion[END_REF][START_REF] Vavrek | Neuronal populations capable of regeneration following a combined treatment in rats with spinal cord transection[END_REF]. RS neurons in the ventral reticular formation can notably sprout across the spinal midline following a hemi-section model and may also have the capacity to regenerate [START_REF] Lu | Motor axonal regeneration after partial and complete spinal cord transection[END_REF]. Medullary 5-HT RS neurons also show plasticity [START_REF] Basso | Descending systems contributing to locomotor recovery after mild or moderate spinal cord injury in rats: experimental evidence and a review of literature[END_REF][START_REF] Vavrek | Neuronal populations capable of regeneration following a combined treatment in rats with spinal cord transection[END_REF]. In fact, increasing 5-HT neurotransmission in the spinal cord improves recovery after experimental SCI and in humans [START_REF] Ghosh | The role of the serotonergic system in locomotor recovery after spinal cord injury[END_REF][START_REF] Ribotta | Activation of locomotion in adult chronic spinal rats is achieved by transplantation of embryonic raphe cells reinnervating a precise lumbar level[END_REF][START_REF] Van Den Brand | Restoring voluntary control of locomotion after paralyzing spinal cord injury[END_REF][START_REF] Wenger | Spatiotemporal neuromodulation therapies engaging muscle synergies improve motor control after spinal cord injury[END_REF]. A critical step for functional recovery after SCI would thus be the restoration of glutamatergic and/or serotonergic innervation beyond the lesion. Finally, an interesting aspect for both RS and 5-HT neurons is that favoring their activity may stimulate their endogenous plasticity [START_REF] Hentall | Promotion of recovery from thoracic spinal cord contusion in rats by stimulation of medullary raphe or its midbrain input[END_REF][START_REF] Lu | Motor axonal regeneration after partial and complete spinal cord transection[END_REF].

Altogether, these observations support the notion that RS neurons, within the medullary reticular formation, control multiple motor actions and relay the motor signal from the cognitive/integrative to the executory centers. However, the exact position, identity, and connectivity of the cells implicated in controlling locomotion, and/or other motor actions are not identified yet. The complexity of the reticular formation has been a major lock for such investigations. We should stress here that although serotonergic, noradrenergic and dopaminergic neurons are often organized in clustered nuclei (therefore not literally belonging to the reticular formation per se, with the exception of the Ppy serotonergic neurons) and represent small populations, the glutamatergic and inhibitory ones are largely spatially intermingled and represent considerably larger populations that might even be heterogeneous. This makes the characterization of a single neurochemical pathway and its partners within the executory circuits a daunting task with classical system neuroscience approaches. Electrical recordings for instance cannot easily discriminate excitatory from inhibitory cell types. Electrical activations are similarly blind to cell-types, and also come with a poor control of the current spread and with the impossibility to disambiguate responses obtained from activating a neuronal somata from passing fibers of distant origin. Mechanical ablations or toxins do not help to resolve cellular specificity and have poor spatio-temporal control. In the next chapter, I will hence cover how novel methods of investigation based on developmental genetics can help to make huge progresses in investigating the medullary reticular formation and its involvement in controlling multiple motor actions by accessing smaller, presumably homogeneous, cellular ensembles.

Novel approaches to cellular diversity in motor circuits

Spinal cord development

The studies I mentioned in the previous chapters investigated the brain by mostly taking in consideration the location of the neuronal somata or the neurotransmitter released. In the field of spinal cord, one of the main strategies to investigate the function of smaller, and presumably homogeneous neuronal ensembles, has been to imprint tools from developmental biology, and in particular the concept that cells sharing a similar developmental history might share largely similar phenotypic properties (neurotransmitter, localization, electrical properties, connectivity), and thus a similar function. More precisely, reserachers have started to define a "cell-type" by the common history of expression, during development, of transcription factors essential for the cellular development and specification [START_REF] Goulding | Circuits controlling vertebrate locomotion: moving in a new direction[END_REF][START_REF] Kiehn | Decoding the organization of spinal circuits that control locomotion[END_REF]. By using specific transgenic mouse lines engineered on the basis of such markers, scientists can now see, trace and manipulate a given cell type within a complex and heterogenous structure. This approach has largely been applied to the spinal cord, and I will hence describe its main fundamentals and benefits below. Nevertheless, a similar developmental coding of neuronal diversity applies to the brainstem and should allow new access to cellular diversity in the reticular formation. In fact, my worked investigated the only type of RS neurons defined by such considerations.

The main concept is that during the development of the spinal cord, the levels of expression in progenitor cells of specific developmental genes, namely transcription factors, vary along the dorso-ventral axis of the neural tube. Indeed, the expression oftranscription factors is dependent principally from the protein Sonic hedgehog (Shh) and bone morphogenetic protein (BMP). The concentration of Shh is higher ventrally, while BMP is more concentrated dorsally [START_REF] Jessell | Neuronal specification in the spinal cord: inductive signals and transcriptional codes[END_REF] (Figure 11). These Shh and BMP gradients along the dorsoventral are fundamental in defining the neural fate of the cells along the axis [START_REF] Barth | Bmp activity establishes a gradient of positional information throughout the entire neural plate[END_REF][START_REF] Briscoe | Specification of neuronal fates in the ventral neural tube[END_REF][START_REF] Briscoe | A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube[END_REF][START_REF] Ericson | Graded sonic hedgehog signaling and the specification of cell fate in the ventral neural tube[END_REF][START_REF] Roelink | Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis[END_REF]. In particular, the gradient of Shh induces the expression of different transcription factors that can be grouped in two distinct classes. Class I proteins (Pax6, Bbx2) are repressed by Shh signaling, while Class II proteins (Nkx.2.2, Nkx6.1) are induced by Shh [START_REF] Briscoe | A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube[END_REF][START_REF] Briscoe | Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling[END_REF]. The selective cross repression interaction between Class I and Class II proteins along the dorsoventral axis defines five domains of progenitors in the ventral spinal cord: p0, p1, p2, PMN, P3 [START_REF] Briscoe | A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube[END_REF]. Patterning along the dorsoventral axis of the spinal cord is dependent on signaling from early developmental organizing centers. Sonic Hedgehog (Shh) that is secreted ventrally from the notochord and floor plate (fp) triggers the differentiation of ventral cell types. Similarly, Bone morphogenic proteins (BMPs) and Wingless-Ints (Wnts), secreted from the dorsal ectoderm and the roof plate (rp), have an important role in the specification of dorsal cell fates. These secreted proteins act as long-range morphogens, which establish a concentration gradient. In the neural progenitors, a given concentration of morphogens translates in the expression of a given set of transcription factors. Thus, progenitors will organize in "domains" along the dorso-ventral axis, with each domain expressing a unique combination of transcription factors. Dorsally-born progenitors are labelled dI1 to dI6, while ventrally born ones are labelled p0 to p3 for interneurons, and pMN for motoneuronal progenitors. Each progenitor domain will give rise to a discrete set of neuronal cell types, with neurons belonging to one cell type sharing similar properties (i.e. neurotransmitter phenotype, axonal projection...). Neuronal cell types themselves can again be recognized by their specific expression profiles of transcription factors. Interestingly, the final position of the differentiated neurons does not reliably reflect the position of the progenitor domain from which they are born. Cell-type specific manipulations can thus be achieved by driving the expression of reporter proteins or interfering tools under genetic control a TF (or a combination of TF) that is specific to the cell-type of interest.

When progenitors exit the cell cycle and differentiate into neurons, the dorsal p0 and p1 progenitors generate ventral 0 (V0) and ventral 1 (V1) cells, while the p2, pMN and p3 ventral progenitors generate Ventral 2 (V2) neurons, the somatic motoneurons (sMN) and ventral 3 (V3) neurons [START_REF] Briscoe | A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube[END_REF][START_REF] Briscoe | Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling[END_REF]. Within these five classes of neurons, scientists determined further subpopulations based on the expression of identified transcription factors [START_REF] Briscoe | Specification of neuronal fates in the ventral neural tube[END_REF].

Benefits to the investigation of spinal executory centers

Thanks to the new insights on the spinal cord development, different research have extensively been using the corresponding genetic tools to manipulate such geneticallydefined cell types of the spinal cord. As a result, enormous progresses have been made in understanding how the executory centers within the spinal cord control locomotion.

To understand how the locomotor CPG control locomotion there are hierarchical features that are necessary to take in account: the left-right alternation system, the rhythm generating system and the flexor-extensor patterning system [START_REF] Kiehn | Locomotor circuits in the mammalian spinal cord[END_REF]. On the one hand, the coordination between the two sides of the body is under the control of commissural neurons. These are characterized by axons that cross the midline and make synapses with neurons on the contralateral side of the spinal cord. Several studies converged on the fact that a most important class of neurons for left/right coordination are V0 neurons.

They are located in the ventral spinal cord and are born from DBX1-expressing progenitors of the p0 domain [START_REF] Pierani | Control of interneuron fate in the developing spinal cord by the progenitor homeodomain protein Dbx1[END_REF]. It has been showed that the ablation of V0 neurons disrupt left-right alternation [START_REF] Lanuza | Genetic identification of spinal interneurons that coordinate left-right locomotor activity necessary for walking movements[END_REF]. The V0 neurons are subdivided in inhibitory V0d neurons, that coordinate limb alternations at low locomotor frequencies, and excitatory V0v neurons, that control left right alternation at higher frequencies [START_REF] Talpalar | Dual-mode operation of neuronal networks involved in left-right alternation[END_REF]. From these studies, it has been proposed that V0d neurons are active at low speed of locomotion, while the V0v neurons are active at high speed of locomotion [START_REF] Bellardita | Phenotypic characterization of speed-associated gait changes in mice reveals modular organization of locomotor networks[END_REF]. An intriguing conclusion of these studies is that part of the contralateral inhibition relies on excitatory neurons (the V0v at high speed). This is counterintuitive for excitatory neurons, but it illustrates very well my earlier postulate that, in complex and heterogeneous structures, neurotransmitter identity is not always accurate to predict function. It also illustrates the importance of post-synaptic targets to appreciate function: that V0d and V0v neurons both mediate contralateral inhibition requires that they each contact different cell types on the opposite side.

On the other hand, the generation of the locomotor rhythm was known to be dependent from excitatory spinal interneurons [START_REF] Goulding | Circuits controlling vertebrate locomotion: moving in a new direction[END_REF][START_REF] Hagglund | Activation of groups of excitatory neurons in the mammalian spinal cord or hindbrain evokes locomotion[END_REF][START_REF] Roberts | Origin of excitatory drive to a spinal locomotor network[END_REF]. However, the identity of the underlying neurons remains to be fully elucidated. Experiments on excitatory V2a neurons (expressing the transcription factor Chx10) demonstrated that this population does not affect locomotor rhythm [START_REF] Crone | Genetic ablation of V2a ipsilateral interneurons disrupts left-right locomotor coordination in mammalian spinal cord[END_REF]. In contrast, experiments on spinal excitatory neurons expressing the transcription factor SHOX2+ (expressed in some V2a neurons and some non-V2a neurons) demonstrate that the non-V2a SHOX2+ population has a role in generating the locomotor rhythm [START_REF] Crone | Genetic ablation of V2a ipsilateral interneurons disrupts left-right locomotor coordination in mammalian spinal cord[END_REF]. Silencing the activity of the non-V2a SHox2+ population indeed reduces the rhythmogenicity, however without completely blocking it [START_REF] Dougherty | Locomotor rhythm generation linked to the output of spinal shox2 excitatory interneurons[END_REF]. Another neural population investigated in the control of rhythm are the neurons expressing HB9, that are located in the ventral spinal cord, and demonstrated that they have rhythmogenic cellular properties supporting a role in rhythm generation [START_REF] Brownstone | Strategies for delineating spinal locomotor rhythm-generating networks and the possible role of Hb9 interneurones in rhythmogenesis[END_REF][START_REF] Caldeira | Spinal Hb9::Cre-derived excitatory interneurons contribute to rhythm generation in the mouse[END_REF].

