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While respiratory adaptation to exercise is compulsory to cope with the increased metabolic supply to body tissues and with the necessary clearing of metabolic waste, the neural apparatus at stake remains poorly identified. Using viral tracing, ex vivo and in vivo optogenetic and chemogenetic interference strategies in mice, we unravel interactive locomotor and respiratory networks’ nodes that mediate the respiratory rate increase that accompanies a running exercise. We show that the mesencephalic locomotor region (MLR) and the lumbar spinal locomotor pattern generator (lumbar CPG), which respectively initiate and execute the locomotor behavior, access the respiratory network through distinct entry points. The MLR directly projects onto the inspiratory rhythm generator, the preBötzinger complex (preBötC), and can trigger a moderate increase of respiratory frequency, prior to, or even in the absence of, locomotion. In contrast, the lumbar CPG projects onto the retrotrapezoid nucleus (RTN) that in turn contacts the preBötC to enforce, during effective locomotion, higher respiratory frequencies. These data expand, on the one hand, the functional implications of the MLR beyond locomotor initiation to a bona fide respiratory modulation. On the other hand, they expand the adaptive respiratory ambitions of the RTN beyond chemoception to “locomotor- ception”.

International audience; 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.

Calcium imaging of genetically defined neurons in the ventral brainstem during locomotion reveals unexpected functional diversity., 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.

International audience; 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.

Examining whether and how the rhythms of limb and breathing movements interact is highly informative about the mechanistic origin of hyperpnoea to exercise. However, studies have failed to reveal regularities. In particular, whether breathing frequency is inherently proportional to limb velocity and/or imposed by a synchronization of breaths to strides is still unclear. Here, we examined the specifications of respiratory changes during running in mice, the premier model for investigating, in a standardized manner, complex integrative tasks including adaptive breathing. We show that respiratory rate increases during running to a fixed and stable value, irrespective of trotting velocities and of inclination. Yet, respiratory rate was further enhanced during gallop. We also demonstrate the absence of temporal coordination of breaths to strides at any speed, intensity or gait. Our work thus highlights a hardwired mechanism that sets respiratory frequency independently of limb movements but in relation with the engaged locomotor program.

International audience; Locomotion is a fundamental motor function common to the animal kingdom. It is implemented episodically and adapted to behavioural needs, including exploration, which requires slow locomotion, and escape behaviour, which necessitates faster speeds. The control of these functions originates in brainstem structures, although the neuronal substrate(s) that support them have not yet been elucidated. Here we show in mice that speed and gait selection are controlled by glutamatergic excitatory neurons (GlutNs) segregated in two distinct midbrain nuclei: the cuneiform nucleus (CnF) and the pedunculopontine nucleus (PPN). GlutNs in both of these regions contribute to the control of slower, alternating-gait locomotion, whereas only GlutNs in the CnF are able to elicit high-speed, synchronous-gait locomotion. Additionally, both the activation dynamics and the input and output connectivity matrices of GlutNs in the PPN and the CnF support explorative and escape locomotion, respectively. Our results identify two regions in the midbrain that act in conjunction to select context-dependent locomotor behaviours.

International audience; 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.

International audience; 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.

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. DOI: http://dx.doi.org/10.7554/eLife.23011.001

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 512) with SIAS and without arterial occlusion. The perfusion (dynamic susceptibil- ity 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 cere- bral 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. Hum Brain Mapp 00:000-000, 2014. V C 2014 Wiley Periodicals, Inc.

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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.

International audience; 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.

International audience; The proper development and maturation of neuronal circuits require precise migration of component neurons from their birthplace (germinal zone) to their final positions. Little is known about the effects of aberrant neuronal position on the functioning of organized neuronal groups, especially in mammals. Here, we investigated the formation and properties of brainstem respiratory neurons in looptail (Lp) mutant mice in which facial motor neurons closely apposed to some respiratory neurons fail to migrate due to loss of function of the Wnt/Planar Cell Polarity (PCP) protein Vangl2. Using calcium imaging and immunostaining on embryonic hindbrain preparations, we found that respiratory neurons constituting the embryonic parafacial oscillator (e-pF) settled at the ventral surface of the medulla in Vangl2(Lp/+) and Vangl2(Lp/Lp) embryos despite the failure of tangential migration of its normally adjacent facial motor nucleus. Anatomically, the e-pF neurons were displaced medially in Lp/+ embryos and rostro-medially Lp/Lp embryos. Pharmacological treatments showed that the e-pF oscillator exhibited characteristic network properties in both Lp/+ and Lp/Lp embryos. Furthermore, using hindbrain slices, we found that the other respiratory oscillator, the preBötzinger complex, was also anatomically and functionally established in Lp mutants. Importantly, the displaced e-pF oscillator established functional connections with the preBötC oscillator in Lp/+ mutants. Our data highlight the robustness of the developmental processes that assemble the neuronal networks mediating an essential physiological function.

Breathing is a spontaneous, rhythmic motor behavior critical for maintaining O2, CO2, 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 CO2in 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 toxicPHOX2B27Alamutation 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 CO2at 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 PCO2within the normal range. Input from peripheral chemoreceptors that sense PO2in the blood appears to compensate for the missing CO2response since silencing them by high O2abolishes rhythmic breathing. CO2chemosensitivity partially recovered in adulthood. Hence, during the early life of rodents, the excitatory input normally afforded by elevated CO2is dispensable for life-sustaining breathing and maintaining CO2homeostasis in the blood.

