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Understanding the neural basis for locomotion is of critical importance since it subserves many behaviours necessary for survival. The spinal cord contains all the elements required to produce the basic locomotor pattern. These elements which compose the central pattern generator for locomotion are activated and sculpted by descending inputs from the brainstem, subcortical and cortical structures. In this review, we examine the aspects of descending control of spinal cord circuits, focusing on the spinal cord, brainstem, and the diencephalon–hypothalamus. In this short review, we discuss recent data and consider opportunities for incorporating connectomics and optogenetic advances to continue the progress in deciphering the descending locomotor connectome.

Developing spinal motor networks produce a diverse array of outputs, including episodic and continuous patterns of rhythmic activity. Variation in excitability state and neuromodulatory tone can facilitate transitions between episodic and continuous rhythms; however, the intrinsic mechanisms that govern these rhythms and their transitions are poorly understood. Here, we tested the capacity of a single central pattern generator (CPG) circuit with tunable properties to generate multiple outputs. To address this, we deployed a computational model composed of an inhibitory half-center oscillator (HCO). Following predictions of our computational model, we tested the contributions of key properties to the generation of an episodic rhythm produced by isolated spinal cords of the newborn mouse. The model recapitulates the diverse state-dependent rhythms evoked by dopamine. In the model, episodic bursting depended predominantly on the endogenous oscillatory properties of neurons, with Na

Dopamine is well known to regulate movement through the differential control of direct and indirect pathways in the striatum that express D1 and D2 receptors respectively. The spinal cord also expresses all dopamine receptors however; how the specific receptors regulate spinal network output in mammals is poorly understood. We explore the receptor-specific mechanisms that underlie dopaminergic control of spinal network output of neonatal mice during changes in spinal network excitability. During spontaneous activity, which is a characteristic of developing spinal networks operating in a low excitability state, we found that dopamine is primarily inhibitory. We uncover an excitatory D1-mediated effect of dopamine on motoneurons and network output that also involves co-activation with D2 receptors. Critically, these excitatory actions require higher concentrations of dopamine; however, analysis of dopamine concentrations of neonates indicates that endogenous levels of spinal dopamine are low. Because endogenous levels of spinal dopamine are low, this excitatory dopaminergic pathway is likely physiologically-silent at this stage in development. In contrast, the inhibitory effect of dopamine, at low physiological concentrations is mediated by parallel activation of D2, D3, D4 and α2 receptors which is reproduced when endogenous dopamine levels are increased by blocking dopamine reuptake and metabolism. We provide evidence in support of dedicated spinal network components that are controlled by excitatory D1 and inhibitory D2 receptors that is reminiscent of the classic dopaminergic indirect and direct pathway within the striatum. These results indicate that network state is an important factor that dictates receptor-specific and therefore dose-dependent control of neuromodulators on spinal network output and advances our understanding of how neuromodulators regulate neural networks under dynamically changing excitability.Significance statementMonoaminergic neuromodulation of neural networks is dependent not only on target receptors but also on network state. We studied the concentration-dependent control of spinal networks of the neonatal mouse, in vitro, during a low excitability state characterized by spontaneous network activity. Spontaneous activity is an essential element for the development of networks. Under these conditions, we defined converging receptor and cellular mechanisms that contribute to the diverse, concentration-dependent control of spinal motor networks by dopamine, in vitro. These experiments advance understanding of how monoamines modulate neuronal networks under dynamically changing excitability conditions and provide evidence of dedicated D1 and D2 regulated network components in the spinal cord that are consistent with those reported in the striatum.

Developing spinal motor networks produce a diverse array of outputs, including episodic and continuous patterns of rhythmic activity. Variation in excitability state and neuromodulatory tone can facilitate transitions between episodic and continuous rhythms; however, the intrinsic mechanisms that govern these rhythms and their transitions are poorly understood. Here, we tested the capacity of a single central pattern generator (CPG) circuit with tunable properties to generate multiple outputs. To address this, we deployed a computational model composed of an inhibitory half-centre oscillator (HCO). Following predictions of our computational model, we tested the contributions of key properties to the generation of an episodic rhythm produced by isolated spinal cords of the newborn mouse. The model recapitulates the diverse state-dependent rhythms evoked by dopamine. In the model, episodic bursting depended predominantly on the endogenous oscillatory properties of neurons, with Na+/K+ ATPase pump (IPump) and hyperpolarization-activated currents (Ih) playing key roles. Modulation of either IPumpMax or Ih produced transitions between episodic and continuous rhythms and silence. As IPump increased, the episode duration and period increased along with a reduction in interepisode interval. Increasing Ih increased the episode period along with an increase in episode duration. Pharmacological manipulations of Ih with ZD7288 and IPump with ouabain or monensin in isolated spinal cords produced findings consistent with the model. Our modelling and experimental results highlight key roles of Ih and IPump in producing episodic rhythms and provide insight into mechanisms that permit a single CPG to produce multiple patterns of rhythmicity.Significance statementThe ability of a single CPG to produce and transition between multiple rhythmic patterns of activity is poorly understood. We deployed a complementary computational half-centre oscillator model and an isolated spinal cord experimental preparation to identify key currents whose interaction produced episodic and continuous rhythmic activity. Together, our experimental and modelling approaches suggest mechanisms in spinal networks that govern diverse rhythms and transitions between them. This work sheds light on the ability of a single CPG to produce episodic bouts observed in behavioural and pathological contexts.

Identifying the spinal circuits controlling locomotion is critical for unravelling the mechanisms controlling the production of gaits. Development of the circuits governing left-right coordination relies on axon guidance molecules such as ephrins and netrins. To date, no other class of proteins have been shown to play a role during this process. Here we have analyzedhopmice, which walk with a characteristic hopping gait using their hind legs in synchrony. Fictive locomotion experiments suggest that a local defect in the ventral spinal cord contributes to the aberrant locomotor phenotype.Hopmutant spinal cords had severe morphological defects, including the absence of the ventral midline and a poorly defined border between white and grey matter. Thehopmice represent the first model where the left and right central pattern generators (CPGs) are fused to form one central CPG, with a synchronous gait as a functional consequence. These defects were exclusively found in the lumbar domain and were associated with abnormal developmental processes, including a misplaced notochord and reduced induction of ventral progenitor domains. While the underlying mutation inhopmice has been suggested to lie withinTtc26, other genes in close vicinity have been associated with gait defects. By replicating the point mutation withinTtc26, employing CRISPR technology, we observed mice with an identical phenotype, thereby verifying the hop mutation. Thus, we show that the assembly of the lumbar CPG network is dependent on a fully functional TTC26 protein.