Finally, the coordination between the activity of motoneurons controlling antagonist muscles (flexor and extensor muscles) of a given limb is another crucial trait of the locomotor behavior. An essential role in regulating this function is made by reciprocal-Ia-inhibitory interneurons, that are activated by the stretch-activated group Ia afferent from muscle spindles. Furthermore, the reciprocal-Ia-inhibitory interneurons inhibit the motoneuron that is antagonist to the motoneuron activated by Ia afferent neurons [START_REF] Hultborn | Transmission in the pathway of reciprocal Ia inhibition to motoneurones and its control during the tonic stretch reflex[END_REF][START_REF] Talpalar | Identification of minimal neuronal networks involved in flexor-extensor alternation in the mammalian spinal cord[END_REF]. Another study demonstrated that the ablation of V1 interneurons inhibit flexor activity, while ablation of V2b neurons inhibit extensor activity [START_REF] Zhang | V1 and v2b interneurons secure the alternating flexor-extensor motor activity mice require for limbed locomotion[END_REF].

As we can conclude from these investigations on the spinal cord network for controlling locomotor activity, the identification of a cell types by their developmental history allowed to describe its function and anatomy. Therefore, it is a common thought that this method of investigation might crucial for studying the medullary reticular formation and its role in motor control. In the next chapter, I will introduce the only RS neural population that has been genetically identified and that represents the starting point of my investigations.

The case of V2a reticular neurons

The concepts from developmental biology that I described in the previous section have seldom been applied to the brainstem reticular formation, although similar principles and combination of transcription factors apply [START_REF] Briscoe | Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling[END_REF]. My supervisor was one of the firsts to benefit from these concepts and apply them to the identification of functional cell types in the reticular formation. He and others notably identified the vital role of brainstem V0 neurons for inspiratory rhythm generation in the preBötC [START_REF] Bouvier | Hindbrain interneurons and axon guidance signaling critical for breathing[END_REF][START_REF] Gray | Modulation of respiratory frequency by peptidergic input to rhythmogenic neurons in the preBotzinger complex[END_REF]. Interestingly, these V0 neurons also mediate the characteristic left/right synchronicity of diaphragmatic contractions. This illustrates that developmentally-defined cell types in the brainstem and spinal cord share some similarities (V0 neurons are commissural in both cases) but their functional outcome is obviously distinct (breathing versus locomotion). Even more interestingly, some follow-up work revealed that the same neuronal class (V0 neurons) in the brainstem also constitute the pre-motor output of the preBötC to the phrenic motor neurons. The V0 neuronal class hence has reticulospinal projections, but seems overall tuned for inspiratory control. Other studies have revealed genetic determinants of other important components of the respiratory CPG, including in the PFRG region. Indeed there, the subgroup of neurons relevant for modulating respiratory rhythm generation, at least in the context of central chemoception are best identified by a combined history of expression of the transcription factors Phox2b and Atoh1 [START_REF] Ramanantsoa | Breathing without CO(2) chemosensitivity in conditional Phox2b mutants[END_REF][START_REF] Ruffault | The retrotrapezoid nucleus neurons expressing Atoh1 and Phox2b are essential for the respiratory response to CO(2)[END_REF]. Other studies have identified crucial neurons of the brainstem reticular formation and linked them to a function, mostly in orofacial control [START_REF] Hernandez-Miranda | Genetic identification of a hindbrain nucleus essential for innate vocalization[END_REF].

In contrast, the identification of specific neurons for the control of limb and body movements has progressed slowly. To date, the only genetically-identified class of RS neurons are the V2a neurons, neurons that have expressed Lhx3 and Chx10 [START_REF] Al-Mosawie | Heterogeneity of V2-derived interneurons in the adult mouse spinal cord[END_REF]. The V2a neurons form a column that span throughout the brainstem and the spinal cord. As already described in previous chapters, V2a neurons located within the spinal cord have been investigated, but less was known about their function in the brainstem. First studies on the V2a neurons of the brainstem were done almost simultaneously on both zebrafish and mice (Bretzner and Brownstone, 2013;Kimura et al., 2013). Kimura and colleagues showed that chx10-expressing neurons in the hindbrain of the zebrafish are active during swimming and when activated with optogenetics, they induce the bending of the body that make the zebrafish swimming (Figure 12 D). Therefore, in Zebrafish, Chx10 neurons seem to trigger locomotion. Furthermore, they identified that there are at least two different types of Chx10 neurons based on electrophysiological properties. In parallel, a research conducted by Bretzner and Brownstone on mice, demonstrated that Chx10 neurons in mice are glutamatergic and projections were found at the level of the spinal cord. Furthermore, they demonstrated that V2a neurons were active during locomotor tasks and they have heterogeneous electrophysiological properties. Moreover, investigations of the calcium transients during MLR stimulation suggest that V2a neurons might receive inputs from this region in order to promote the ongoing locomotion.

A more recent study by my supervisor confirmed the reticulospinal status of V2a neurons at all levels of the medullary reticular formation (Figure 12 A). It also investigated the role these cells in mice during locomotion both in vitro and in in vivo (Bouvier et al., 2015). Surprisingly, the optogenetics activation of the medullary V2a neurons at rest did not evoke any locomotor activity. In contrast, it induced an immediate stop of the ongoing locomotion if applied during displacement (Figure 12C). Conversely, silencing V2a neurons' activity led to hyperactive mice that could barely stop in an open field paradigm. The locomotor arrest driven by V2a RS neurons was ascribed to a functional inhibition of locomotor-generating properties of the locomotor CPG, rather than to a freezing response.

This effect was also limited to a most rostral fraction of V2a neurons, residing in the Gi nucleus. Therefore, these V2a "stop neurons" represent another example (the spinal V0v (Bouvier et al., 2015;Kimura et al., 2013). (D) Activation of hindbrain V2a neurons elicits swimming in zebrafish. A flash of blue light was applied to the boxed area (left panel). This evoked swimming-like behavior (right panels).

ones being the first, see section 1.3.2) of an excitatory population that does not act to favor movement. Yet, the physiological role of this population was mysterious. As I will detail in the objectives, this observation prompted by PhD project.

I should note that my first publication on these V2a reticulospinal neurons was in 2020. Therefore, this is the current state of the art that was available to me until publication.

Yet, for the sake of completeness, I should also mention here the work that have been published since. For instance, another project run in the lab in parallel than mine was to investigate the endogenous activity of V2a RS neurons in the mouse, during freely-moving displacement. Using calcium imaging performed on in vivo freely moving mice, my colleagues confirmed that V2a neurons are indeed active when the animal arrests its locomotion, but that this was only true for a fraction of V2a neurons. Others were active during different motor behaviors (Schwenkgrub et al., 2020). This suggested that the V2a neurons might control not only locomotor arrest, which was also my lead. Another study, this time led by an independent group, demonstrated that the unilateral activation of the medullary V2a neurons induces an ipsilateral turning of the mouse during ongoing locomotion (Cregg et al., 2020). As I will explain in the discussion, this investigation was performed almost in concert with mine, although I had no information about it. The findings are, at least at first glance, similar to mine but we will see that we reach substantially different conclusions on the circuit and mechanistic levels.

The medullary V2a neurons were hence, and still are nowadays, the only medullary RS neurons that are defined genetically and thus tractable. Since they form a subtype of glutamatergic neurons, and since their function seem to diverge from the classical view that excitatory circuits favor movement, they thus represent an exquisite entry-point to study diversity and specialization of RS neurons.

OBJECTIVES

The brainstem reticular formation is key for controlling motor actions. Indeed, reticular neurons interface multiple sensory and cognitive modalities upstream, with executory motor circuits downstream. However, how the reticular formation selects, initiates and combines multiple motor actions is largely unknown.

My PhD project is prompted by the above observation. We decided to start my exploration from a cell type of the reticular formation that has been genetically defined, the V2a neurons, but whose intrinsic diversity and function besides arresting locomotion, were not known. From this, my PhD project followed 3 objectives, that were run in parallel:

1. To start replacing these V2a neurons in larger behavioral circuits, and shine light on the behavioral relevance of their "locomotor stop" function, I have explored anatomically the origins of their inputs. For this, I deployed using cutting-edge, rabies-based viral tracing to label neurons located one, and only one synapse upstream the V2a RS neurons and high-throughput microscopy. By this, I aimed at deciphering what are the integrative/cognitive centers sending the command to the V2a neurons. 2. To test whether the V2a neurons control other motor behaviors besides locomotion, I took advantage from optogenetic techniques to unilaterally photoactivate the V2a medullary neurons and examined the consequences on multiple body-scale and orofacial movements. Furthermore, using retrograde tracings and circuit optogenetics, I investigated whether additional actions would be attributed to collaterals of a unique V2a population, or instead to distinct subsets that differ by projection.

3. To start investigating the postsynaptic targets of medullary V2a neurons in different executory centers, I started from the possibility, prompted by our behavioral recordings and some anatomical findings, that some might be directly connected to specific motoneuronal pools. To demonstrate it, I aimed at revealing the presynaptic connectome of candidate muscles, using intramuscular deliveries of rabies viral vectors on newborn mice. Furthermore, I participate at the research of another PhD student of the laboratory, Coralie Hérent. Her project aims to investigate the central circuits interfacing locomotor and respiratory centers. In particular, it focuses on the locomotor and respiratory' nodes that mediate the increase of the respiratory rate during running exercise. Indeed, one important request of the reviewers, after the initial submission, was to improve the anatomical mapping of transfected neurons. Since Coralie has left the lab for her postdoc, I got involved in some experiments to address this critic. I perfused animals, imaged brains and mapped neurons for analysis.

My PhD altogether aimed, taking V2a neurons as an entry-point, at decoding the extent to which the genetic classification of RS neurons is sufficient to approach neuronal diversity in the reticular formation. Combined with my contribution to the respiratory/locomotor interaction project, it also aimed at understanding better the neuronal basis of action diversification.

V2a medullary premotoneurons orchestrate upper trunk and orofacial orienting movements

For the following results, that are still not published, I took the initiative to present them here in a paper format.

However, I am fully aware that these data and analysis are not sufficient to complete a peer-reviewed article. Indeed, as the reader will see, the results are not exhaustive, especially for the quantification and analysis.

Moreover, other experiments, that I am currently performing, are required to make these results sufficient for a peer-reviewed article. Some of them indeed are explained in the short discussion that I inserted at the end.

Introduction

Body movements are necessary for all animals in order to explore the external environment, obtain food, escape for predators and interact with other animals. Most studies have focused on the neuronal circuits for either sensory integration or motor execution. However, the intermediate relay, i.e. the cells that link integrative centers with the executive circuits in the spinal cord, is still poorly documented. This yet represents a fundamental challenge and a prerequisite for designing rehabilitative therapies for patients after spinal cord injuries.

The brainstem reticular formation (RF) is a candidate relay of sensory and goaldirected modalities from multiple higher brain areas to the adequate executive motor circuits of the spinal cord that perform motor actions (Arber and Costa, 2018;Drew et al., 1986;Grillner and El Manira, 2020;Jordan et al., 2008;Leiras et al., 2022;Lemon, 2008;Ruder and Arber, 2019). Indeed, the RF receives inputs from multiple brain regions and hosts reticulospinal (RS) neurons, characterized by long axonal projections contacting different regions of the spinal cord (Caggiano et al., 2018;Ferreira-Pinto et al., 2018;Josset et al., 2018). Furthermore, reticular neurons comprise several cell types differing in their neurotransmitter identity, morphology, developmental history and gene expression (Brownstone and Chopek, 2018;Deliagina et al., 2002;Liu and Jordan, 2005;Valverde, 1961). This cellular heterogeneity suggests that i) each cell type could control a specific motor function, and ii) that different reticular neuronal subtypes might be part of distinct/parallel long-range neural circuits in different descending circuits. However, the functional diversity of reticular neurons and their efferent connectivity to the spinal cord is poorly characterized.

