Descending Command Neurons in the Brainstem that Halt Locomotion.

The episodic nature of locomotion is thought to be controlled by descending inputs from the brainstem. Most studies have largely attributed this control to initiating excitatory signals, but little is known about putative commands that may specifically determine locomotor offset. To link identifiable brainstem populations to a potential locomotor stop signal, we used developmental genetics and considered a discrete neuronal population in the reticular formation: the V2a neurons. We find that those neurons constitute a major excitatory pathway to locomotor areas of the ventral spinal cord. Selective activation of V2a neurons of the rostral medulla stops ongoing locomotor activity, owing to an inhibition of premotor locomotor networks in the spinal cord. Moreover, inactivation of such neurons decreases spontaneous stopping in vivo. Therefore, the V2a "stop neurons" represent a glutamatergic descending pathway that favors immobility and may thus help control the episodic nature of locomotion.

Genetic identification of a hindbrain nucleus essential for innate vocalization.

Vocalization in young mice is an innate response to isolation or mechanical stimulation. Neuronal circuits that control vocalization and breathing overlap and rely on motor neurons that innervate laryngeal and expiratory muscles, but the brain center that coordinates these motor neurons has not been identified. Here, we show that the hindbrain nucleus tractus solitarius (NTS) is essential for vocalization in mice. By generating genetically modified newborn mice that specifically lack excitatory NTS neurons, we show that they are both mute and unable to produce the expiratory drive required for vocalization. Furthermore, the muteness of these newborns results in maternal neglect. We also show that neurons of the NTS directly connect to and entrain the activity of spinal (L1) and nucleus ambiguus motor pools located at positions where expiratory and laryngeal motor neurons reside. These motor neurons control expiratory pressure and laryngeal tension, respectively, thereby establishing the essential biomechanical parameters used for vocalization. In summary, our work demonstrates that the NTS is an obligatory component of the neuronal circuitry that transforms breaths into calls.

Dual-mode operation of neuronal networks involved in left-right alternation.

All forms of locomotion are repetitive motor activities that require coordinated bilateral activation of muscles. The executive elements of locomotor control are networks of spinal neurons that determine gait pattern through the sequential activation of motor-neuron pools on either side of the body axis. However, little is known about the constraints that link left-right coordination to locomotor speed. Recent advances have indicated that both excitatory and inhibitory commissural neurons may be involved in left-right coordination. But the neural underpinnings of this, and a possible causal link between these different groups of commissural neurons and left-right alternation, are lacking. Here we show, using intersectional mouse genetics, that ablation of a group of transcriptionally defined commissural neurons--the V0 population--leads to a quadrupedal hopping at all frequencies of locomotion. The selective ablation of inhibitory V0 neurons leads to a lack of left-right pattern at low frequencies, mixed coordination at medium frequencies, and alternation at high locomotor frequencies. When ablation is targeted to excitatory V0 neurons, left-right alternation is present at low frequencies, and hopping is restricted to medium and high locomotor frequencies. Therefore, the intrinsic logic of the central control of locomotion incorporates a modular organization, with two subgroups of V0 neurons required for the existence of left-right alternating modes at different speeds of locomotion. The two molecularly distinct sets of commissural neurons may constrain species-related naturally occurring frequency-dependent coordination and be involved in the evolution of different gaits.

MRI Assessment of Ischemic Lesion Evolution within White and Gray Matter.

BACKGROUND: In acute ischemic stroke (AIS), gray matter (GM) and white matter (WM) have different vulnerabilities to ischemia. Thus, we compared the evolution of ischemic lesions within WM and GM using MRI. METHODS: From a European multicenter prospective database (I-KNOW), available T1-weighted images were identified for 50 patients presenting with an anterior AIS and a perfusion weighted imaging (PWI)/diffusion weighted imaging (DWI) mismatch ratio of 1.2 or more. Six lesion compartments were outlined: initial DWI (b = 1,000 s/mm2) lesion, initial PWI-DWI mismatch (Tmax >4 s and DWI-negative), final infarct mapped on 1-month fluid-attenuated inversion recovery (FLAIR) imaging, lesion growth between acute DWI and 1-month FLAIR, DWI lesion reversal at 1 month and salvaged mismatch. The WM and GM were segmented on T1-weighted images, and all images were co-registered within subjects to the baseline MRI. WM and GM proportions were calculated for each compartment. RESULTS: Fifty patients were eligible for the study. Median delay between symptom onset and baseline MRI was 140 min. The percentage of WM was significantly greater in the following compartments: initial mismatch (52.5 vs. 47.5%, p = 0.003), final infarct (56.7 vs. 43.3%, p < 0.001) and lesion growth (58.9 vs. 41.2%, p < 0.001). No significant difference was found between GM and WM percentages within the initial DWI lesion, DWI reversal and salvaged mismatch compartments. CONCLUSIONS: Ischemic lesions may extend preferentially within the WM. Specific therapeutic strategies targeting WM ischemic processes may deserve further investigation.