Background Gait analysis forms a critical part of many lab workflows, ranging from those interested in preclinical neurological models to others who use locomotion as part of a standard battery of tests. Unfortunately, while paw detection can be semi-automated, it becomes generally a time-consuming process with error corrections. Improvement in paw tracking would aid in better gait analysis performance and experience. New Method Here we show the use of Visual Gait Lab (VGL), a high-level software with an intuitive, easy to use interface, that is built on DeepLabCut™. VGL is optimized to generate gait metrics and allows for quick manual error corrections. VGL comes with a single executable, streamlining setup on Windows systems. We demonstrate the use of VGL to analyze gait. Results Training and evaluation of VGL were conducted using 200 frames (80/20 train-test split) of video from mice walking on a treadmill. The trained network was then used to visually track paw placements to compute gait metrics. These are processed and presented on the screen where the user can rapidly identify and correct errors. Comparison with existing methods Gait analysis remains cumbersome, even with commercial software due to paw detection errors. DeepLabCut™ is an alternative that can improve visual tracking but is not optimized for gait analysis functionality. Conclusions VGL allows for gait analysis to be performed in a rapid, unbiased manner, with a set-up that can be easily implemented and executed by those without a background in computer programming.

Over the last decade, there has been a resurgence of work using mammalian systems in vivo. We build on the idea that there were three pillars of motor control built on invertebrate and vertebrate models. We discuss the translational aspect of animal models and provide examples of how basic science using multiple species developed robust concepts that led to successful translation. Finally, with advances in biological and computational tools, we discuss some ideas regarding the future for understanding motor networks.

During development of the spinal cord, a precise interaction occurs between descending projections and sensory afferents, with spinal networks that lead to expression of coordinated motor output. In the rodent, during the last embryonic week, motor output first occurs as regular bursts of spontaneous activity, progressing to stochastic patterns of episodes that express bouts of coordinated rhythmic activity perinatally. Locomotor activity becomes functionally mature in the 2nd postnatal wk and is heralded by the onset of weight-bearing locomotion on the 8th and 9th postnatal day. Concomitantly, there is a maturation of intrinsic properties and key conductances mediating plateau potentials. In this review, we discuss spinal neuronal excitability, descending modulation, and afferent modulation in the developing rodent spinal cord. In the adult, plastic mechanisms are much more constrained but become more permissive following neurotrauma, such as spinal cord injury. We discuss parallel mechanisms that contribute to maturation of network function during development to mechanisms of pathological plasticity that contribute to aberrant motor patterns, such as spasticity and clonus, which emerge following central injury.

Background The effects of exercise on brain function are widely known; however, there is a need for inexpensive, practical solutions for monitoring and metering the activity of multiple mice. New method A contoured running wheel that has a built-in radio-frequency identification (RFID) receiver to monitor the activity of several mice in a single cage is presented. This system is scalable , the interface is easy to use, and the wheel can be dynamically locked so that each group-housed mouse receives a set exercise regimen. Results We were able to reliably monitor three mice that were group-housed. We were able to reliably meter the amount of exercise performed by the mice using the servo-controlled lock. Comparison with existing methods Current methods allow a wheel to be locked when a set distance is reached. However, an issue with this method is that the set distance includes the cumulative activity of all mice in the cage so one mouse could contribute a disproportionate amount to the total distance. Our solution ensures that the wheel is locked when an individual mouse reaches the target distance, but remains unlocked for individuals that have not reached the programmed distance. Conclusions The dynamic locking wheel (DynaLok) is designed to allow a researcher to provide individually designed exercise plans for multi-housed mice; therefore, users are able to house mice conventionally rather than in individual cages. DynaLok reduces animal housing costs, allows for new experimental exercise regimens to be developed, and is scalable and cost-effective.

Key points Inflammatory kinins are released following spinal cord injury or neurotrauma. The effects of these kinins on ongoing locomotor activity of central pattern generator networks are unknown. In the present study, kinins were shown to have short- and long-term effects on motor networks. The short-term effects included direct depolarization of interneurons and motoneurons in the ventral horn accompanied by modulation of transient receptor potential vanilloid 1-sensitive nociceptors in the dorsal horn. Over the long-term, we observed a bradykinin-mediated effect on promoting plasticity in the spinal cord. In a model of spinal cord injury, we observed an increase in microglia numbers in both the dorsal and ventral horn and, in a microglia cell culture model, we observed bradykinin-induced expression of glial-derived neurotrophic factor. Abstract The expression and function of inflammatory mediators in the developing spinal cord remain poorly characterized. We discovered novel, short and long-term roles for the inflammatory nonapeptide bradykinin (BK) and its receptor bradykinin receptor B2 (B2R) in the neuromodulation of developing sensorimotor networks following a spinal cord injury (SCI), suggesting that BK participates in an excitotoxic cascade. Functional expression of B2R was confirmed by a transient disruptive action of BK on fictive locomotion generated by a combination of NMDA, 5-HT and dopamine. The role of BK in the dorsal horn nociceptive afferents was tested using spinal cord attached to one-hind-limb (HL) preparations. In the HL preparations, BK at a subthreshold concentration induced transient disruption of fictive locomotion only in the presence of: (1) noxious heat applied to the hind paw and (2) the heat sensing ion channel transient receptor potential vanilloid 1 (TRPV1), known to be restricted to nociceptors in the superficial dorsal horn. BK directly depolarized motoneurons and ascending interneurons in the ventrolateral funiculus. We found a key mechanism for BK in promoting long-term plasticity within the spinal cord. Using a model of neonatal SCI and a microglial cell culture model, we examined the role of BK in inducing activation of microglia and expression of glial-derived neurotrophic factor (GDNF). In the neonatal SCI model, we observed an increase in microglia numbers and increased GDNF expression restricted to microglia. In the microglia cell culture model, we observed a BK-induced increased expression of GDNF via B2R, suggesting a novel mechanism for BK spinal-mediated plasticity.