Previous works investigated one class of RS neurons of the Gigantocellular reticular nucleus (Gi) (Franklin and Paxinos, 2007), termed the V2a neurons (defined by the expression of the transcription factor Chx10) (Bretzner and Brownstone, 2013;Kimura et al., 2013). V2a neurons represent a genetically-circumscribable subgroup of excitatory RS neurons in the Gi that, when activated bilaterally, arrests ongoing locomotion (Bouvier et al., 2015). Such "V2a stop neurons" were then seen as divergent from the classical view that excitatory descending circuits favor movements. Recent works showed that the V2a neurons in the Gi receive inputs from multiple motor-related areas among which the contralateral superior colliculus is the main source (Cregg et al., 2020;Usseglio et al., 2020). This midbrain structure is well-known for its role in spatial orientation (Basso et al., 2021;Basso and May, 2017;Isa et al., 2021;Masullo et al., 2019;May, 2006). Functional investigations indeed confirmed that the activity of V2a neurons control orienting maneuvers (Usseglio et al., 2020). Yet, the same study demonstrated the existence of at least two subsets of V2a neurons controlling different motor components of orientation: one that projects to the lumbar spinal cord arrests locomotion and one that projects to the cervical spinal cord induces a head rotation and change in the trajectory. Yet V2a neurons in the medullary RF are exclusively glutamatergic. Hence, the opposite functional outcome of the two subsets must therefore reflect different targeted cell types in the spinal cord. While lumbarprojecting V2a neurons may exert their function via spinal inhibitory neurons (Bouvier et al., 2015), the post-synaptic targets of cervical projecting V2a neurons remained to be defined.

What's more, the orienting behavior in the large sense is known to mobilize additional motor components, including eye and snout movements (Basso and May, 2017;Isa et al., 2021;Isa and Sasaki, 2002;May, 2006), actions that are piloted by supra-spinal brainstem circuits. Interestingly, it was recently shown that V2a medullary neurons might comprise a non-spinally projecting population (Chopek et al., 2021), but its functional role was not directly examined. In general, the global efferent connectivity of medullary V2a neurons, as well as their role in controlling other motor actions besides locomotion, head movements and breathing (Crone et al., 2012) are not known.

Our present work demonstrate that the medullary V2a neurons control not only spinal-related motor actions, but also eye movements, an orienting response that is known to be controlled by the superior colliculus (Basso and May, 2017;Isa et al., 2021;Isa and Sasaki, 2002;May, 2006). Furthermore, we demonstrate that there is a subset of V2a neurons contacting the facial motor nucleus (7N), where reside the motoneurons controlling nose and whiskers movements (Guthrie, 2007). Importantly, the activation of the 7Nprojecting V2a subset does not trigger spinal-related movements nor eye movements.

Conversely, the C2-projecting and the L2-projecting V2a subsets do not control supraspinal motor actions (snout and eye movements). The different motor outcomes observed during medullary V2a neurons' activation (promoting movements, for the case of neck, eyes, snout movements and arresting motor action, in the case of the stop of locomotion) suggest that there might be distinct post synaptic targets. Indeed, while in the lumbar spinal cord the medullary V2a neurons might exert the stop of locomotion activating inhibitory neurons in the lumbar spinal cord (Bouvier et al., 2015), we demonstrated that the 7N and C2 projecting subsets directly form monosynaptic connections with the motoneurons controlling snout and neck muscles, respectively.

Our work shows that the V2a neurons control both spinal and brainstem-related motor functions, demonstrating a multifunctional ambition of this cell class for orchestrating components of the orienting behavior. Moreover, our results place V2a neurons as an essential premotor population for upper trunk and orofacial orienting actions. Altogether, our study contributes to propel forward our understanding of the complex and multifunction ambition of reticular neurons in the control of composite motor behaviors.

Results

V2a medullary neurons control multiple supraspinal-related motor actions

Previous work showed that the V2a neurons in the Gi control multiple motor components of orientation (Usseglio et al., 2020), including an ipsilateral snout displacement. We hypothesized here that V2a Gi neurons might in addition control eye movements, also typical for orienting behavior and commonly associated to superior colliculus functions (Basso et al., 2021;Basso and May, 2017;Isa et al., 2021). To investigate this, we delivered in Chx10-Cre mice the ChR2 in the Gi ipsilaterally and we implanted an optic fiber on the same side (Figure 1A,B). Then, we placed the mouse in a head-fixed setup and, using infrared illumination, we tracked the pupil movements during the light activation (Figure 1C) as previously done (Meyer et al., 2020;Michaiel et al., 2020;Sakatani and Isa, 2007). During the photo-activation (1 single pulse, 500 ms), we observed a horizontal deflection of the ipsilateral eye laterally, away from the nasal edge (Figure 1F,G).

This movement was accompanied by a conjugate displacement of the contralateral eye towards the nasal edge (data not shown). In contrast, the pupil movement was virtually absent in the vertical direction (Figure 1).

Furthermore, we also tested whether the eye displacement could be trigger by the photo-activation of spinally-projecting V2a neurons (C2 or L2-projecting). To selectively manipulate spinal-projecting V2a Gi neurons, we took advantage of the INTRSECT viral strategy (Fenno et al., 2014). We injected, in the C2, or L2, spinal cord segement of a Chx10-Cre mouse, a retrogradely-transported AAV coding for the Flp recombinase (AAVretro-flp), and another AAV, activated by both Cre and Flp coding a fluorescent protein and an excitatory channelrhodopsin (CreON-FlpON-ChR2-YFP) in the Gi (Figure 2A,D). In this way, the implantation of an optic fiber above the Gi, allows to activate selectively 7N-projecting V2a neurons (Figure 2A,D). Interestingly, we found no effect (Figure 2). These results showed that V2a neurons control horizontal movements of the pupil, a known a component of orienting, and that such eye movements are not controlled by spinally-projecting (C2 and L2) V2a neurons.

7N-projecting V2a medullary neurons trigger ipsilateral snout movements, but do not control eye movements nor arrest locomotion

We previously revealed that activating V2a Gi neurons, but not those with projections to the cervical or lumbar cord, evokes ipsilateral snout movements, suggesting that this function is a property of locally-projecting V2a Gi neurons. We therefore hypothesized that the snout movement is driven by a dedicated subset of V2a Gi neurons with projections to the 7 th facial motor nucleus (7N) and that horizontal eye movements might be controlled by axonal collaterals of this latter cell class. To selectively manipulate 7N-projecting V2a Gi neurons, we took again advantage of the INTRSECT viral strategy (Fenno et al., 2014). We injected, in the 7N of a Chx10-Cre mouse, a retrogradely-transported AAV coding for the Flp recombinase (AAVretro-flp), and another AAV, activated by both Cre and Flp coding a fluorescent protein and an excitatory channelrhodopsin (CreON-FlpON-ChR2-YFP) in the Gi (Figure 3A). In this way, the implantation of an optic fiber above the Gi, allows to activate selectively 7N-projecting V2a neurons (Figure 3A). We delivered the light when the mouse was in a head-fixed set-up and we recorded the snout activity. The light activation (50 ms) triggers a robust ipsilateral snout movement (Figure 3C). Activating the 7N-projecting V2a neurons therefore engages a similar response of the snout as that observed when activating the entire population of V2a Gi neurons ( Usseglio et al., 2020). Then, we investigated whether activating the 7N-projecting V2a neurons triggers also eye movements. With the same head-fixed set-up, we hence tracked the pupil of the ipsilateral eye during light activation as explained above. Surprisingly, and in stark contrast to photo-activating all V2a Gi neurons, we found here no movement of the eye (Figure 3D,E). These results reveal that the 7N-projecting V2a neurons are responsible for the snout displacement, but that another subset is responsible for eye movements.

Hence, the eye movement is likely controlled by a population of locally-projecting V2a neurons that differ from that controlling the snout.

V2a neurons target specific pool of motoneurons to control facial and neck muscles

The observations above suggest that the V2a neurons control different motor components related to orientation through multiple subtypes and project to different regions of the brainstem and spinal cord. It is therefore possible that distinct V2a subsets projecting to different brain regions may contact different postsynaptic targets. The lumbarprojecting V2a RS neurons might contact inhibitory interneurons inducing the arrest of the ongoing locomotion (Bouvier et al., 2015). In contrast, our previous work raised the possibility that cervical-projecting V2a RS neurons might contact directly specific motoneurons of the neck and upper trunk muscles, allowing to rapidly control axial musculature (Usseglio et al., 2020). This might also apply to the 7N-projections. To start addressing such hypotheses, we performed dedicated experiments that employ a transneuronal labelling strategy from selected muscle pools (Skarlatou et al., 2020;Stepien et al., 2010).

Firstly, to test our ability to target the motoneurons that control neck movements, we injected a retrogradely-transported Herpes virus coding a fluorophore (HSV-FP), in the dorsal neck muscle (splenius capitis) of a newborn mice at P2 (Figure 4A). As expected, this led to the labelling of numerous motoneurons, confined to ventral laminae of the spinal cord and the lower brainstem. On average, we found a total of 12 motoneurons per set of section per animal (only 1 out of 4 sections were imaged), which gives an estimate of 48 motoneurons per animal. The majority of labelled motoneurons were confined within the first segment of the cervical spinal cord (63%), while less were detected within the second cervical segment (26%) and at the level of the segmental level of the pyramidal decussation where the motoneurons of the 11 th cranial nerve are located (11%) (Figure 3). This positioning is in line with the known location of motoneurons of the splenius capitis, the main horizontal head flexor.

After having ascertained our capacity to transfect a selected muscle pool with a retrograde HSV, we next took advantage of transsynaptic rabies viral strategies that are widely used to identify the presynaptic connectome of the corresponding motoneurons. The aim was to identify whether some premotor neurons are of V2a identity, and if applicable, to register their position and examine their degree of collateralization to other motor pools.

There are multiple strategies used for a pre-motor labelling, and each has its own advantages and caveats and that they all have different rates of success that are summarized in a recent, still not peer-reviewed, review (Ronzano et al., 2022). I should also mention that such strategies had not been used in my lab before, and it hence took time for me to optimize injections, survival, and collect meaningful results. At this stage, we obtained the tools (in terms of viruses and mouse line) to perform two of these complementary strategies, that are represented in figure 5. In Figure 5 A is represented a strategy consisting in injecting in the selected muscle 2 viruses simultaneously: a retrograde virus to deliver the G protein (HSV-G) and a G-deleted-Rabies virus coding for a fluorescent protein. The transfected motoneurons, contacting the targeted muscle, thus express the G protein that allows the rabies to "jump" one synapse upstream and transfect the premotor neurons (Wickersham et al., 2007). However, with this strategy, there is a risk of transfecting the bipolar sensory neurons that contact the targeted muscle. As the rabies could potentially contaminate the distribution of pre-motoneurons via the anterograde ability to transfect the postsynaptic cells of the afferent neurons (Zampieri et al., 2014), there will be therefore a little fraction of cells expressing the fluorescent protein that are not part of the presynaptic connectome of the motoneurons (Figure 5B). On the other hand the second strategy (Figure 5C), consisting in injecting a G-deleted rabies virus in the selected muscle in a Chat-Cre;RGT mouse (expressing the G protein in the cholinergic cells), allows to avoid to transfect postsynaptic cell of the sensory neurons. However, the limitation of such strategy is represented by the presence of cholinergic spinal interneurons contacting the motoneurons, the V0c (cholinergic V0) that will therefore allow the rabies virus to transfect their presynaptic cells (Figure 5D). Therefore, both strategies come with experimental limitations.

Thus, both of them are required to validate the data obtained. Firstly, using a monosynaptic Rabies viral strategy (Stepien et al., 2010) consisting in injecting, unilaterally, an HSV-oG and a G-deleted Rabies virus in the neck muscles on newborn mice, we transfected the motoneurons controlling neck muscles and revealed their presynaptic neurons (Figure 6A). Indeed, a large fraction of neurons were found outside of the known location of motoneurons we described in the previous section. In particular, we observed abundant fluorescent neurons in the Gi nucleus (Figure 6B). To question their V2a identity, sections we co-stained with an anti-Chx10 antibody. We indeed observed Chx10positive neurons co-expressing expressing the fluorescent protein coded by the rabies virus (Figure 6C), i.e., neck premotoneurons of V2a identity. Hence, some V2a RS neurons in the Gi form monosynaptic connections with motoneurons in the upper cervical spinal cord controlling the neck muscles. Moreover, to confirm such data without the potentially confounding contribution of sensory afferences, we used an alternative approach consisting in injecting G-deleted rabies viruses coding for a fluorescent protein in the ipsilateral neck muscles of a newborn Chat-Cre;RGT mouse (Figure 6F). Here again, within the presynaptic connectome of the motoneurons of the neck we observed V2a neurons (Figure 6G). These results demonstrate that some V2a Gi neurons directly control the axial musculature of the neck, presumably allowing a rapid ipsilateral head displacement.