Reduced CMRO2 and cerebrovascular reserve in patients with severe intracranial arterial stenosis: a combined multiparametric qBOLD oxygenation and BOLD fMRI study.

Multiparametric quantitative blood oxygenation level dependent (mqBOLD) magnetic resonance Imaging (MRI) approach allows mapping tissular oxygen saturation (StO2 ) and cerebral metabolic rate of oxygen (CMRO2 ). To identify hemodynamic alteration related to severe intracranial arterial stenosis (SIAS), functional MRI of cerebrovascular reserve (CVR BOLD fMRI) to hypercapnia has been proposed. Diffusion imaging suggests chronic low grade ischemia in patients with impaired CVR. The aim of the present study was to evaluate how oxygen parameters (StO2 and CMRO2 ), assessed with mqBOLD approach, correlate with CVR in patients (n = 12) with SIAS and without arterial occlusion. The perfusion (dynamic susceptibility contrast), oxygenation, and CVR were compared. The MRI protocol conducted at 3T lasted approximately 1 h. Regions of interest measures on maps were delineated on segmented gray matter (GM) of middle cerebral artery territories. We have shown that decreased CVR is spatially associated with decreased CMRO2 in GM of patients with SIAS. Further, the degree of ipsilateral CVR reduction was well-correlated with the amplitude of the CMRO2 deficit. The altered CMRO2 suggests the presence of a moderate ischemia explained by both a decrease in perfusion and in CVR. CVR and mqBOLD method may be helpful in the selection of patients with SIAS to advocate for medical therapy or percutaneous transluminal angioplasty-stenting.

Sensory feedback and central neuronal interactions in mouse locomotion.

Locomotion is a complex process involving specific interactions between the central neural controller and the mechanical components of the system. The basic rhythmic activity generated by locomotor circuits in the spinal cord defines rhythmic limb movements and their central coordination. The operation of these circuits is modulated by sensory feedback from the limbs providing information about the state of the limbs and the body. However, the specific role and contribution of central interactions and sensory feedback in the control of locomotor gait and posture remain poorly understood. We use biomechanical data on quadrupedal locomotion in mice and recent findings on the organization of neural interactions within the spinal locomotor circuitry to create and analyse a tractable mathematical model of mouse locomotion. The model includes a simplified mechanical model of the mouse body with four limbs and a central controller composed of four rhythm generators, each operating as a state machine controlling the state of one limb. Feedback signals characterize the load and extension of each limb as well as postural stability (balance). We systematically investigate and compare several model versions and compare their behaviour to existing experimental data on mouse locomotion. Our results highlight the specific roles of sensory feedback and some central propriospinal interactions between circuits controlling fore and hind limbs for speed-dependent gait expression. Our models suggest that postural imbalance feedback may be critically involved in the control of swing-to-stance transitions in each limb and the stabilization of walking direction.

Upregulation of breathing rate during running exercise by central locomotor circuits in mice.

While respiratory adaptation to exercise is compulsory to cope with the increased metabolic demand, the neural signals at stake remain poorly identified. Using neural circuit tracing and activity interference strategies in mice, we uncover here two systems by which the central locomotor network can enable respiratory augmentation in relation to running activity. One originates in the mesencephalic locomotor region (MLR), a conserved locomotor controller. Through direct projections onto the neurons of the preBötzinger complex that generate the inspiratory rhythm, the MLR can trigger a moderate increase of respiratory frequency, prior to, or even in the absence of, locomotion. The other is the lumbar enlargement of the spinal cord containing the hindlimb motor circuits. When activated, and through projections onto the retrotrapezoid nucleus (RTN), it also potently upregulates breathing rate. On top of identifying critical underpinnings for respiratory hyperpnea, these data also expand the functional implication of cell types and pathways that are typically regarded as "locomotor" or "respiratory" related.