Spinal cord injury and peripheral nerve injuries are traumatic events that greatly impact quality of life. One factor that is being explored throughout patient care is the idea of diet and the role it has on patient outcomes. But the effects of diet following neurotrauma need to be carefully explored in animal models to ensure that they have beneficial effects. The ketogenic diet provides sufficient daily caloric requirements while being potentially neuroprotective and analgesic. In this study, animals were fed a high-fat, low-carbohydrate diet that led to a high concentration of blood ketone that was sustained for as long as the animals were on the diet. Mice fed a ketogenic diet had significantly lower levels of tyrosine and tryptophan, but the levels of other monoamines within the spinal cord remained similar to those of control mice. Mice were fed a standard or ketogenic diet for 7 d before and 28 d following the injury. Our results show that mice hemisected over the T10–T11 vertebrae showed no beneficial effects of being on a ketogenic diet over a 28 d recovery period. Similarly, ligation of the common peroneal and tibial nerve showed no differences between mice fed normal or ketogenic diets. Tests included von Frey, open field, and ladder-rung crossing. We add to existing literature showing protective effects of the ketogenic diet in forelimb injuries by focusing on neurotrauma in the hindlimbs. The results suggest that ketogenic diets need to be assessed based on the type and location of neurotrauma.

Hemodynamic parameters, such as tissue oxygen saturation and blood volume fraction, are important markers of brain physiology. They are also widely used surrogate markers of electrophysiological activity. Here, we present a single fiber spectroscopic (SFS) system for monitoring cerebral oxygen saturation in localized, non-line-of-sight brain regions in freely-moving rodents. We adapted the implantation ferrule and patch cable design from commercialized optogenetics and fiber photometry systems, enabling stereotaxic fiber implantation, longitudinal tissue access and measurement from freely-moving animals. The optical system delivers and collects light from the brain through a 200 µm-core-diameter, 0.39NA multimode fiber. We robustly measured oxygen saturation from phantoms with different optical properties mimicking brain tissue. In mice, we demonstrated, for the first time, measurements of oxygen saturation from a highly-localized, targeted brain region over 31 days and continuous measurements from a freely-moving animal for over an hour. These results suggest that single fiber spectroscopy has enormous potential for functional brain monitoring and investigating neurovascular coupling in freely-moving animals. In addition, this technique can potentially be combined with fiber photometry systems to correct for hemodynamic artifacts in the fluorescence detection.

The role of orexin during development, and especially in terms of spinal cord function, is not well understood. It is for this reason that we focused on the network actions of orexin during the first week of development. We found that orexinergic fibers were present in the lumbar spinal cord of postnatal day 0 (P0) to P3 mice. The fibers were expressed mainly in the dorsal horn, but occasional fibers were observed in the ventral horn. Both orexin (OX) A and OXB increased the motoneurons (MNs) tonic neurogram discharge. However, only OXA was found to significantly increase spontaneous bursting activity and the frequency of fictive locomotor bursts. We show that OXA is able to act directly on MNs. To test the contribution of the recurrent MN collaterals, we blocked the nicotinic cholinergic drive and observed that OXA retained its ability to increase fictive locomotor activity. Additionally, we recorded neurograms from ventral lateral funiculi, where OXA had no effect on population discharge. These effects were also confirmed by recording from descending commissural interneurons via patch recordings. The loci of the effects of OXA were further investigated in a dorsal horn-removed preparation where OXA also shows an increase in the discharge from ventral root neurograms but no increase in the frequency of spontaneous or fictive locomotion burst activity. In summary, multiple lines of evidence from our work demonstrate the robust effects of orexins on spinal cord networks and MNs at the time of birth.

The mesencephalic locomotor region (MLR) is an important integrative area for the initiation and modulation of locomotion. Recently it has been realized that dopamine (DA) projections from the substantia nigra pars compacta project to the MLR. Here we explore DA projections from an area of the medial zona incerta (ZI) known for its role in motor control onto the MLR. We provide evidence that dopaminergic (DAergic) A13 neurons have connectivity to the cuneiform nucleus (CnF) and pedunculopontine tegmental nucleus (PPTg) of the MLR. No ascending connectivity to the dorsolateral striatum was observed. On the other hand, DAergic A13 projections to the medullary reticular formation (MRF) and the lumbar spinal cord were sparse. A small number of non-DAergic neurons within the medial ZI projected to the lumbar spinal cord. We then characterized the DA A13 cells and report that these cells differ from canonical DA neurons since they lack the Dopamine Transporter (DAT). The lack of DAT expression, and possibly the lack of a dopamine reuptake mechanism, points to a longer time of action compared to typical dopamine neurons. Collectively our data suggest a parallel descending DAergic pathway from the A13 neurons of the medial ZI to the MLR, which we expect is important for modulating movement.

Summary Ependymal cells are multi-ciliated cells that form the brain's ventricular epithelium and a niche for neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ). In addition, ependymal cells are suggested to be latent NSCs with a capacity to acquire neurogenic function. This remains highly controversial due to a lack of prospective in vivo labeling techniques that can effectively distinguish ependymal cells from neighboring V-SVZ NSCs. We describe a transgenic system that allows for targeted labeling of ependymal cells within the V-SVZ. Single-cell RNA-seq revealed that ependymal cells are enriched for cilia-related genes and share several stem-cell-associated genes with neural stem or progenitors. Under in vivo and in vitro neural-stem- or progenitor-stimulating environments, ependymal cells failed to demonstrate any suggestion of latent neural-stem-cell function. These findings suggest remarkable stability of ependymal cell function and provide fundamental insights into the molecular signature of the V-SVZ niche.