Secondly, we use the rabies transsynaptic strategy on newborn mice to interrogate whether the V2a neurons form a monosynaptic connection with the motoneurons controlling nose muscles (in the 7N motor nucleus). As we did for the motoneurons controlling neck muscles, we targeted the motoneurons that control snout musculature. We injected a retrogradely-transported Herpes virus coding a fluorophore (HSV-FP), in the nose muscles of a newborn mice at P2 (Figure 7A). As expected, this led to the labelling of numerous motoneurons, confined to the 7N. We found a total of 30 motoneurons per set of sections (estimating to 120 for one animal).

Therefore, we injected rabies G-deleted rabies viruses coding for a fluorescent protein in the snout muscles of a newborn Chat-Cre;RGT mouse (Figure 8A). We observe V2a

Rabies-positive neurons at all antero-posterior levels of the RF (figure 8B,C) on both the ipsilateral and contralateral sides. Importantly, we found V2a Rabies-positive neurons at different rostrocaudal positions of the brainstem, not only in the Gi and associated nuclei (GiA, GiV, LPGi), and in the spinal cord (Figure 8D,E,F,G).

These data reveal that some V2a neurons directly contact the motoneurons located within the facial motor nucleus in order to control snout movements.

Discussion

As the superior colliculus is well known to control multiple orienting movements, including eye and orofacial movements (Basso and May, 2017;Isa et al., 2021), and it has been previously shown that the medullary V2a neurons receive inputs from the superior colliculus (Cregg et al., 2020;Usseglio et al., 2020), we focused on whether the V2a neurons might control orienting motor actions associated to supraspinal/brainstem executory circuits. Notably, we observed, with dedicated experiments we designed along the way, that the unilateral activation of medullary V2a neurons triggers an ipsilateral snout movement, as we already demonstrated in our previous study, and a horizontal deflection of the pupil toward the stimulated side. After such observations, we immediately asked whether the V2a neurons control these motor outcomes with dedicated subsets, as the case for the spinallyprojecting V2a neurons (Usseglio et al., 2020). Indeed, nose and eye movements were only observed when activating V2a Gi neurons collectively, but when activating any of the spinally-projecting subset we did not observe any orofacial and eye movements. Moreover, we observed that the V2a neurons projecting to the facial motor nucleus, where reside the motoneurons controlling snout musculature, control selectively nose displacement, but no spinal related motor functions nor eye movements.

A crucial statement is that different V2a subsets can support opposing outcomes at the muscular level: promotion of movement (ipsilateral head rotation, eye and snout movements) for the cervical-projecting and supraspinal subsets, and motor arrest (of the hindlimbs) for the lumbar-projecting subset. These opposite functional outcomes could be due to the different cell-types contacted by the distinct V2a subsets. Whilst lumbarprojecting V2a neurons may exert their function via spinal inhibitory neurons (Bouvier et al., 2015), the post-synaptic targets of cervical-and facial-projecting V2a neurons remains to be defined. Our investigations, so far, reveal that cervical-projecting V2a RS neurons contact directly specific motoneurons of the neck and upper trunk muscles, known to reside in the upper cervical spinal segments and to be under control from the RF (Isa and Sasaki, 2002;Peterson et al., 1978;Sasaki et al., 2004). However, V2a neurons represent only a fraction of the RS neurons contacting the motoneurons that control neck muscles. It might be of interest to identify the other RS neurons in order to understand how they concur together with V2a neurons to finely control head movements. For example, different RS neurons might be recruited for different motor behaviors, or it could be that they can be recruited simultaneously to regulate the speed and the degrees of the head displacement.

Moreover, as we already mentioned, our study demonstrated that the medullary V2a neurons control eye movements. Up to now, the identification of a V2a subset that controls selectively eye movements has not been provided yet. However, the absence of pupil displacement during the activation of the C2, L2 and 7N-projecting V2a subsets, suggests the existence of a dedicated subset of V2a neurons controlling eye movements. Notably, we observed that the displacement of the ipsilateral pupil during the unilateral activation of the V2a Gi neurons was accompanied by the movement of the contralateral pupil toward the nasal edge. It might be interesting to understand how the V2a neurons control circuits for conjugate eye movements. Investigations could be done for example starting from the analysis of the V2a projections. Indeed, in the brainstem are located the motor nuclei (3 rd , 4 th , 5 th facial motor nucleus) where reside the motoneurons contacting eye muscles (Guthrie, 2007).

Furthermore, we observed that the V2a neurons projecting to the 7N contact directly the motoneurons controlling nose musculature. Therefore, it could be that the V2a subsets are premotor for promoting only certain motor actions (neck and snout movements). The cell type contacted at different levels of the brainstem and the spinal cords might differ based on the subset' function. Thus, it will be extremely interesting to explore further the postsynaptic targets of the V2a neurons in different regions of the brainstem and spinal cord, and discover what are the molecular and developmental basis that induce such heterogeneity.

As already explained within the results, the experimental rabies strategies we use have limits. Therefore, next step will be to use a third strategy (Figure 9) consisting in injecting a retrograde virus in the selected muscle to deliver the G protein and the TVA receptor. In a second step, the G-deleted Rabies pseutodyped with EnvA (that can transfect only cells expressing TVA receptor) will be injected centrally, within the spinal cord at the level of the motoneurons controlling the selected muscle. Importantly, crucial points remain to address in order to deeply understand the functional connectivity of the V2a neurons: 1) it is necessary indeed to investigate whether the V2a neurons, at different levels of the spinal cord and brainstem, contact distinct cell targets and 2) it is necessary to demonstrate whether the V2a neurons contact motoneurons only at the level of the cervical spinal cord and the 7N or also at the level of other brain regions. This might elucidate whether the postsynaptic connectivity of the medullary V2a neurons differs at distinct regions of the brainstem and spinal cord, in order to evoke specific responses: promoting (in the case of neck, eye and head movements) and preventing (in the case of the arrest of locomotion) motor action.

Another important open question related to the V2a network is whether the different subsets are interconnected. Recent work from Brownstone and colleagues demonstrated that the locally-projecting V2a neurons contact V2a RS neurons and they observed that these two groups of V2a neurons differ from soma size and electrophysiological properties.

However, it is not demonstrated yet whether there is connectivity between the other V2a subsets.

Moreover, the orienting behavior is associated to multiple motor actions including forelimb movements. Therefore, to study forelimb movements during medullary V2a neurons and whether they control extensor and flexor muscles of the forelimb might carry a more detailed overview about the functional and anatomical connectivity of the medullary V2a neurons.

Finally, it is essential to highlight that the V2a neurons represent only one population of the RF. Therefore, future investigations focusing in the other cells of the RF are crucial to understand the neural network within the RF. In particular, an important point is to decipher whether the organization of the V2a neurons is specific and unique for this cell type, or whether the other reticular neuronal types share similar organization in projection-defined subsets controlling unitary motor actions of complex behaviors.

Altogether, these data, combined with results obtained within previous studies (Chopek et al., 2021;Crone et al., 2012;Schwenkgrub et al., 2020;Usseglio et al., 2020), reveal a functional diversity in V2a RS neurons that can be subdivided in two macrocategories, anatomically and functionally, i) one that controls ocular, orofacial and respiratory movements through supra-spinal projections, and ii) another one that controls trunk and limb movements through spinal projections. Within each, a further specialization by projection site and postsynaptic targets supports the control of individual motor components.

Methods

Mice

The Chx10-Cre was kindly provided by S. Crone, K. Sharma, L. Zagoraiou, and T.M.

Jessell (Azim et al., 2014;Bouvier et al., 2015;Romer et al., 2017). C57BL6 wild-type mice were obtained by Janvier Labs (Le Genest-Saint-Isle, France). The Chat-Cre was obtained from Jackson Laboratories. Animals were group-housed with free access to food and water in controlled temperature conditions and exposed to a conventional 12-h light/dark cycle.

Experiments were performed on animals of either sex, aged 2 to 3 months at the time of first injection. All procedures were approved by the French Ethical Committee (authorization 2020-022410231878) and conducted in accordance with EU Directive 2010/63/EU. All efforts were made to reduce animal suffering and minimize the number of animals.

Viruses used

For Cre-dependent expression of ChR2 in the Gi we injected unilaterally 100 to 200 nL of an AAV9-Ef1a-DIO-hChR2(E123T/T159C)-eYFP (addgene #35509, titer 3.2 x 10 12 vp/ml (Mattis et al., 2011)). For mapping the motoneurons, we used intramuscular injections of 1 µL of HSV-mcherry, obtained from Dr. Rachael Neve (Gene Delivery Technology Core, Massachusetts General Hospital, USA). For premotor labelling from the muscules, we mixed 1µl of HSV-oG with 1µL of G-Rabies made in house (N. Zampieri & E. Toscano) as previously published (Skarlatou et al., 2020).

Surgical procedures for injections and implants in the brainstem

Animals were anesthetized with isoflurane throughout the surgery (4 % at 1 L/min for induction, and 2-3 % at 0.3 L/min for maintenance). Buprenorphine (0,025 mg/kg) was administered subcutaneously for analgesia before the surgery. The temperature of the mice was maintained at 36 °C with a feedback-controlled heating pad. Anesthetized animals were placed on a stereotaxic frame (Kopf) and the skull was exposed. Viral vectors were delivered using a pulled glass pipette connected to a syringe pump (Legato 130, KD Scientific, customized by Phymep, France). The infusion flow was set to 100 nL/min. Coordinates (in mm) used to target V2a neurons were: -6.0 from bregma, 0.8 lateral, and 4.5 from the dorsal brain surface. Coordinates (in mm) used to target 7N-projecting V2a neurons were: -6.0 from bregma, 1.4 lateral, and 5.1 from the dorsal brain surface. After the injection, the pipette was held in place for 5 min before being slowly retracted. For optogenetic activations, a 200 µm core 0.39 NA optic fiber (Thorlabs) connected to a 1.25 mm diameter ferrule (Thorlabs)

was implanted ~500 µm above the injected site. This operation was performed during the same surgery as the viral injection when both were targeted to the brainstem. For activating spinally-projecting V2a neurons, the spinal injection was performed first (see below) and the optic fiber was implanted 5 to 7 days later. Dental cement (Tetric Evoflow) was used to secure the implanted ferrules. Animals were followed daily after the surgery.

Surgical procedures for muscle injection

Animals were anesthetized with isoflurane throughout the surgery (with a variable flow depending on their size. Buprenorphine (0,025 mg/kg) was administered subcutaneously for analgesia before the surgery. Rabies virus was injected through a pulled glass capillary (Harvard apparatus, 1.2 OD x 0.69 x 100L mm), using 10 ms pressure air puffs, delivered by a Picospritzer.

Histology

Behavioral experiments

Optogenetic activations

Behavioral experiments started 15 to 21 days after the viral injection. Implanted animals were connected to a laser source (473 nm DPSS system, LaserGlow Technologies, Toronto, Canada) through a mating sleeve (Thorlabs). In all conditions light was delivered in trains of pulses of 15 ms at 40 Hz frequency for a duration of 500 ms. We used the minimal laser power sufficient to evoke a response, which was measured to be between 5-12 mW at the fiber tip using a power meter (PM100USB with S120C silicon power head, Thorlabs) to restrict photo-activations unilaterally, prevent heat, and exclude an unintentional silencing by over-activation.

Behavioral setting and video recordings

For analyzing changes in snout and eye movements, animals were placed in a custom made head-fixed set-up for a maximum of 10 minutes without prior habituation and filmed from above (snout) or the side (eye) at 400 images/sec using a CMOS camera (JaiGO 2400 USB). For analyzing eye movements during optogenetic activations, animals were place in a dark room and we used an IR light to detect pupil displacements. Images were streamed to disk on a computer using 2 nd Look (IO Industries). Timings of photo-activations were recorded using the TTL output of the laser connected to a National Instruments acquisition card (USB-6211) and the LabScribe NI software (iWorxs). Both recordings were synchronized using hardware trigger. Photo-activations were delivered manually using the NI MAX tool with a minimal interval of 30 seconds between two consecutive activations. Throughout the manuscript, one trial correspond to one photo-activation.