Sensory Feedback and Central Neuronal Interactions in Mouse Locomotion.

Locomotion is a complex process involving specific interactions between the central neural controller and the mechanical components of the system. The basic rhythmic activity generated by locomotor circuits in the spinal cord defines rhythmic limb movements and their central coordination. The operation of these circuits is modulated by sensory feedback from the limbs providing information about the state of the limbs and the body. However, the specific role and contribution of central interactions and sensory feedback in the control of locomotor gait and posture remain poorly understood. We use biomechanical data on quadrupedal locomotion in mice and recent findings on the organization of neural interactions within the spinal locomotor circuitry to create and analyze a tractable mathematical model of mouse locomotion. The model includes a simplified mechanical model of the mouse body with four limbs and a central controller composed of four rhythm generators, each operating as a state machine controlling the state of one limb. Feedback signals characterize the load and extension of each limb as well as postural stability (balance). We systematically investigate and compare several model versions and compare their behavior to existing experimental data on mouse locomotion. Our results highlight the specific roles of sensory feedback and some central propriospinal interactions between circuits controlling fore and hind limbs for speed-dependent gait expression. Our models suggest that postural imbalance feedback may be critically involved in the control of swing-to-stance transitions in each limb and the stabilization of walking direction.

Breathing without CO(2) chemosensitivity in conditional Phox2b mutants.

Breathing is a spontaneous, rhythmic motor behavior critical for maintaining O(2), CO(2), and pH homeostasis. In mammals, it is generated by a neuronal network in the lower brainstem, the respiratory rhythm generator (Feldman et al., 2003). A century-old tenet in respiratory physiology posits that the respiratory chemoreflex, the stimulation of breathing by an increase in partial pressure of CO(2) in the blood, is indispensable for rhythmic breathing. Here we have revisited this postulate with the help of mouse genetics. We have engineered a conditional mouse mutant in which the toxic PHOX2B(27Ala) mutation that causes congenital central hypoventilation syndrome in man is targeted to the retrotrapezoid nucleus, a site essential for central chemosensitivity. The mutants lack a retrotrapezoid nucleus and their breathing is not stimulated by elevated CO(2) at least up to postnatal day 9 and they barely respond as juveniles, but nevertheless survive, breathe normally beyond the first days after birth, and maintain blood PCO(2) within the normal range. Input from peripheral chemoreceptors that sense PO(2) in the blood appears to compensate for the missing CO(2) response since silencing them by high O(2) abolishes rhythmic breathing. CO(2) chemosensitivity partially recovered in adulthood. Hence, during the early life of rodents, the excitatory input normally afforded by elevated CO(2) is dispensable for life-sustaining breathing and maintaining CO(2) homeostasis in the blood.

Absent phasing of respiratory and locomotor rhythms in running mice.

Examining whether and how the rhythms of limb and breathing movements interact is highly informative about the mechanistic origin of hyperpnoea during running exercise. However, studies have failed to reveal regularities. In particular, whether breathing frequency is inherently proportional to limb velocity and imposed by a synchronization of breaths to strides is still unclear. Here, we examined respiratory changes during running in the resourceful mouse model. We show that, for a wide range of trotting speeds on a treadmill, respiratory rate increases to a fixed and stable value irrespective of trotting velocities. Respiratory rate was yet further increased during escape-like running and most particularly at gallop. However, we found no temporal coordination of breaths to strides at any speed, intensity, or gait. Our work thus highlights that exercise hyperpnoea can operate, at least in mice and in the presently examined running regimes, without phasic constraints from limb movements.

Deep imaging in the brainstem reveals functional heterogeneity in V2a neurons controlling locomotion.

V2a neurons are a genetically defined cell class that forms a major excitatory descending pathway from the brainstem reticular formation to the spinal cord. Their activation has been linked to the termination of locomotor activity based on broad optogenetic manipulations. However, because of the difficulties involved in accessing brainstem structures for in vivo cell type-specific recordings, V2a neuron function has never been directly observed during natural behaviors. Here, we imaged the activity of V2a neurons using micro-endoscopy in freely moving mice. We find that as many as half of the V2a neurons are excited at locomotion arrest and with low reliability. Other V2a neurons are inhibited at locomotor arrests and/or activated during other behaviors such as locomotion initiation or stationary grooming. Our results establish that V2a neurons not only drive stops as suggested by bulk optogenetics but also are stratified into subpopulations that likely contribute to diverse motor patterns.