The adult decerebrate mouse model (a mouse with the cerebrum removed) enables the study of sensory-motor integration and motor output from the spinal cord for several hours without compromising these functions with anesthesia. For example, the decerebrate mouse is ideal for examining locomotor behavior using intracellular recording approaches, which would not be possible using current anesthetized preparations. This protocol describes the steps required to achieve a low-blood-loss decerebration in the mouse and approaches for recording signals from spinal cord neurons with a focus on motoneurons. The protocol also describes an example application for the protocol: the evocation of spontaneous and actively driven stepping, including optimization of these behaviors in decerebrate mice. The time taken to prepare the animal and perform a decerebration takes ∼2 h, and the mice are viable for up to 3-8 h, which is ample time to perform most short-term procedures. These protocols can be modified for those interested in cardiovascular or respiratory function in addition to motor function and can be performed by trainees with some previous experience in animal surgery.

Compared to proprioceptive afferent collateral projections, less is known about the anatomical, neurochemical, and functional basis of nociceptive collateral projections modulating lumbar central pattern generators (CPG). Quick response times are critical to ensure rapid escape from aversive stimuli. Furthermore, sensitization of nociceptive afferent pathways can contribute to a pathological activation of motor circuits. We investigated the extent and role of collaterals of capsaicin-sensitive nociceptive sacrocaudal afferent (nSCA) nerves that directly ascend several spinal segments in Lissauer's tract and the dorsal column and regulate motor activity. Anterograde tracing demonstrated direct multisegmental projections of the sacral dorsal root 4 (S4) afferent collaterals in Lissauer's tract and in the dorsal column. Subsets of the traced S4 afferent collaterals expressed transient receptor potential vanilloid 1 (TRPV1), which transduces a nociceptive response to capsaicin. Electrophysiological data revealed that S4 dorsal root stimulation could evoke regular rhythmic bursting activity, and our data suggested that capsaicin-sensitive collaterals contribute to CPG activation across multiple segments. Capsaicin's effect on S4-evoked locomotor activity was potent until the lumbar 5 (L5) segments, and diminished in rostral segments. Using calcium imaging we found elevated calcium transients within Lissauer's tract and dorsal column at L5 segments when compared to the calcium transients only within the dorsal column at the lumbar 2 (L2) segments, which were desensitized by capsaicin. We conclude that lumbar locomotor networks in the neonatal mouse spinal cord are targets for modulation by direct multisegmental nSCA, subsets of which express TRPV1 in Lissauer's tract and the dorsal column. J. Comp. Neurol. 521:2870-2887, 2013. © 2013 Wiley Periodicals, Inc.

Exposure to a stressor sensitizes behavioral and hormonal responses to future stressors. Stress-associated release of noradrenaline enhances the capacity of central synapses to exhibit plasticity (metaplasticity). We report noradrenaline-dependent metaplasticity at GABA synapses in the paraventricular nucleus of the hypothalamus in rat and mouse that control the hypothalamic-pituitary-adrenal axis. Previously, work indicates these GABA synapses become excitatory following acute stress. The current study finds that in vivo stress exposure is also required for these synapses to undergo activity-dependent long-term potentiation (LTPGABA). The activation of β-adrenergic receptors (β-ARs) during stress functionally upregulates metabotropic (mGluR1) glutamate receptors allowing for mGluR1-dependent LTPGABA during afferent bursts. LTPGABA is expressed postsynaptically and manifests as the emergence of new functional synapses. Our findings provide the first demonstration that noradrenaline release during an in vivo challenge alters information storage capacity at GABA synapses. These changes may contribute to neuroendocrine sensitization to stress.

Serotonin has a myriad of central functions involving mood, appetite, sleep, and memory and while its release within the spinal cord is particularly important for generating movement, the corresponding role on cortical movement representations (motor maps) is unknown. Using adult rats we determined that pharmacological depletion of serotonin (5-HT) via intracerebroventricular administration of 5,7 dihydroxytryptamine resulted in altered movements of the forelimb in a skilled reaching task as well as higher movement thresholds and smaller maps derived using high-resolution intracortical microstimulation (ICMS). We ruled out the possibility that reduced spinal cord excitability could account for the serotonin depletion-induced changes as we observed an enhanced Hoffman reflex (H-reflex), indicating a hyperexcitable spinal cord. Motor maps derived in 5-HT1Areceptor knock-out mice also showed higher movement thresholds and smaller maps compared with wild-type controls. Direct cortical application of the 5-HT1A/7agonist 8-OH-DPAT lowered movement thresholdsin vivoand increased map size in 5-HT-depleted rats. In rats, electrical stimulation of the dorsal raphe lowered movement thresholds and this effect could be blocked by direct cortical application of the 5-HT1Aantagonist WAY-100135, indicating that serotonin is primarily acting through the 5-HT1Areceptor. Next we developed a novelin vitroICMS preparation that allowed us to track layer V pyramidal cell excitability. Bath application of WAY-100135 raised the ICMS current intensity to induce action potential firing whereas the agonist 8-OH-DPAT had the opposite effect. Together our results demonstrate that serotonin, acting through 5-HT1Areceptors, plays an excitatory role in forelimb motor map expression.

Degos disease is characterized as a rare systemic vaso-occlusive disorder, although the exact pathophysiology is uncertain. Fewer than 200 patients have been reported in the literature, and only two reports describe the course of the disease during pregnancy. Here, we present the first reported case of the course of pregnancy in a woman with the systemic form of Degos disease. The patient had been diagnosed with Degos prior to pregnancy and was monitored throughout the duration of the pregnancy. Her presentation and treatment are described. There was no further exacerbation secondary to the pregnancy itself; the pregnancy course was uncomplicated and the baby unaffected to date.

As wild-type and genetically modified mice are progressively becoming the predominant models for studying locomotor physiology, the technical ability to record sensory and motor components from adult mice, in vivo, are expected to contribute to a better understanding of sensorimotor spinal cord networks. Here, specific technical and surgical details are presented on how to produce an adult decerebrate mouse preparation that can reliably produce sustained bouts of stepping, in vivo, in the absence of anesthetic drugs. Data are presented demonstrating the ability of this preparation to produce stepping during treadmill locomotion, adaptability in its responses to changes in the treadmill speed, and left-right alternation. Furthermore, intracellular recordings from motoneurons and interneurons in the spinal cord are presented from preparations where muscle activity was blocked. Intraaxonal recordings are also presented demonstrating that individual afferents can be recorded using this preparation. These data demonstrate that the adult decerebrate mouse is a tractable preparation for the study of sensorimotor systems.