Quantification and statistical analysis

Pose estimation using DeepLabCut and quantifications of snout and eye movements

To compute changes in eye movements, we labelled manually 3 points of interest (POIs) from 437 frames taken across 22 videos using DeepLabCut version 2.1.5.2. We then used 95% of the labelled frames to train the network using a ResNet-101-based neural network with default parameters for 2 training iteration. We validated with 1 shuffles in the second training, and found that the test error was: 1.79 pixels, train: 1,51 pixels (image size was 120 by 110). We then used a p-cutoff of 0,6 to condition the X,Y coordinates for future analyses. This network was then used to analyze all other videos taken with same experimental settings.

To compute changes in snout movements, we labelled manually 3 points of interest (POIs) from 28 frames taken across 2 videos using DeepLabCut version 2.1.5.2. We then used 95% of the labelled frames to train the network using a ResNet-50-based neural network with default parameters for 1 training iteration. We validated with 1 shuffle, and found that the test error was: 1.26 pixels, train: 1,48 pixels (image size was 928 by 450). We then used a p-cutoff of 0,1 to condition the X,Y coordinates for future analyses. This network was then used to analyze all other videos taken with same experimental settings.

Custom scripts were written in MATLAB (Mathworks) and used for computation. The displacement of the pupil of the animal is defined as the movement of the pupil on the median to lateral axis (Fig 1E).

The snout rotation is defined by the angle β between two instances of the vector from R to N at the first frame and at the current frame. R is the right side of the nose, while N is the tip of the nose.

𝜃 𝑖 =

DISCUSSION

Our first interrogation focused on the behavioral significance of the locomotor arrests driven by V2a RS neurons (Bouvier et al., 2015). The results we obtained along the way raised new crucial questions that lead me to design specific experiments. To start exploring how the V2a neurons are engaged in arresting locomotion, first, we characterized anatomically their neuronal inputs with the highest selectivity. This showed that V2a Gi neurons receive most of their inputs from the contralateral Superior Colliculus. The inputs from the superior colliculus from the contralateral side made the V2a neurons candidates for relaying lateralized commands pertinent for orienting behaviors. We therefore performed unilateral photo-activations of V2a neurons in the Gi and found that the most striking response was a robust rotation of the head in the yaw axis and a displacement of the body orientation towards the stimulated side. We also observed an immediate horizontal deflection of the snout and the pupils tip towards the stimulated side. All these motor responses are known components of oriented attention in rodents [START_REF] Kurnikova | Coordination of Orofacial Motor Actions into Exploratory Behavior by Rat[END_REF]Masullo et al., 2019;[START_REF] Sahibzada | Movements resembling orientation or avoidance elicited by electrical stimulation of the superior colliculus in rats[END_REF]. Remarkably, when light stimulation was given during ongoing locomotion, the above orienting responses were accompanied by a bilateral arrest of all four limbs, followed by a pronounced change of locomotor trajectory towards the stimulated side. Moreover, we revealed the existence of at least three distinct V2a subsets: a lumbarprojecting subset whose activation arrests locomotion without changes in orientation nor pupil and snout movements, a cervical-projecting subset that evokes head rotation movements and whose activation alone imposes a change of locomotor direction, a facialprojecting subset that triggers an ipsilateral snout movement, but not head turning nor arrests of locomotion. Furthermore, we questioned the possibility that the divergent outcomes across muscular groups (e.g., ipsilateral excitation for the head and snout, bilateral inhibition for the hindlimbs) might reflect differences in V2a efferent connectivity at each executory module. Hence, using a rabies-based transsynaptic strategy on newborn mice, we documented that the cervical and facial-projecting V2a neurons directly contact the motoneurons controlling neck and snout musculature, respectively.

Therefore, our work highlights that V2a Gi neurons orchestrate orienting motor actions and can be subdivided in at least two macro-categories, i) one controlling ocular and orofacial movements through supra-spinal projections, and ii) one controlling trunk and limb movements through spinal projections. Within each, a further specialization by projection site and postsynaptic target supports the control of individual motor components of a coherent multi-faceted behavior.

Additionally to my main PhD project, I participated to the main research of a former PhD student of the laboratory [START_REF] Hérent | Upregulation of breathing rate during running exercise by central locomotor circuits[END_REF]. Results revealed that glutamatergic neurons of the MLR, and in particular that of the Cuneiform nucleus projects onto the PreBötC, the brainstem center that paces the inspiratory rhythm [START_REF] Del Negro | Breathing matters[END_REF].

Moreover, the activation of the CnF induces an increase of respiratory rate in a two-step sequential manner. Firstly, we observed a modest increase immediately at light onset but before the first locomotor movements. Secondly, the average respiratory rate was further augmented when the animal effectively engages in locomotion. These data suggest that glutamatergic CnF neurons can modulate respiratory activity independently of their action on limb movements, in line with what was reported in the lamprey [START_REF] Gariepy | Specific neural substrate linking respiration to locomotion[END_REF].

Yet the fact that the engagement in actual locomotor movements is associated with a stronger increase in breathing may reflect the mobilization, during actual running, of an additional activatory signal which could originate in executive lumbar locomotor circuits. We indeed show that the glutamatergic cells located ventrally in the lumbar spinal cord, send ascending projections to the brainstem. Furthermore, the activation of the lumbar segments increased the frequency of respiratory-like activities. However, in contrast to the anterograde tracings from the CnF glutamatergic neurons, these ascending projections were not targeted to the preBötC but rather to the parafacial respiratory region, another candidate of respiratory regulation during metabolic challenges including effort. Targeted chemogenetic silencing of these neurons demonstrated that they are required for setting the adapted ventilatory frequency during running exercise.

Altogether, these results thus uncover here two systems by which the central locomotor network can enable respiratory rate to be augmented in relation to running activity. These data expand, on the one hand, the functional implications of the MLR and the locomotor CPG beyond locomotor initiation and execution to bona fide respiratory modulation and on the other hand, the adaptive respiratory ambitions of the RTN beyond central C02 chemoception to "locomotor-ception".

The head turning steers locomotor trajectory

The discovery of the cervical-projecting V2a subset that control head turning and its activation suffices to steer the animals' directional heading, placing the head orientation as the prime driver of locomotor trajectory is very important since the mechanical substrate for trajectory changes during ongoing locomotion, whether it is head orientation [START_REF] Dollack | Head Anticipation During Locomotion With Auditory Instruction in the Presence and Absence of Visual Input[END_REF][START_REF] Grasso | The predictive brain: anticipatory control of head direction for the steering of locomotion[END_REF][START_REF] Grasso | Eye-head coordination for the steering of locomotion in humans: an anticipatory synergy[END_REF], an asymmetric descending drive to limb controllers [START_REF] Fagerstedt | Lateral turns in the Lamprey. I. Patterns of motoneuron activity[END_REF][START_REF] Oueghlani | Brainstem Steering of Locomotor Activity in the Newborn Rat[END_REF] or a combination of both, has remained speculative. Indeed, our investigations demonstrated that the unilateral activation of the entire population of the medullary V2a Gi neurons or the cervical-projecting

V2a neurons leads to a change in direction, but that this change cannot be reproduced when activating only the lumbar-projecting V2a neurons. This argues that the impact of V2a neurons on locomotor trajectory is primarily supported by their capacity to impose the head orientation. It will be extremely interesting to examinate whether mice could change direction without the lead of the head. This might help to comprehend what are the neural circuits for the steering of the locomotion, which is a topic that is poorly studied. Indeed, while the control of forward locomotion had progressed substantially over the last decades, our investigations provided here the first identification of a cellular substrate for the control of locomotor direction. Notably, as the head was already proposed and studied as an essential drive for steering trajectory in humans [START_REF] Grasso | The predictive brain: anticipatory control of head direction for the steering of locomotion[END_REF][START_REF] Grasso | Eye-head coordination for the steering of locomotion in humans: an anticipatory synergy[END_REF], the neural circuits controlling the head turning is poorly understood. Most of the studies focused on the superior colliculus/optic tectum (Basso and May, 2017;Isa et al., 2021;[START_REF] Saitoh | Tectal control of locomotion, steering, and eye movements in lamprey[END_REF]. Here, we uncover a large-scale neural circuit that connect the superior colliculus, the V2a neurons that reach the cervical spinal cord, and the motoneurons controlling neck musculature, which contraction induces the head turning and the consequently change of the trajectory while the animal is locomoting (Figure 13A). For future research, as we observed also RS neurons that are not Chx10-positive contacting the motoneurons controlling axial musculature of the neck, it might be important to understand whether there are other neural substrates that control the head movements in order to change the trajectory (Figure 13B). One possibility is that such "parallel" neural circuits might be used for different behavioral situations, including escaping or hunting, that are controlled by different cognitive/integrative centers. Discovering whether these parallel neural circuits might be also reciprocally connected could be important (Figure 13B). Indeed, this anatomical finding could allow to understand how distinct behaviors are associated.

Notably, the steering of the locomotor trajectory is an excellent example of composite/complex motor behavior that requires the involvement of multiple higher/cognitive and executive centers. Indeed, as the change of the trajectory might be due to avoid an obstacle or to a change of the destination, in both of the cases it is induced by a rapid integration of sensory inputs (vision, audition) to modify a motor behavior that was already ongoing (locomotion). Moreover, it requires further movements and postural adjustments of multiple parts of the body to avoid losing the equilibrium and fall.

During the second year of my PhD, we realized that another important laboratory was studying the same topic (Cregg et al., 2020). This situation represented an important point of my career since I found myself in a situation of "concurrency". Although it is clearly not optimal, it is common in research. It represented for me a difficult moment but the same time an important situation for my professional training. Thanks to the motivation of my PhD supervisor for me to continue on my own path and to the support of the whole team, I continued my research and I am persuaded that this was an important step for my PhD formation. Indeed, I from one side lit forced me to be more efficient, on the other side, and probably this is the most important point, I learnt how to question my own research and to be more critical about my own work. Furthermore, even if some of the results we obtained coincided with the results acquired from the other laboratory, which is important sign of reproducibility, the two works diverged on many aspects. Indeed, we both demonstrated that the unilateral activation of V2a Gi neurons induces a change in direction. While the other laboratory claim that the steering of the trajectory is regulated by an asymmetric control of V2a neurons on lumbar locomotor circuits, we rather argued, and demonstrated, that this change cannot be reproduced when activating only the lumbar-projecting subset, even unilaterally. Furthermore, we demonstrated that the activation of the cervicalprojecting V2a neurons, which induces the ipsilateral head turning, suffices to evoke a change of the trajectory toward the direction imposed by the head. This argues that the impact of the V2a neurons on locomotor trajectory is primarily supported by their capacity to impose the head orientation, rather than by their regulation of hindlimb controllers. The hindlimb asymmetry that indeed occurs during turning thus does not appear to be a primary consequence of activated descending V2a neurons but may rather be an adaptive adjustment to the displacement of the body axis, primed by the head, which is driven by cervical-projecting V2a neurons.

V2a neurons are organized in projection-based subsets

The mobilization of different motor actions controlled by medullary V2a neurons raises several questions: how are these motor actions controlled by the V2a neurons? Do the V2a neurons have access to the cervical and lumbar segments of the spinal cord where reside the circuits for the control of the head and hindlimb movements, respectively? Do they have access to the brainstem motor circuits in order to evoke the orofacial movements observed? Do the medullary V2a neurons control all these motor responses as a unique entity through a wide collateralization of their axons, or instead through distinct subpopulations? Do the opposite motor outcomes triggered by this cell class (promoting effect for head/eye, or arrest for locomotion) reflect different cell types (inhibitory/excitatory) contacted at different executory centers along the brainstem and the spinal cord?

These questions are crucial for understanding the reasoning of our experiments during the past four years. Indeed, we proceeded our investigations starting from the idea that the V2a neurons might have access to the distinct brainstem and spinal cord regions for the control of the diversity of motor actions observed during their activation. However, and that is one of the most striking findings of my work, these distinct motor spinal cord segments are not predominantly contacted by a unique population of V2a neurons with branched collaterals. Conversely, as I described before, V2a neurons are organized in subsets controlling distinct motor actions (Figure 13A).