Control of Orienting Movements and Locomotion by Projection-Defined Subsets of Brainstem V2a Neurons.

Spatial orientation requires the execution of lateralized movements and a change in the animal's heading in response to multiple sensory modalities. While much research has focused on the circuits for sensory integration, chiefly to the midbrain superior colliculus (SC), the downstream cells and circuits that engage adequate motor actions have remained elusive. Furthermore, the mechanisms supporting trajectory changes are still speculative. Here, using transneuronal viral tracings in mice, we show that brainstem V2a neurons, a genetically defined subtype of glutamatergic neurons of the reticular formation, receive putative synaptic inputs from the contralateral SC. This makes them a candidate relay of lateralized orienting commands. We next show that unilateral optogenetic activations of brainstem V2a neurons in vivo evoked ipsilateral orienting-like responses of the head and the nose tip on stationary mice. When animals are walking, similar stimulations impose a transient locomotor arrest followed by a change of trajectory. Third, we reveal that these distinct motor actions are controlled by dedicated V2a subsets each projecting to a specific spinal cord segment, with at least (1) a lumbar-projecting subset whose unilateral activation specifically controls locomotor speed but neither impacts trajectory nor evokes orienting movements, and (2) a cervical-projecting subset dedicated to head orientation, but not to locomotor speed. Activating the latter subset suffices to steer the animals' directional heading, placing the head orientation as the prime driver of locomotor trajectory. V2a neurons and their modular organization may therefore underlie the orchestration of multiple motor actions during multi-faceted orienting behaviors.

Afadin Signaling at the Spinal Neuroepithelium Regulates Central Canal Formation and Gait Selection.

Afadin, a scaffold protein controlling the activity of the nectin family of cell adhesion molecules, regulates important morphogenetic processes during development. In the central nervous system, afadin has critical roles in neuronal migration, axonal elongation, and synapse formation. Here we examine the role of afadin in development of spinal motor circuits. Afadin elimination in motor neuron progenitors results in striking locomotor behavior: left-right limb alternation is substituted by synchronous activation, characteristic of bound gait. We find that afadin function at the neuroepithelium is required for structural organization of the spinal midline and central canal morphogenesis. Perturbation of afadin results in formation of two central canals, aberrant contralateral wiring of different classes of spinal premotor interneurons, and loss of left-right limb alternation, highlighting important developmental principles controlling the assembly of spinal motor circuits.

Brain-derived neurotrophic factor enhances fetal respiratory rhythm frequency in the mouse preBötzinger complex in vitro.

Brain-derived neurotrophic factor (BDNF) is required during the prenatal period for normal development of the respiratory central command; however, the underlying mechanisms remain unknown. To approach this issue, the present study examined BDNF regulation of fetal respiratory rhythm generation in the preBötzinger complex (preBötC) of the mouse, using transverse brainstem slices obtained from prenatal day 16.5 animals. BDNF application (100 ng/mL, 15 min) increased the frequency of rhythmic population activity in the preBötC by 43%. This effect was not observed when preparations were exposed to nerve growth factor (100 ng/mL, 30 min) or pretreated with the tyrosine kinase inhibitor K252a (1 h, 200 nm), suggesting that BDNF regulation of preBötC activity requires activation of its cognate tyrosine receptor kinase, TrkB. Consistent with this finding, single-cell reverse transcription-polymerase chain reaction experiments showed that one third of the rhythmically active preBötC neurons analysed expressed TrkB mRNA. Moreover, 20% expressed BDNF mRNA, suggesting that the preBötC is both a target and a source of BDNF. At the network level, BDNF augmented activity of preBötC glutamatergic neurons and potentiated glutamatergic synaptic drives in respiratory neurons by 34%. At the cellular level, BDNF increased the activity frequency of endogenously bursting neurons by 53.3% but had no effect on basal membrane properties of respiratory follower neurons, including the Ih current. Our data indicate that BDNF signalling through TrkB can acutely modulate fetal respiratory rhythm in association with increased glutamatergic drive and bursting activity in the preBötC.

A V0 core neuronal circuit for inspiration.