Abnormal respiratory chemosensitivity is implicated in recurrent apnea syndromes, with the peripheral chemoreceptors, the carotid bodies, playing a particularly important role. Previous work suggests that supraphysiological concentrations of the endocannabinoid endovanilloid and TASK channel blocker anandamide (ANA) excite carotid bodies, but the mechanism(s) and physiological significance are unknown. Given that carotid body output is temperature-sensitive, we hypothesized that ANA stimulates carotid body chemosensory afferents via temperature-sensitive vanilloid (TRPV1) receptors. To test this hypothesis, we used the dual-perfused in situ rat preparation to confirm that independent perfusion of carotid arteries with supraphysiological concentrations of ANA strongly excites carotid sinus nerve afferents and that this activity is sufficient to increase phrenic activity. Next, using ex vivo carotid body preparations, we demonstrate that these effects are mediated by TRPV1 receptors, not CB1 receptors or TASK channels: in CB1-null mouse preparations, ANA increased afferent activity across all levels of Po2, whereas in TRPV1-null mouse preparations, the stimulatory effect of ANA was absent. In rat ex vivo preparations, ANA's stimulatory effects were mimicked by olvanil, a nonpungent TRPV1 agonist, and suppressed by the TRPV1 antagonist AMG-9810. The specific CB1 agonist oleamide had no effect. Physiological levels of ANA had no effect alone but increased sensitivity to mild hyperthermia. AMG-9810 blocked ANA's effect on the temperature response. Immunolabeling and RT-PCR demonstrated that TRPV1 receptors are not expressed in carotid body glomus cells but reside in petrosal sensory afferents. Together, these results suggest that ANA plays a physiological role in augmenting afferent responses to mild hyperthermia by activating TRPV1 receptors on petrosal afferents.

Rhythmic activity is responsible for numerous essential motor functions including locomotion, breathing and chewing. In the case of locomotion, it has been realized for some time that the spinal cord contains sufficient circuitry to produce a sophisticated stepping pattern. However, the central pattern generator for locomotion in mammals has remained a ‘black box’ where inputs to the network were manipulated and the outputs interpreted. Over the last decade, new genetic approaches and techniques have been developed that provide ways to identify and manipulate the activity of classes of interneurons. The use of these techniques will be critically discussed and related to current models of network function.

During early postnatal development, between birth and postnatal days 8–11, mice start to achieve weight-bearing locomotion. In association with the progression of weight-bearing locomotion there are presumed developmental changes in the intrinsic electrical properties of spinal α-motoneurons. However, these developmental changes in the properties of α-motoneuron properties have not been systematically explored in mice. Here, data are presented documenting the developmental changes of selected intrinsic motoneuron electrical properties, including statistically significant changes in action potential half-width, intrinsic excitability and diversity (quantified as coefficient of variation) of rheobase current, afterhyperpolarization half-decay time, and input resistance. In various adult mammalian preparations, the maintenance of intrinsic motoneuron electrical properties is dependent on activity and/or transmission-sensitive motoneuron–muscle interactions. In this study, we show that botulinum toxin–induced muscle paralysis led to statistically significant changes in the normal development of intrinsic motoneuron electrical properties in the postnatal mouse. This suggests that muscle activity during early neonatal life contributes to the development of normal motoneuron electrical properties.

Serotonin (5-HT) can potently activate and modulate spinal locomotor circuits in a variety of species. Many of these findings have been obtained by applying serotonin exogenously to the isolated spinal cord of in vitro preparations, which has the drawback of indiscriminately activating extrasynaptic receptors and neurons. To investigate the role of endogenously released serotonin in modulating locomotor networks, the selective serotonin reuptake inhibitor citalopram was used. Fictive locomotion was elicited by either electrical stimulation of the brainstem or the sacral 4 (S4) dorsal root. The addition of 20 μm of citalopram caudal to thoracic segment 5 (T5) had an overall inhibitory effect on the lumbar central pattern generator (CPG). Left–right and flexor–extensor coupling were significantly decreased, and there was also a phase shift in the flexor–extensor relationship. In addition, there was a significant decrease in burst amplitude. These effects were observed during both afferent and brainstem evoked fictive locomotion. When citalopram was added in the presence of 5-HT1A and 5-HT1B antagonists, the inhibitory effects were largely reversed. The remaining excitatory effects were mediated by 5-HT7 and 5-HT2 receptors. These results suggest that endogenous 5-HT release can modulate locomotor-like activity early in neonatal development.

It is well recognized that proprioceptive afferent inputs can control the timing and pattern of locomotion. C and Adelta afferents can also affect locomotion but an unresolved issue is the identity of the subsets of these afferents that encode defined modalities. Over the last decade, the transient receptor potential (TRP) ion channels have emerged as a family of non-selective cation conductances that can label specific subsets of afferents. We focus on a class of TRPs known as ThermoTRPs which are well known to be sensor receptors that transduce changes in heat and cold. ThermoTRPs are known to help encode somatosensation and painful stimuli, and receptors have been found on C and Adelta afferents with central projections onto dorsal horn laminae. Here we show, using in vitro neonatal mouse spinal cord preparations, that activation of both spinal and peripheral transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential melastatin 8 (TRPM8) afferent terminals modulates central pattern generators (CPGs). Capsaicin or menthol and cooling modulated both sacrocaudal afferent (SCA) evoked and monoaminergic drug-induced rhythmic locomotor-like activity in spinal cords from wild type but not TRPV1-null (trpv1(-/-)) or TRPM8-null (trpm8(-/-)) mice, respectively. Capsaicin induced an initial increase in excitability of the lumbar motor networks, while menthol or cooling caused a decrease in excitability. Capsaicin and menthol actions on CPGs involved excitatory and inhibitory glutamatergic mechanisms, respectively. These results for the first time show that dedicated pathways of somatosensation and pain identified by TRPV1 or TRPM8 can target spinal locomotor CPGs.