Our results reveal that multiple motor components of orientation are controlled by distinct reticular neurons that share a common genetic identity but that differ in their efferent connectivity. Therefore, our investigations underscore the organization of the reticular formation in functional modules defined by transcription factors, and at the same time argues for a further segregation within modules whereby a functional connectivity organized by muscle or muscle groups may underlie the execution of individual motor actions of a coherent and multi-faceted behavior (Fig 13 C,D). Indeed, similar specialization by projection has been proposed for other brain structures including cerebello-spinal neurons [START_REF] Sathyamurthy | Cerebellospinal Neurons Regulate Motor Performance and Motor Learning[END_REF], cortico-spinal neurons [START_REF] Nelson | Corticospinal populations broadcast complex motor signals to coordinated spinal and striatal circuits[END_REF][START_REF] Sahni | Corticospinal neuron subpopulation-specific developmental genes prospectively indicate mature segmentally specific axon projection targeting[END_REF] and for the MLR [START_REF] Ferreira-Pinto | Functional diversity for body actions in the mesencephalic locomotor region[END_REF]. Moreover, in our study on exercise hyperpnoea, we demonstrated that the MLR does not control only locomotion, but also respiratory centers. Whether these functional outcomes are controlled by one neural population sending collaterals to the distinct executory centers or there are distinct subpopulations each controlling one functions, is still unknown. We postulate for the second hypothesis. Indeed, it has been showed, in lampreys that the MLR contacts the locomotor and respiratory centers with distinct subpopulations [START_REF] Gariepy | Specific neural substrate linking respiration to locomotion[END_REF].

Such specialization and diversification of motor actions controlled by specific projections could represent an important entry point to further decipher the neuronal basis for the diversity of motor repertoires and how the brain elaborates complex movements.

Indeed, this diversification might allow the nervous system to control one motor component independently from the others, when needed, and to increase the degree and versatility in the execution of composite motor behaviors. Furthermore, these subsets might be interconnected. Indeed, it has been shown that locally-projecting V2a neurons contact spinally-projecting V2a neurons (Chopek et al., 2021). However, whether different spinallyprojecting V2a subsets are connected, is still not known. If so, there could be a reciprocal control between different subsets. Moreover, as the V2a neurons represent only a fraction of the RS neurons, it is crucial to focus in addition to the other types of RS neurons on order to decipher whether the organization of the V2a neurons is unique for this cell type, or whether the other cell types share similar configuration in controlling actions of composite motor behaviors. Indeed, studies on other brainstem and spinal cell types defined by transcription factors demonstrated that they might contain distinct functional subtypes [START_REF] Sweeney | Origin and Segmental Diversity of Spinal Inhibitory Interneurons[END_REF][START_REF] Talpalar | Dual-mode operation of neuronal networks involved in left-right alternation[END_REF][START_REF] Wu | A V0 core neuronal circuit for inspiration[END_REF]. On this basis, from our study on the V2a neurons, we hypothesize that there might be a further specialization also in other RS cell types, that still remain to identify genetically (Fig 13E).

An approach that might be potentially groundbreaking in the investigation of the genetic diversity of the reticular formation is represented by the single cell RNA-sequencing.

Indeed, as we saw above, the identification and the study of a class of neurons by transcription factors has limitations. Indeed, transcription factors might be expressed only transiently at birth or with different degree of expression at early postnatal stages and adult animal. There might even be cells sharing the same genetic history with different levels of expression of the common transcription factor, which seems to be the case of the V2a neurons [START_REF] Hayashi | Graded Arrays of Spinal and Supraspinal V2a Interneuron Subtypes Underlie Forelimb and Hindlimb Motor Control[END_REF]. Furthermore, this classification with one single transcription factor cannot recapitulate a further functionally and anatom²ical classification within a so-called "cell type". Regarding V2a neurons, the diversification highlighted throughout my doctoral work is bound to reflect differences in gene expression that go well beyond the classification by the expression of Chx10. Moreover, single cells RNA sequencing can help to identified cell types by sequencing the RNA of hundreds of cells simultaneously [START_REF] Jaitin | Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types[END_REF], in order to reveal and explore heterogeneity in complex structures. For instance, such strategy has been used to delineate cell types with the spinal cord [START_REF] Blum | Single-cell transcriptomic analysis of the adult mouse spinal cord reveals molecular diversity of autonomic and skeletal motor neurons[END_REF][START_REF] Sathyamurthy | Massively Parallel Single Nucleus Transcriptional Profiling Defines Spinal Cord Neurons and Their Activity during Behavior[END_REF]. In our case, we could use it not only to reveal a possible genetic code underlying the diversity of the V2a neurons, but help study RS neurons in general. We might, first, identify a genetic signature that is associated to all reticulospinal neurons but that may be not shared with neurons of the reticular formation that do not project to the spinal cord. We could also reveal genetic determinants of other RS neurons besides the V2a ones. We might also identify the genetic underpinnings for the existence of different subsets of V2a neurons that differ by their projections. Applying the scRNA-seq on investigating the transcriptomic profiles of the reticular neurons, together with the study of projection profiles, soma position, developmental origin, might be fundamental to allow scientists to further progress in the understanding the neuronal heterogeneity of the reticular formation, and in general the entire brainstem, and its functions. Moreover, scRNA-seq strategy might allow scientists to identify the homologous cellular population in primate, and make comparison between rodents and primates, including humans.

Impact on clinical research

An important follow up of all the research focusing on reticulospinal neurons and their role in motor control, is represented by the investigation of the neural plasticity (sprouting and regeneration) of the descending fibers occurring after spinal cord injuries.

The term "sprouting" typically refers to new connections established by an axon that was not itself damaged. This is notably illustrated in hemi-section models of SCI, where descending axons reaching the non-lesioned side often branch new collaterals to the lesioned side [START_REF] Engmann | The Gigantocellular Reticular Nucleus Plays a Significant Role in Locomotor Recovery after Incomplete Spinal Cord Injury[END_REF][START_REF] Raineteau | Plasticity of motor systems after incomplete spinal cord injury[END_REF][START_REF] Zorner | Chasing central nervous system plasticity: the brainstem's contribution to locomotor recovery in rats with spinal cord injury[END_REF]. On the other hand, "regeneration" relates to the growth of cut axons and their extension into or beyond the lesioned tissue. There is general agreement that the greatest hope for functional recovery after spinal cord injury involves regeneration of descending motor tracts [START_REF] Siddiqui | Translating mechanisms of neuroprotection, regeneration, and repair to treatment of spinal cord injury[END_REF][START_REF] Tuszynski | Concepts and methods for the study of axonal regeneration in the CNS[END_REF]. Addressing the capacity of medullary descending tracts to sprout and/or regenerate is therefore essential and, together with understanding the anatomy of the RS neurons and their correlation with motor actions, represents a major challenge and a crucial necessary background to developed new efficient therapies aimed to motor recoveries after spinal cord injuries. Therefore, unveiling the link from the upper motor center involved in action planning and circuits in the brainstem and spinal cord leading the execution of body movements will help design protocols to restore post-traumatic motor disabilities. In particular, such protocols could led to locomotor recovery following spinal cord injury (SCI), an incurable condition in which the interruption or weakening of axonal tracts leads to highly debilitating loss of motor and sensory functions, including the ability to stand and walk, [START_REF] Sekhon | Epidemiology, demographics, and pathophysiology of acute spinal cord injury[END_REF][START_REF] Varma | Spinal cord injury: a review of current therapy, future treatments, and basic science frontiers[END_REF][START_REF] Wilson | Clinical predictors of neurological outcome, functional status, and survival after traumatic spinal cord injury: a systematic review[END_REF]. Indeed, following SCI, the locomotor CPG in the spinal cord is denervated from its obligatory descending signals but it retains much of the capacity to produce a locomotor-like output if activated adequately. In fact, some degree of motor function is often regained spontaneously from the first clinical assessment. This is thought, at least from correlations studies, to owe to spared descending tracts and/or a potential regrowth of the severed ones [START_REF] Fouad | Adaptive changes in the injured spinal cord and their role in promoting functional recovery[END_REF][START_REF] Hilton | Neuroprotection and secondary damage following spinal cord injury: concepts and methods[END_REF][START_REF] Zorner | Chasing central nervous system plasticity: the brainstem's contribution to locomotor recovery in rats with spinal cord injury[END_REF].

Establishing means of providing locomotor-relevant descending inputs below the lesion is thus an absolute requirement.

CONCLUSION

Our investigations reveal therefore that the V2a Gi neurons form a functional module of the reticular formation that globally controls orienting motor actions but that, within this module, projection-defined subsets are in charge of the individual component motor actions. This possibility is well in line with the idea of the reticular formation as a key getaway for the regulation of motor actions and its organization into functional "clusters" with specialized outputs [START_REF] Humphries | Is there a brainstem substrate for action selection?[END_REF]. These results contribute to the elucidation of the organization of the reticular formation, in line with the proposition that the brainstem is stratified in channels that must be combined to generate the motor signal to the execution of a composed behavior [START_REF] Arber | Networking brainstem and basal ganglia circuits for movement[END_REF]. A crucial point for next investigations might be to focus on the local connectivity within the reticular formation, whether there is a crosstalk between different modules, or even between different subsets at the level of the reticular formation in order to uncover whether the reticular formation has a role in refining motor programs, in action selection and commitment to action. Importantly, to understand the functioning of the reticular formation in controlling motor behaviors, it will be essential to consider the reticular formation part of a system-wide network that is connected to other supraspinal brain areas that control motor actions. Indeed, I am persuaded that uncovering how individual motor areas are bound into coordinated networks to perform the desired movements, is essential to understand how the brain control motor behaviors. Altogether, these supraspinal motor-related areas might concur to the high level of diversity, versatility and flexibility of the execution of the motor repertoires.

If we go back to the shaolin monk represented within the first page of this manuscript, we can now, comprehend the enormous efforts that are done by his nervous system to correctly accomplish such composite motor behavior. Indeed, as we saw, performing multiple motor actions simultaneously, requires the activity of multiple neural centers that span throughout the brain, and a crucial role is played by the reticular formation. However, we are just at the beginning of understanding how all these centers are connected to form large-scale neuronal circuits, and how the reticular formation, with its high neural heterogeneity, relays the motor signal and permits the execution of composite motor behaviors.

ENGLISH ABSTRACT

An essential function of the brain is to perform the desired motor actions. Our nervous system controls complex movements to execute coherent motor behaviors in response to our needs at every specific moment. Most studies focus on the executory centers in the brain and the spinal cord, or on the integrative/cognitive centers upstream.

The main structure that conveys the motor command from the upstream centers to the downstream centers is the reticular formation. It is known that it acts as a relay and that it is involved in multiple motor actions. However, whether it is organized in different cell types controlling individual motor actions or whether it works as a unique entity controlling synergistically multiple movements has to be addressed.

My PhD project attempts to understand how the reticular formation selects, initiates and combines multiple motor actions To address this we examined, in mice, the function and diversity of a genetically circumscribed class of glutamatergic neurons, the V2a neurons (Chx10-expressing).

Firstly, using rabies-based transsynaptic tracings, we reveal that V2a neurons receive abundant synaptic inputs from the contralateral superior colliculus, making them a candidate relay of orienting commands. Furthermore, we show that unilateral photoactivations of V2a neurons evoke multiple orienting-like motor responses, including a yaw rotation of the head and a displacement of the body axis towards the stimulated side. During locomotion, such activations lead to a transient locomotor arrest followed by a striking change in path trajectory.

Secondly, using retrograde tracers and circuit optogenetics we reveal that these multiple motor actions are supported by distinct projection-defined V2a subsets, with at least 1) a lumbar-projecting subset whose activation, even unilaterally, arrests locomotion but neither impacts trajectory nor evokes orienting movements, and 2) a cervical-projecting subset dedicated to head orientation and whose activation suffices to change the animal's trajectory. This argues that the impact of V2a RS neurons on path trajectory owes to their capacity to impose the head orientation.