Breathing in mammals relies on permanent rhythmic and bilaterally synchronized contractions of inspiratory pump muscles. These motor drives emerge from interactions between critical sets of brainstem neurons whose origins and synaptic ordered organization remain obscure. Here, we show, using a virus-based transsynaptic tracing strategy from the diaphragm muscle in the mouse, that the principal inspiratory premotor neurons share V0 identity with, and are connected by, neurons of the preBötzinger complex that paces inspiration. Deleting the commissural projections of V0s results in left-right desynchronized inspiratory motor commands in reduced brain preparations and breathing at birth. This work reveals the existence of a core inspiratory circuit in which V0 to V0 synapses enabling function of the rhythm generator also direct its output to secure bilaterally coordinated contractions of inspiratory effector muscles required for efficient breathing.The developmental origin and functional organization of the brainstem breathing circuits are poorly understood. Here using virus-based circuit-mapping approaches in mice, the authors reveal the lineage, neurotransmitter phenotype, and connectivity patterns of phrenic premotor neurons, which are a crucial component of the inspiratory circuit.

Spatiotemporal correlation of spinal network dynamics underlying spasms in chronic spinalized mice.

Spasms after spinal cord injury (SCI) are debilitating involuntary muscle contractions that have been associated with increased motor neuron excitability and decreased inhibition. However, whether spasms involve activation of premotor spinal excitatory neuronal circuits is unknown. Here we use mouse genetics, electrophysiology, imaging and optogenetics to directly target major classes of spinal interneurons as well as motor neurons during spasms in a mouse model of chronic SCI. We find that assemblies of excitatory spinal interneurons are recruited by sensory input into functional circuits to generate persistent neural activity, which interacts with both the graded expression of plateau potentials in motor neurons to generate spasms, and inhibitory interneurons to curtail them. Our study reveals hitherto unrecognized neuronal mechanisms for the generation of persistent neural activity under pathophysiological conditions, opening up new targets for treatment of muscle spasms after SCI.

Effect of Cyclosporine on Lesion Growth and Infarct Size within the White and Gray Matter.

BACKGROUND: In a recent trial, cyclosporine A (CsA) failed to reduce infarct size in acute stroke patients treated with intravenous thrombolysis. White matter (WM) and gray matter (GM) may have distinct vulnerability to ischemia and response to therapy. Using final infarct size and lesion growth as endpoints, our objectives were to (1) investigate any tissue-specific effect of CsA and (2) compare WM and GM response to thrombolysis. MATERIALS AND METHODS: We analyzed 84 patients from the randomized and placebo-controlled CsA-Stroke trial, who underwent MRI both on admission and at 1 month. Lesion growth was defined voxel-wise as infarcted tissue at 1 month with no visible lesion on baseline diffusion-weighted imaging. After automatic segmentation of GM/WM, final infarct size and lesion growth were compared within the GM and WM. RESULTS: Occlusion level was distal (>M1) in 51% of cases. No significant difference in GM/WM proportions was observed within final infarcts between treatment groups (P = 0.21). Infarct size within the GM or WM was similar between the CsA and control groups [GM: 9.2 (2.4; 22.8) with CsA vs 8.9 (3.7; 28.4) mL with placebo, P = 0.74; WM: 9.9 (4.7; 25.4) with CsA vs 14.1 (5.6; 34.1) mL with placebo, P = 0.26]. There was no significant effect of CsA on lesion growth in either the GM or WM. Pooling all patients, a trend for increased relative lesion growth in WM compared to GM was observed [49.0% (14.7; 185.7) vs 43.1% (15.4; 117.1), respectively; P = 0.12]. CONCLUSION: No differential effect of CsA was observed between WM and GM. Pooling all patients, a trend toward greater lesion growth in WM was observed.

Acute role of the brain-derived neurotrophic factor (BDNF) on the respiratory neural network activity in mice in vitro.

In humans, several pathologies are associated with disturbances of the respiratory control, some of them including alteration in the brain-derived neurotrophic factor (BDNF) signalling pathway. BDNF has long been known as a neurotrophic factor involved in survival, differentiation and maintenance of neuronal populations in the peripheral and central nervous system. More recently BDNF has also been discovered to be a potent neuromodulator with acute effects on neuronal excitability and synaptic plasticity. Animals deleted for the gene encoding BDNF exhibit respiratory alteration suggesting an important but yet undefined role of the neurotrophin in respiratory rhythmogenesis either by a trophic and/or an acute action. The possibility that BDNF might exert an acute regulatory role on the rhythmic activity of the respiratory generator of the pre-Bötzinger complex has been recently examined in newborn mice in vitro. Results obtained, reviewed in the present paper, will help getting insights in respiratory rhythm regulatory mechanisms that involve BDNF signalling.