At birth, thoracosacral spinal cord networks in mouse can produce a coordinated locomotor-like pattern. In contrast, less is known about the cervicothoracic networks that generate forelimb locomotion. Here we show that cervical networks can produce coordinated rhythmic patterns in the brain stem-spinal cord preparation of the mouse. Segmentally the C5 and C8 neurograms were each found to be alternating left-right, and the ipsilateral C5 and C8 neurograms also alternated. Collectively these patterns were suggestive of locomotor-like activity. This pattern was not dependent on the presence of thoracosacral segments because they could be evoked following a complete transection of the spinal cord at T5. We next demonstrated that activation of thoracosacral networks either pharmacologically or by stimulation of sacrocaudal afferents could produce rhythmic activity within the C5 and C8 neurograms. On the other hand, pharmacological activation of cervical networks did not evoke alternating cervical rhythmic activity either in isolated cervicothoracic or -sacral preparations. Under these conditions, we found that activation of cervicothoracic networks could alter the timing of thoracosacral locomotor-like patterns. When thoracosacral networks were not activated pharmacologically but received rhythmic drive from cervicothoracic networks, a pattern of slow bursts with superimposed fast synchronous oscillations became the dominant lumbar neurogram pattern. Our data suggest that in neonatal mice the cervical CPG is capable of producing coordinated rhythmic patterns in the absence of input from lumbar segments, but caudorostral drive contributes to cervical patterns and rhythm stability.

During development, descending projections to the spinal cord are immature. Available data suggest that even though these projections are not fully formed, they contribute to activation of spinal circuitry and promote development of network function. Here we examine the modulation of sacrocaudal afferent-evoked locomotor activity by descending pathways. We first examined the effects of brainstem transection on the afferent evoked locomotor-like rhythm using an isolated brainstem‐spinal cord preparation of the mouse. Transection increased the frequency and stability of the locomotor-like rhythm while the phase remained unchanged. We then made histologically verified lesions of the ventrolateral funiculus and observed similar effects on the stability and frequency of the locomotor rhythm. We next tested whether these effects were due to downstream effects of the transection. A split-bath was constructed between the brainstem and spinal cord. Neural activity was suppressed in the brainstem compartment using cooled high sucrose solutions. This manipulation led to a reversible change in frequency and stability that mirrored our findings using lesion approaches. Our findings suggest that spontaneous brainstem activity contributes to the ongoing modulation of afferent-evoked locomotor patterns during early postnatal development. Our work suggests that some of the essential circuits necessary to modulate and control locomotion are at least partly functional before the onset of weight-bearing locomotion.

Monoaminergic projections are among the first supraspinal inputs to innervate spinal networks. Little is known regarding the role of monoamines in modulating ongoing locomotor patterns evoked by endogenous release of neurotransmitter. Here we activate a locomotor-like rhythm by electrical stimulation of afferents and then test the modulatory effects of monoamines on the frequency, pattern, and quality of the rhythm. Stimulation of the cauda equina induced a rhythm consisting of left-right and ipsilateral alternation indicative of locomotor-like activity. First, we examined the effects of noradrenaline (NA), serotonin (5-HT), or dopamine (DA) at dose levels that did not elicit locomotor activity. Bath application of NA and DA resulted in a depression of the cauda-equina-evoked rhythm. Conversely, bath-applied 5-HT increased both the amplitude and cycle period of the evoked rhythm, an effect that was mimicked by the addition of 5-HT2 agonists to the bath. Application of 5-HT7 agonists disrupted the evoked rhythmic behavior. Next, we examined the effects of NA α1 and α2 agonists and found that the suppressive effects of NA on the rhythm could be reproduced by adding the α2 agonist, clonidine, to the bath. In contrast, bath applying the α1 agonist, phenylephrine, increased the amplitude and duration of the cycle period. Finally, the suppressive effects of DA were not replicated by the administration of D1, D2, or D3 agonists although application of NA α2 antagonists reversed the effects of DA. Application of D1 agonists, increased the amplitude of the bursts but did not affect the cycle period. Our results indicate that monoamines can control the expression, pattern, and timing of cauda-equina-evoked locomotor patterns in developing mice.

Locally projecting inhibitory interneurons play a crucial role in the patterning and timing of network activity. However, because of their relative inaccessibility, little is known about their development or incorporation into circuits. In this study, we characterized the functional onset, neurotransmitters, rostrocaudal spread, and funicular distribution of one such spinal interneuronal circuit during development. The R-interneuron is the avian homologue of the mammalian Renshaw cell. Both cell types receive input from motoneuron recurrent collaterals and make direct connections back onto motoneurons. By stimulating motoneurons projecting in a given ventral root and recording the response in adjacent ventral roots, we demonstrate that the R-interneuron circuit becomes functional between embryonic day 6 (E6) and E7. This ventral root response is observed at E11 and at E14 until it can no longer be detected at E16. Using bath-applied neurotransmitter receptor antagonists, we were able to demonstrate that the circuit is predominately nicotinic and GABAergic from E7.5 to E15. We also found a glutamatergic component to the pathway throughout this developmental period. The R-interneuron projects three or more segments both rostrally and caudally through the ventrolateral funiculus. The distribution of this circuit may become more locally focused between E7.5 and E15.

Recently, it has been shown that bath-applied 5-HT can elicit fictive locomotion from perinatal mouse preparations. Since 5-HT acts on multiple receptor subtypes, the focus of this study was to examine which receptor families contribute to the genesis and modulation of locomotor activity. Blockade of 5-HT2 (ketanserin or N-desmethylclozapine) or 5-HT7 receptors (SB-269970) could reversibly block or modulate the locomotor-like pattern. A 5-HT2 agonist (α-methyl-5-HT) was shown to be capable of activating the rhythm. Bath application of 5-HT7 agonists (5-CT) generally led to a tonic increase in neurogram discharge, accompanied by bouts of rhythmic activity. Blockade of dopaminergic receptors {D1 [ R-(+)-SCH-23390 or LE 300]/D2 [(±)-sulpiride or L-741,626] } could reversibly disrupt the rhythm and most effectively did so when the D1 and D2 antagonists were added together. Conversely, 5-HT2 and D1/D2 agonists can interact to evoke locomotor activity. Overall, our data show that, in the neonatal mouse preparation, 5-HT evoked locomotion is partly dependent on activation of 5-HT2, 5-HT7, and dopaminergic receptor subtypes.