Thirdly, we reveal that V2a reticular neurons also control other motor components of orienting, including snout and eye, whose executory circuits are located in the brainstem.

Importantly, these functions are supported by brainstem-projecting V2a subsets rather than by collaterals of the spinally-projecting ones. Indeed, we identified a V2a subset projecting to the facial motor nucleus (7N) whose activation elicits an ipsilateral snout displacement, but does not control neck and eye movements nor arrests locomotion.

Fourthly, we question the possibility that the divergent outcomes across muscular groups (e.g., ipsilateral excitation for the head and snout, bilateral inhibition for the hindlimbs) reflect differences in V2a efferent connectivity at each executory center. Hence, using a rabies-based transsynaptic strategy on newborn mice, we document the identity of the postsynaptic cells of the V2a neurons at different brainstem and spinal levels.

Specifically, we demonstrate that some V2a reticulospinal neurons make monosynaptic connection with motoneurons controlling axial muscle of the neck, at the level of the upper cervical spinal cord. We also demonstrate that the V2a neurons directly contact facial motoneurons that control snout and orofacial movements.

Our work highlights that V2a Gi neurons orchestrate orienting motor actions and can be subdivided in at least two macro-categories, i) one controlling ocular and orofacial movements through supra-spinal projections, and ii) one controlling trunk and limb movements through spinal projections. Within each, a further specialization by projection site and postsynaptic target supports the control of individual motor components of the multi-faceted orienting behavior. We identified the specific cell type in the caudal brain (the V2a neurons) that forms the link between an important sensory center in the brain and the spinal cord which dictates muscular contractions. We also found that these neurons show an unexpected diversity: there are several categories and each contacts a single region of the spinal cord for independently controlling the limbs, the head, the nose, and eye movements.

FRENCH ABSTRACT

Our study provides important new information on descending nerve signals for coordinating movements. The interruption or weakening of these descending connections is a main cause of loss of motor autonomy after spinal cord injury. Our findings should therefore ultimately help to develop targeted motor restoration strategies.

  Lastly, I would like to thank Dr. Daniel Shulz, Dr. Claire Wyart, Dr. Matilde Cordero-Erausquin, Dr. Micaela Galante, Dr. Stéphane Dieudonné and Dr. Pierre-Paul Vidal to have accepted to be members of the jury and dedicate their time and energy to evaluate my PhD work.

Figure 1 .

 1 Figure 1. Organization of the executory centers. (A) and (B): the executory centers are distributed in a somatotopic manner throughout the antero-posterior axis of the spinal cord; their position reflects the muscles that they control. (C) Schematic representation of the executory centers (in green) within the spinal cord. The shaded areas represents the circuit that contacts the motoneuron controlling the muscle fiber. (D) Representation of the twelve cranial nerves of the human brainstem. Realized using BioRender.

Figure 2 .

 2 Figure 2. Schematic representation of the brain, highlighting the location of the superior colliculus in two mammalian species, a monkey and a mouse (not to scale). The dashed line indicates an axial cut through the colliculus to reveal the layers. Stratum griseum superficiale (SGS) and stratum opticum (SO)

Figure 3 .

 3 Figure 3. Overview of the central pattern generator for breathing. Core rhythm-generating circuits appear to have two distinct brainstem oscillators: the endogenously active preBotzinger Complex (preBotC) (red box) and the conditionally active retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) (blue box). The preBotC drives inspiratory activity by projections to various premotor populations [rostral ventral respiratory group (rVRG), parahypoglossal (pXII)] that in turn project both to inspiratory muscles that pump air, e.g., the diaphragm and external intercostals, and to inspiratory muscles that modulate airflow resistance, e.g., laryngeal and tongue muscles. The RTN/pFRG has a similar functional path to expiratory muscles. preBotC and RTN/pFRG progenitors and neurons express distinct transcription factors (Dbx1 or Phox2b) and other genes (right; italics). Numerous neuromodulatory (left), suprapontine (top), and sensory (right) influences are shown. (Left) Neuromodulation. Respiratory pattern is highly labile. When animals go from quiet sitting to slow walking, the O2 consumption increases approximately threefold, and if ventilation does not increase rapidly, animals will probably pass out within 100 m. Peptides, serotonin, norepinephrine, and other endogenous neuromodulatorsoriginating in projections from, for example, the raphe, locus ceruleus, and hypothalamus-can affect rhythmogenesis. These actions are essential for normal regulation and may go awry in diseases affecting breathing. The dorsolateral pons, aka pontine respiratory group, including the Kolliker-Fuse and parabrachial nuclei, is also an important modulatory source. (Top) Suprapontine inputs are related to volition and emotion. (Right) Sensory inputs are essential for the proper regulation of blood gases and for mechanical adjustments related to posture, body mechanics, and likely metabolic efficiency. (Feldman et al., 2013) (B) Breathing rate augments during trotting independently of limb velocity or surface inclination. Diaphragm activity recordings in control condition (CTL), trotting at 25 and 50 cm/s and during recovery after the run (REC). Raw (Dia. EMG) and integrated (Int.) signals are illustrated in each condition.

Figure 4 .

 4 Figure 4. The neuronal control of locomotion in vertebrates. The selection and initiation of locomotion involves various regions of the brain and brainstem. Output neurons of the basal ganglia (BG) project both to the thalamus (Tha) -which sends projections to the motor cortex (MCtx) and other cortical areas -and to areas in the brainstem, including the mesencephalic locomotor region (MLR). Initiation of locomotion is thought to be mediated by the activity of neurons in the MLR including the cuneiform nucleus (CNf) and the pedunculopontine nucleus (PPN). MLR neurons project to neurons in the reticular formation in the brainstem. Neurons in the reticular formation project to locomotor networks in the spinal cord that execute locomotion (the locomotor CPG). Descending fibers from the vestibular and rubrospinal spinal pathways (brainstem nuclei (BSN)) maintain posture and modulatory signals that regulate the ongoing locomotor activity. The cerebellum coordinates locomotor behavior by mediating movement-generated feedback and internal feedback, as well as by modulating the activity in the descending pathways. Proprioceptive sensory feedback modulates the activity of the spinal locomotor network. Cortical activity (MCtx) provides visuomotor (VCtx) correction of locomotion via the posterior parietal cortex (pPCtx).[START_REF] Kiehn | Decoding the organization of spinal circuits that control locomotion[END_REF].

Figure 5 .

 5 Figure 5. Cranial nerves and their targets in humans.(Guthrie 2007)

Figure 6 .

 6 Figure 6. The main descending tracts. (A) Schematic representation of the origin of the five main descending tracts: the corticospinal tract (in green), the reticulospinal tract (in blue), the cerebellospinal tract (in black), the vestibulospinal tract (yellow) and the rubrospinal tract (in yellow).

Figure 7 .

 7 Figure 7. The complex organization of the reticular formation. A, transverse sections at equal distances through the pons and medulla oblongata indicating the size and the grouping of the cells of the reticular

Figure 8 .

 8 Figure 8. Mesencephalic Locomotor Region (MLR) connections with reticulospinal targets. Schematic view of the MLR that projects to the medullary reticular formation (MRF) with reticulospinal axons that descend to local, executory locomotor networks in the spinal cord through the ventrolateral funiculus.

Figure 9 .

 9 Figure 9. Schematic diagram illustrating the close spatial proximity of brainstem neurons implicated in orofacial and respiratory behaviors. (A) Top-down anatomical depiction of the BötC, preBötC, rVRG, IRt, and PCRt. Excitatory vGlut2 (teal) and inhibitory vGAT (purple) neurons, as well as Dbx1 (blue) neurons, are shown. The rostrocaudal boundary between MRF and PRF is indicated along with relevant cranial motor nuclei (gray). (B) Depiction of licking, breathing, and whisking behaviors with the implicated brainstem structures, and how rhythms between these behaviors can be synchronized. The breathing rhythm can entrain the whisking rhythm, indicating a network between relevant circuit elements.

Figure 10 .

 10 Figure 10. Schematic representation of distinct RS neurons of the medullary reticular formation. The glutamatergic RS neurons (in red) are commonly thought to promote locomotion, while inhibitory RS neurons (in blue) to prevent or block locomotion. The serotonergic RS neurons (in green) are considered to favor the ongoing locomotion. The noradrenergic RS neurons (in yellow) do not alter locomotor activity.

Figure 11 .

 11 Figure 11. Patterning and cell specification by transcription-factors in the developing spinal cord.

Figure 12 .

 12 Figure 12. V2a neurons are reticulospinal and their function has been investigated in two species. (A) Left Panel: transverse hemi-sections of the caudal reticular formation stained for V2a neurons (Tdtomato reporter mice line). Small panel on the top-right: schematic representation of the injections of the retrograde marker CTB at the 2nd lumbar segment of the spinal cord. Right panel: transverse hemisections of the caudal reticular formation stained for CTB and V2a neurons. (B) Transverse hemi-section in the Gi indicating Vglut2+ glutamatergic (red) V2a neurons (YFP). Bar-graphs show the average percentage of Vglut2+ that are V2a neurons within different regions of the reticular formation. Insets in B' and B'' are magnified views of Vglut2 expression alone (left) and merged with YFP (right) of medially (B') and laterally positioned (B'') V2a neurons. White arrowheads indicate co-expression. (C) In mice, the ability to stop locomotion is regulated by V2a neurons that project to the spinal cord where they depress locomotor rhythm generation.(Bouvier et al., 2015; Kimura et al., 2013). (D) Activation of hindbrain V2a

Figure 1 .

 1 Figure 1. Optogenetic activation of medullary V2a neurons evokes a horizontal displacement of the pupil. (A) Experimental strategy for photo-activating V2a Gi neurons. OF, optic fiber. The injection pipette is depicted with an angle for clarity, but the injection was performed without angle. (B) Left: transverse section at the level of the Gi showing transfected V2a neurons (eYFP, green) on a Nissl background (magenta), the position of the OF, and the theoretical light cone at the fiber tip (dashed blue lines). Scale bar, 1 mm. (C) Schematic representation of the experimental set up. The infrared camera, linked with an infrared light, was placed close to the eye, in a dark room to detect pupil movements. (D) The snapshot on the left shows the position of the pupil before the photo-activation. The snapshot in the centers shows the position of the pupil during the photo-activation (the light is represented with the blue border) The snapshot on the right shows the position of the pupil after the photo-activation. (E) A single frame with three references used for Deep Lab Cut analysis: a median point (in blue close to the nasal edge), the center of the pupil (red point) and the lateral point (green point, away from the nasal edge). (F) Graph showing the horizontal deflection of the pupil during the light activation (blue line). (G) Mean ± SEM changes in pupil deflection during light activation across trials (2 mice, 35 trials).

Figure 2 .

 2 Figure 2. Optogenetic activation of L2 and C2-projecting V2a neurons does not evoke a horizontal displacement of the pupil. (A, D) Experimental strategy for photo-activating V2a Gi neurons projecting to the L2 (A) and C2 (D). OF, optic fiber. The injection pipettes are depicted with an angle for clarity, but the injections were performed without angle. (B, E) Measurement of the horizontal deflection of the pupil during a single event. The snapshots above show the position of the pupil before (left snapshot), during (central snapshot with blue border) and after the light stimulation (right snapshot). (C, F) Mean ± SEM changes in pupil displacement during the light activation across trials (2 animals per experiments).

Figure 3 .

 3 Figure 3. Optogenetic activation of the 7N-projecting V2a neurons evokes snout movement. (A) Experimental strategy for photo-activating V2a Gi neurons projecting to the 7N. OF, optic fiber. The injection pipettes are depicted with an angle for clarity, but the injections were performed without angle. (B) Left: transverse section at the level of the Gi showing transfected 7N-projecting V2a neurons (eYFP, green) on a Nissl background (blue) and the beads (red) to confirm the injection site of the AAVretro-flp at the level of the 7N, the position of the OF, and the theoretical light cone at the fiber tip (dashed blue lines). Scale bar, 1 mm. Right: transverse section at the level of the Gi showing the transfected 7Nprojecting V2a neurons (eYFP, green) on a Nissl background (magenta). (C) Mean angular deflection ± SEM of the snout normalized to the angle during the same time interval before the light onset. Snapshots above show the displacement of the snout before (left snapshot), during (central snapshot with blue border) and after the light stimulation (right snapshot). (D) Measurement of the horizontal deflection of the pupil during a single event. The snapshots above show the position of the pupil before (left snapshot), during (central snapshot with blue border) and after the light stimulation (right snapshot). (E) Mean ± SEM changes in pupil displacement during the light activation across trials (2 mice).