International audience; Foetal breathing in mice results from prenatal activity of the two coupled hindbrain oscillators considered to be responsible for respiratory rhythm generation after birth: the pre-Bötzinger complex (preBötC) is active shortly before the onset of foetal breathing; the parafacial respiratory group (e-pF in embryo) starts activity one day earlier. Transcription factors have been identified that are essential to specify neural progenitors and lineages forming each of these oscillators during early development of the neural tube: Hoxa1, Egr2 (Krox20), Phox2b, Lbx1 and Atoh1 for the e-pF; Dbx1 and Evx1 for the preBötC which eventually grow contralateral axons requiring expression of Robo3. Inactivation of the genes encoding these factors leads to mis-specification of these neurons and distinct breathing abnormalities: apneic patterns and loss of central chemosensitivity for the e-pF (central congenital hypoventilation syndrome, CCHS, in humans), complete loss of breathing for the preBötC, right-left desynchronized breathing in Robo3 mutants. Mutations affecting development in more rostral (pontine) respiratory territories change the shape of the inspiratory drive without affecting the rhythm. Other (primordial) embryonic oscillators start in the mouse three days before the e-pF, to generate low frequency (LF) rhythms that are probably required for activity-dependent development of neurones at embryonic stages; in the foetus, however, they are actively silenced to avoid detrimental interaction with the on-going respiratory rhythm. Altogether, these observations provide a strong support to the previously proposed hypothesis that the functional organization of the respiratory generator is specified at early stages of development and is dual in nature, comprising two serially non-homologous oscillators.

International audience; 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 preB?nger complex (preB? 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 preB?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 preB?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 preB?neurons analysed expressed TrkB mRNA. Moreover, 20% expressed BDNF mRNA, suggesting that the preB?is both a target and a source of BDNF. At the network level, BDNF augmented activity of preB?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 preB?

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.

Recent advances in the neurobiology of breathing indicate that the Respiratory Rhythm Generator (RRG) relies on the coupling of two distinct oscillators ventrally located in the brainstem: the para-facial respiratory group (pFRG), comprising pre-inspiratory neurons, and the PreBotzinger complex (PreBotC) playing a dominant role in pacing inspiration. Although vital for life ex utero, the prenatal development of the RRG remains poorly understood. Particularly little is known about the origin in the neural tube of interneurons forming these oscillators. We are addressing this issue considering homeodomain proteins defining distinct progenitor domains along the dorsal ventral (DV) axis of the neural tube. We here report on the role of Dbx1, expressed in the domain of neural progenitors giving rise to the V0 class of interneurons in the mouse. Electrophysiological recordings performed on en bloc and preBotC slice preparations from Dbx1 null (Dbx1LacZ/LacZ) embryos show that the preBotC, but not the pFRG, fails to become active. At birth, Dbx1 null neonates immediately die from a complete failure to breathe. Anatomical analysis in Dbx1LacZ/+ embryos revealed the presence of beta-Gal expressing cells in the preBotC but not the pFRG area. Optical recording of these cells in the Dbx1GFP/GFP line using the calcium indicator Calcium-green-1 AM ascertained their rhythmic status. The majority of beta-Gal cells in the preBotC expressed the mRNA for the type 2 vesicular glutamate transporter (vGlut2) suggesting their role in excitatory synaptic processing. Indeed, the absence of rhythmic activity in Dbx1LacZ/LacZ embryos was associated to a massive loss of vGlut2 expressing cells in the preBotC Glutamatergic synaptic transmission (i) within the preBotC on one side and (ii) over the midline across the left and right preBotC was found impaired in the mutant. We conclude that Dbx1-derived V0 interneurons constitute an obligatory component of the preBotC oscillator.

Photoluminescence of strained Si1-x-yGexCy alloy layer and Si1-x-yGexCy /Si1-xGex structures selectively grown by RT-CVD is investigated. Spectroscopic PL at low temperature (14K) and integrated PL at room temperature were performed. We report the effect of C atoms on SiGe photoluminescence spectra features, especially intensity ratio between the no-phonon (NP) and transverse-optical (TO) transitions. Using dedicated Si1-x-yGexCy /Si1-xGex structures, influence on SiGe PL spectra of C atoms supposed in non-substitutional positions is reported.

Background Neurogenic heterotopic ossification (NHO) is a frequent complication, often involving the hip. The functional impact may require surgical management and pre-surgical imaging assessment is necessary, usually by computed tomography (CT). We aimed to compare the performances of magnetic resonance imaging (MRI) and CT for bone assessment on pre-surgical imaging of the heterotopic ossifications and their features in NHO of the hip. Methods This single-center prospective preliminary study included all patients who underwent surgery for NHO with joint limitation from July 2019 to March 2020. All patients had a CT after biphasic iodinated solution injection and an MRI including T1-weighted, STIR and ZTE sequences. Standardized reports were completed for both exams for each patient, evaluating location, implantation and fragmentation of NHO, relation to the joint capsule and bone mineralization, then were compared. Results Seven patients from 32 to 70 years old (mean = 50.2 ± 17.2 years) were evaluated. NHO were bilateral in 2 patients, for a total of nine hips: six right hips and three left hips. Observed concordance rates between MRI and CT were, respectively, 94.4% for location, 100% for circumferential extension, 87.3% for implantation 88.9% for fragmentation, 77.8% for relation to the joint capsule and 66.7% for bone mineralization. It was 100% for femoral neck fracture and osteonecrosis of the femoral head. Conclusion This preliminary study suggests that pre-surgical MRI imaging should be considered as effective as CT for bone assessment of NHO and their features. Trial registration: ClinicalTrials.gov, NCT03832556. Registered February 6, 2019, https://clinicaltrials.gov/ct2/show/NCT03832556.