The development of motor networks in the spinal cord is partly activity-dependent. We have observed receptor-mediated excitatory effects of two peptides, arginine vasopressin (AVP) and oxytocin (OXT), on motor network activity in the neonate. With the use of an en blocin vitropreparation of mouse spinal cord (2-3 d old), which either was isolated completely or had muscles of the hindlimb left intact, we show that the bath application of AVP or OXT can evoke an increase in population bursting of motoneurons recorded from the lumbar ventral roots. By using antagonists for AVP and OXT, we found that these peptides were binding primarily to V1aand OXT receptors, respectively. Western blot analysis revealed a 48 kDa V1aand a 55 kDa OXT receptor immunoreactive band that was expressed in tissue obtained from L1-L6 sections of spinal cord. AVP, but not OXT, could, on occasion, evoke sustained periods of locomotor-like activity. In addition, when we applied AVP or OXT in combination with a 5-HT2agonist, bouts of locomotor-like activity could be observed in a majority of preparations. Collectively, these data point to a novel role for AVP and OXT in the activation of spinal motor networks.

Electromyography can be used to record activity from sets of muscles in awake, freely moving animals using implanted intramuscular electrodes. As a tool, EMG has a wide range of applications ranging from inferring neural processes to analyzing movement. The amplitude of the rectified and filtered electromyogram (EMG) can be used as an indirect measure of muscle activity. Although it is often tempting to correlate the EMG with muscle force, the fact that force varies more with different activation strategies than with EMG estimates must be taken into account. The purpose of this article is to provide the researcher wishing to introduce the technique of recording EMGs from conscious animals using intramuscular electrodes with a step-by-step guide. It includes details on the manufacture of electromyograph electrodes, recording, and analysis considerations along with a section on solving common problems. For the sake of clarity, this article focuses on using the cat as a model and on the implantation of hindlimb muscles with intramuscular wire electrodes. However, the procedures can be adapted for use on other striated muscles and species.

The neonatal mouse en bloc spinal cord-brainstem preparation used in combination with advances in mouse genomics provides a novel strategy for studying the spinal control of locomotion. How well the mouse en bloc prepara- tion is oxygenated however, is unknown. This is an important consideration given that (a) other superfused mammalian en bloc preparations have anoxic cores and (b) hypoxia can have profound effects on neuronal activity. Here we measure the level of tissue oxygenation in the mouse preparation and determine how neuronal activity within the spinal cord is influenced by poor superfusion and/or low oxygen. To measure tissue oxygenation, oxygen depth profiles were obtained (P0 -1 and P2-3; Swiss Webster mice). At P0 -1, spinal cords were oxygenated throughout under rest- ing conditions. When fictive locomotor activity was evoked (5-HT 10 M, dopamine 50 M, NMA 5 M), there was a substantial reduction in tissue PO2 starting within 5 min of drug application. Following washout, the PO2 slowly returned to control levels over a period of 30 min. The experiments described above were repeated using P2-3 preparations. In this older age group, the spinal cord preparations had a hypoxic/anoxic core that was exacerbated during metaboli- cally demanding tasks such as drug-evoked rhythmic activ- ity. To examine how an anoxic core affects neuronal activity within the spinal cord we either altered the flow-rate or ma- nipulated superfusate PO2. When the flow-rate was reduced a transient disruption in the rhythmicity of drug-induced loco- motion occurred during the first 15 min (P0 -1 preparations). However, the motor output adapted and stabilized. During prolonged superfusion with hypoxic artificial cerebrospinal fluid on the other hand, both the motor bursts in spinal nerves and the activity of most neurons near the center of the tissue were abolished. Overall, this study suggests that while oxygenation of P0 -P1 preparations is adequate for studies of locomotor function, oxygenation of older preparations is more problem- atic. Our data also show that neonatal spinal neurons require oxygen to maintain activity; and the spinal locomotor rhythm generator continues to function providing the peripheral tis- sue of the cord is oxygenated. Together, these results are consistent with the results of a previous study which suggest that the locomotor pattern generator is located close to the surface of the spinal cord. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Dopamine is now well established as a modulator of locomotor rhythms in a variety of developing and adult vertebrates. However, in mice, while all five dopamine receptor subtypes are present in the spinal cord, it is unclear which receptor subtypes modulate the rhythm. Dopamine receptors can be grouped into two families—the D1/5 receptor group and the D2/3/4 group, which have excitatory and inhibitory effects, respectively. Our data suggest that dopamine exerts contrasting dose-dependent modulatory effects via the two receptor families. Our data show that administration of dopamine at concentrations >35 μM slowed and increased the regularity of a locomotor rhythm evoked by bath application of 5-hydroxytryptamine (5-HT) and N-methyl-d(l)-aspartic acid (NMA). This effect was independent of the baseline frequency of the rhythm that was manipulated by altering the NMA concentration. We next examined the contribution of the D1- and D2-like receptor families on the rhythm. Our data suggest that the D1-like receptor contributes to enhancement of the stability of the rhythm. Overall, the D2-like family had a pronounced slowing effect on the rhythm; however, quinpirole, the D2-like agonist, also enhanced rhythm stability. These data indicate a receptor-dependent delegation of the modulatory effects of dopamine on the spinal locomotor pattern generator.

The hypothalamic A11 region has been identified in several species including rats, mice, cats, monkeys, zebrafish, and humans as the primary source of descending dopamine (DA) to the spinal cord. It has been implicated in the control of pain, modulation of the spinal locomotor network, restless leg syndrome, and cataplexy, yet the A11 cell group remains an understudied dopaminergic (DAergic) nucleus within the brain. It is unclear whether A11 neurons in the mouse contain the full complement of enzymes consistent with traditional DA neuronal phenotypes. Given the abundance of mouse genetic models and tools available to interrogate specific neural circuits and behavior, it is critical first to fully understand the phenotype of A11 cells. We provide evidence that, in addition to tyrosine hydroxylase (TH) that synthesizes L-DOPA, neurons within the A11 region of the mouse contain aromatic L-amino acid decarboxylase (AADC), the enzyme that converts L-DOPA to dopamine. Furthermore, we show that the A11 neurons contain vesicular monoamine transporter 2 (VMAT2), which is necessary for packaging DA into vesicles. On the contrary, A11 neurons in the mouse lack the dopamine transporter (DAT). In conclusion, our data suggest that A11 neurons are DAergic. The lack of DAT, and therefore the lack of a DA reuptake mechanism, points to a longer time of action compared to typical DA neurons.