Figure 5 .

 5 Figure 5. Experimental rabies strategy to reveal presynaptic connectomes of the motoneurons controlling a selected muscle. (A, B) Schematic representation of the injection on a newborn mouse (postnatal day 2). The injection is performed unilaterally in the neck muscle with a mix of viruses: a retrograde HSV to deliver the G-protein in the motoneurons together with a G-deleted rabies virus coding for a fluorescent protein to identify the transfected cells. The injection can be done on wild-type mice (WT) and reveals the Chx10-positive neurons with specific antibody, or on a Chx10-Cre;TdTomato mouse line to identify the V2a neurons. The perfusion is performed seven days after the injection (postnatal day 9). (C, D) Schematic representation of the injection on a newborn mouse (postnatal day 2). The injection is performed unilaterally in the neck muscle with a G-deleted rabies virus. In this strategy the injection must

Figure 6 .

 6 Figure 6. Rabies injection reveals that V2a neurons are premotor of the motoneurons controlling neck musculature. (A) Schematic representation of the injection on a newborn mouse (postnatal day 2). The injection is performed unilaterally in the neck muscle with a G-deleted rabies virus and a retrograde HSV. In this strategy the injection has been done on a wild-type (WT) mouse line. The perfusion is performed seven days after the injection (postnatal day 9). (B) Transverse section of the brainstem at the level of the Gi showing rabies positive neurons (in red) on a Nissl background (in blue). Scalebar: 0.5 mm. (C) Close-up view over the boxed area in (B) showing the rabies-transfected neurons (in red) and Chx10-positive neurons (in green). Scale bar: 0.1 mm. (D) Density map representing the position of the rabies-positive V2a neurons in the RF. (E) Percentage of the number of Chx10-positive neurons expressing the rabies virus over the total number of rabies-positive cells within the brain (brainstem and spinal cord), in the brainstem and in the Gi and associated nuclei (GiA, GiV and LPGi). (2 animals) (F) Schematic representation of the injection on a newborn mouse (postnatal day 2). The injection is performed unilaterally in the neck muscle with a G-deleted rabies virus. In this strategy the injection must be done on a Chat-Cre;RGT mouse line that expresses the G protein in all motoneurons. The perfusion is performed seven days after the injection (postnatal day 9). (G) Percentage of the number of Chx10-positive neurons expressing the rabies virus over the total number of rabies-positive cells within the brain (brainstem + spinal cord), in the brainstem and in the Gi and associated nuclei (GiA, GiV and LPGi). (4 animals).

Figure 7 .

 7 Figure 7. Retrograde injection reveals motoneurons controlling the snout musculature. (A) Schematic representation of the unilateral HSV injection in the snout muscles. (B) Transverse section at the level of the 7N showing transfected motoneurons (mCherry) on a Nissl background (Cyan). Scalebar: 0.5 mm. (C) Close-up view over the boxed area in (B) showing the transfected neurons in the 7N. Scale bar: 0.5 mm.

Figure 8 .

 8 Figure 8. Rabies injection reveals that V2a neurons are premotor of the motoneurons controlling snout muscles. (A) Schematic representation of the injection on a newborn mouse (postnatal day 2). The injection is performed unilaterally in the neck muscle with a G-deleted rabies virus. In this strategy the injection must be done on a Chat-Cre;RGT mouse line that expresses the Rabies G protein in all motoneurons. The perfusion is performed seven days after the injection (postnatal day 9). (B) Transverse section of the brainstem at the level of the Gi showing rabies positive neurons (in green) on a Nissl background (in blue). Scalebar: 0.5 mm. (C) close-up view over the boxed area in (B) showing the transfected neurons (in green) and Chx10-positive neurons (in red) on a Nissl background (in blue). Scale bar: 0.2 µm. (D-E-F) density maps representing the position of the rabies-positive V2a neurons in the RF at three different levels along the rostro caudal axis. (G) Percentage of Chx10-positive neurons expressing the rabies virus over the total number of rabies-positive cells within the brain the brainstem and in the Gi and associated nuclei (GiA, GiV and LPGi).

Figure 9 .

 9 Figure 9. Experimental rabies strategy to reveal presynaptic connectomes of the motoneurons controlling a selected muscle. (A) Schematic representation of the injection on a newborn mouse (postnatal day 2). The injection is performed unilaterally in the targeted muscle (in this case neck musle) with a retrograde HSV-oG-TVA. The injection can be done on wild type mice (WT). After two weeks, a second injection is performed in the upper cervical spinal cord with an EnvA-G-deleted-rabies virus coding for a fluorescent protein to identify the transfected cells. The perfusion is performed seven days after the injection. (B) This rabies strategy reveals the presynaptic connectome of the motoneurons without transfecting the sensory neurons or the cholinergic interneurons within the spinal cord.

Figure 13 .

 13 Figure 13. Overview of the circuits revealed in this work. (A) Schematic representation of the large-scale neural circuits: The superior colliculus projects to the V2a neurons (in red) that are organized in subsets controlling different components of orientation. The 7N-projecting subset contacts the motoneurons (in green) controlling snout musculature. The C2-projecting V2a neurons contacts motoneurons (in green) controlling axial musculature of the neck. The L2projecting V2a neurons contacts inhibitory interneurons (in yellow) within the lumbar spinal cord. (B) Schematic representation of a neural circuits composed by non-V2a neurons that might be mobilized in other behavioral contexts. Furtheremore, there might be a reciprocal connection between V2a and non-V2a RS neurons contacting motoneurons that control neck muscles. (C) The V2a neurons (in red) control multiple motor components pertinent for orientation. (D) V2a neurons is a heterogeneous population (different colors) organized in subsets each controlling distinct a specific motor component. (E) RS neurons that are non-V2a might control other motor behaviors and could be organized in subsets. Image created using Biorender.

  La principale fonction du cerveau est d'exécuter les actions motrices nécessaires à nos besoins à chaque instant. La plupart des études se concentrent sur les centres exécutifs de la moelle épinière ou sur les centres intégratifs et cognitifs en amont. Cependant, la structure principale qui transmet les commandes motrices de multiples comportements vers les centres exécutifs spinaux est la formation réticulée. Il n'est toutefois pas connu si la formation réticulée est organisée en différents types cellulaires contrôlant chacun des actions motrices individuelles, ou bien si elle forme une entité unique contrôlant de manière synergique de multiples actions. Mon doctorat vise ainsi à comprendre l'organisation de la formation réticulée pour l'orchestration des multiples composantes d'un comportement global cohérent. Je me suis intéressé en particulier aux neurones V2a, sous-population génétiquement définie de neurones excitateurs de la formation réticulée. Premièrement, nous révélons par traçages transsynaptiques que les neurones V2a du noyau gigantocellulaire (Gi) reçoivent de nombreuses entrées synaptiques du colliculus supérieur contralatéral, ce qui en fait des candidats pour orchestrer les commandes d'orientation. Corroborant cela, nous montrons que la photo-activation unilatérale des neurones V2a induit en effet de multiples réponses typiques de l'orientation, dont une rotation de la tête et un déplacement du corps vers le côté stimulé. Pendant la locomotion, l'activation de ces neurones entraîne un arrêt suivi d'un changement de direction. Deuxièmement, par traçages rétrogrades et optogénétique, nous révélons que ces différentes actions motrices sont soutenues par des sous-ensembles de neurones V2a distincts selon leurs projections efférentes, avec au moins 1) un sous-ensemble qui projette vers la moelle lombaire et dont l'activation, même unilatérale, arrête la locomotion mais n'a pas d'impact sur la trajectoire et n'évoque pas de mouvements d'orientation, et 2) un sousensemble qui projette vers la moelle cervicale et dont l'activation suffit à changer la trajectoire de l'animal. Ceci suggère que l'impact des neurones V2a sur la trajectoire est dû à leur capacité à imposer l'orientation de la tête. Troisièmement, nous révélons que les neurones V2a contrôlent d'autres composantes motrices de l'orientation, notamment le museau et l'oeil, dont les circuits exécutifs sont dans le tronc cérébral. Nous montrons surtout que ces fonctions sont soutenues par des sous-ensembles de V2a projetant localement dans le tronc cérébral plutôt que par des collatérales de ceux projetant dans la moelle épinière. En effet, nous avons identifié un sous-ensemble supraspinal de neurones V2a projetant vers le noyau moteur facial (7N) et dont l'activation déplace le museau, mais ne contrôle ni les mouvements du cou et des yeux, ni la locomotion. Quatrièmement, nous interrogeons la possibilité que les finalités fonctionnelles différentes des neurones V2a entre centres exécutifs (par exemple, excitation ipsilatérale pour la tête et le museau, inhibition bilatérale pour les membres postérieurs) reflètent des différences dans la connectivité efférente. Par traçages transsynaptiques, nous démontrons que certains neurones V2a établissent une connexion monosynaptique avec les motoneurones contrôlant le muscle axial du cou. De plus, nous certains neurones V2a contactent directement les motoneurones contrôlant les mouvements du museau. Notre travail met ainsi en évidence que les neurones V2a de la formation réticulée orchestrent les différentes composantes motrices de l'orientation et peuvent être subdivisés en au moins deux macro-catégories : une contrôlant les mouvements oculaires et orofaciaux par des projections supra-spinales, et une contrôlant les mouvements du tronc et des membres par des projections spinales. Dans chacune d'elles, une spécialisation par site de projection et cible postsynaptique soutient le contrôle indépendant de chaque composante. Titre : Circuits du tronc cérébral pour les mouvements locomoteurs et d'orientation : étude fonctionnelle et connectomique des neurones V2a Mots clés : circuit neuronal, formation réticulée, neurones réticulospinaux, optogénétique, traçage viral, sourisResumé : Ce projet porte sur les mécanismes cérébraux qui nous permettent de nous mouvoir. En particulier, nous avons cherché à comprendre l'origine et le rôle des signaux nerveux qui relient le cerveau et la moelle épinière, dont l'interruption est une cause principale des pertes de motricité après lésion médullaire. Nous avons identifié le type de cellule dans le cerveau postérieur (les neurones V2a) qui forme le lien entre un centre sensoriel du cerveau et la moelle épinière. Nous avons également constaté que ces neurones présentent une diversité inattendue : il en existe plusieurs catégories qui contactent chacune une seule région de la moelle épinière pour contrôler indépendamment les mouvements des membres, de la tête, du nez et des yeux. Notre étude fournit ainsi des informations nouvelles sur les signaux nerveux descendants qui coordonnent les mouvements. Ces résultats devraient, à terme, aider à développer des stratégies ciblées de restauration motrice.Title: Brainstem circuits for locomotor and orienting movements: a functional and connectivity study of V2a neurons Keywords: neural circuit, reticular formation, reticulospinal neurons, optogenetics, viral tracing, mouse Abstract: This project focuses on the brain mechanisms that allow us to move. The nerve signals that connect the brain and the spinal cord are vital for controlling movements. It is therefore essential to understand the origin and role of these signals.

Upregulation of breathing rate during running exercise by central locomotor circuits

	4.3.
	tan -1 (	𝑥 𝑁𝑖 -𝑥 𝑅𝑖 𝑦 𝑁𝑖 -𝑦 𝑅𝑖	) -tan -1 (	𝑥 𝑁0 -𝑥 𝑅0 𝑦 𝑁0 -𝑦 𝑅0	)
	𝑤𝑖𝑡ℎ 𝑅 𝑖 = (	𝑥 𝑅𝑖 𝑦 𝑅𝑖	) , 𝑁 𝑖 (	𝑥 𝑁𝑖 𝑦 𝑁𝑖	) , 𝑖 ∈ [0, 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑓𝑟𝑎𝑚𝑒𝑠]

RESULTS

Control of orienting movements and locomotion by

projection-defined subsets of brainstem V2a neurons