The spinal cord contains networks of neurons that can produce locomotor patterns. To readily respond to environmental conditions, these networks must be flexible yet at the same time robust. Neuromodulators play a key role in contributing to network flexibility in a variety of invertebrate and vertebrate networks. For example, neuromodulators contribute to altering intrinsic properties and synaptic weights that, in extreme cases, can lead to neurons switching between networks. Here we focus on the role of dopamine in the control of stepping networks in the spinal cord. We first review the role of dopamine in modulating rhythmic activity in the stomatogastric ganglion and the leech, since work from these preparations provides a foundation to understand its role in vertebrate systems. We then move to a discussion of dopamine’s role in modulation of swimming in aquatic species such as the larval xenopus, lamprey and zebrafish. The control of terrestrial walking in vertebrates by dopamine is less studied and we review current evidence in mammals with a focus on rodent species. We discuss data suggesting that the source of dopamine within the spinal cord is mainly from the A11 area of the diencephalon, and then turn to a discussion of dopamine’s role in modulating walking patterns from both in vivo and in vitro preparations. Similar to the descending serotonergic system, the dopaminergic system may serve as a potential target to promote recovery of locomotor function following spinal cord injury; evidence suggests that dopaminergic agonists can promote recovery of function following spinal cord injury (SCI). We discuss pharmacogenetic and optogenetic approaches that could be deployed in SCI and their potential tractability. Throughout the review we draw parallels with both noradrenergic and serotonergic modulatory effects on spinal cord networks. In all likelihood, a complementary monoaminergic enhancement strategy should be deployed following spinal cord injury.

We examined the ability of the isolated lumbosacral spinal cord of the neonatal mouse (P0–7) to generate rhythmic motor activity under several different conditions. In the absence of electrical or pharmacological stimulation, we recorded several patterns of spontaneous ventral root depolarization and discharge. Spontaneous, alternating discharge between contralateral ventral roots could occur two to three times over a 10-min interval. We also observed other patterns, including left-right synchrony and rhythmic activity restricted to one side of the cord. Trains of stimuli delivered to the lumbar/coccygeal dorsal roots or the sural nerve reliably evoked episodes of rhythmic activity. During these evoked episodes, rhythmic ventral root discharges could occur on one side of the cord or could alternate from side to side. Bath application of a combination of N-methyl-d,l-aspartate (NMA), serotonin, and dopamine produced rhythmic activity that could last for several hours. Under these conditions, the discharge recorded from the left and right L1–L3 ventral roots alternated. In the L4–L5segments, the discharge had two peaks in each cycle, coincident with discharge of the ipsilateral and contralateral L1–L3 roots. The L6 ventral root discharge alternated with that recorded from the ipsilateral L1–L3 roots. We established that the drug-induced rhythm was locomotor-like by recording an alternating pattern of discharge between ipsilateral flexor and extensor hindlimb muscle nerves. In addition, by recording simultaneously from ventral roots and muscle nerves, we established that ankle flexor discharge was in phase with ipsilateral L1/L2 ventral root discharge, while extensor discharge was in phase with ipsilateral L6 ventral root discharge. Rhythmic patterns of ventral root discharge were preserved following mid-sagittal section of the spinal cord, demonstrating that reciprocal inhibitory connections between the left and right sides of the cord are not essential for rhythmogenesis in the neonatal mouse cord. Blocking N-methyl-d-aspartate receptors, in both the intact and the hemisected preparation, revealed that these receptors contribute to but are not essential for rhythmogenesis. In contrast, the rhythm was abolished following blockade of kainate/AMPA receptors with 6-cyano-7-nitroquinoxalene-2,3-dione. These findings demonstrate that the isolated mouse spinal cord can produce a variety of coordinated activities, including locomotor-like activity. The ability to study these behaviors under a variety of different conditions offers promise for future studies of rhythmogenic mechanisms in this preparation.

Previous studies have reported that stimulation of group I afferents from extensor muscles prolongs stance duration during walking in decerebrate cats. The main objective of this investigation was to determine whether this phenomenon occurs during walking in conscious cats. In conscious cats without lesions of the central nervous system (CNS), stimulation of group I afferents in the lateral gastrocnemius/soleus (LGS) nerve during stance prolonged extensor burst duration and increased the cycle period in five of seven animals. The mean increases in cycle period were modest, ranging from 6 to 22%. In five of six animals that walked both quadrupedally and bipedally at the same rate, the effects on cycle period were stronger during bipedal stepping (18% mean increase in cycle period compared with 9%). The stimulated nerves were transected and the experimental procedure was usually delayed in the conscious animals for 2-3 days following implantation of the stimulating electrodes. To assess whether chronic axotomy of the LGS nerve was a factor in the decreased effectiveness, four of the cats with chronic nerve section were decerebrated and their LGS nerves were stimulated after the animals began to spontaneously walk on a motorized treadmill. In all four of these animals, the effects of stimulating the chronically cut LGS nerve on the step cycle period became stronger following decerebration. However, these effects were not as strong as those produced when an acutely sectioned LGS nerve was stimulated. During both quadrupedal and bipedal walking, stimulation of the LGS nerve increased the amplitude of the medial gastrocnemius (MG) electromyogram. The augmented activity of the MG muscle contributed to an increased extension of the ankle during stimulated steps. The conclusion from these experiments is that stimulation of the group I afferents in extensor nerves can prolong stance in the conscious cat, but this effect is weaker than in decerebrate animals. It is likely that transmission in the polysynaptic group I pathways controlling stance duration is regulated in a complex fashion by descending signals from the brain in the conscious animal.