Current Principles of Motor Control, with Special Reference to Vertebrate Locomotion.

The vertebrate control of locomotion involves all levels of the nervous system from cortex to the spinal cord. Here, we aim to cover all main aspects of this complex behavior, from the operation of the microcircuits in the spinal cord to the systems and behavioral levels and extend from mammalian locomotion to the basic undulatory movements of lamprey and fish. The cellular basis of propulsion represents the core of the control system, and it involves the spinal central pattern generator networks (CPGs) controlling the timing of different muscles, the sensory compensation for perturbations, and the brain stem command systems controlling the level of activity of the CPGs and the speed of locomotion. The forebrain and in particular the basal ganglia are involved in determining which motor programs should be recruited at a given point of time and can both initiate and stop locomotor activity. The propulsive control system needs to be integrated with the postural control system to maintain body orientation. Moreover, the locomotor movements need to be steered so that the subject approaches the goal of the locomotor episode, or avoids colliding with elements in the environment or simply escapes at high speed. These different aspects will all be covered in the review.

A specialized spinal circuit for command amplification and directionality during escape behavior.

In vertebrates, action selection often involves higher cognition entailing an evaluative process. However, urgent tasks, such as defensive escape, require an immediate implementation of the directionality of escape trajectory, necessitating local circuits. Here we reveal a specialized spinal circuit for the execution of escape direction in adult zebrafish. A central component of this circuit is a unique class of segmentally repeating cholinergic V2a interneurons expressing the transcription factor Chx10. These interneurons amplify brainstem-initiated escape commands and rapidly deliver the excitation via a feedforward circuit to all fast motor neurons and commissural interneurons to direct the escape maneuver. The information transfer within this circuit relies on fast and reliable axo-axonic synaptic connections, bypassing soma and dendrites. Unilateral ablation of cholinergic V2a interneurons eliminated escape command propagation. Thus, in vertebrates, local spinal circuits can implement directionality of urgent motor actions vital for survival.

Motor neurons control locomotor circuit function retrogradely via gap junctions.

Motor neurons are the final stage of neural processing for the execution of motor behaviours. Traditionally, motor neurons have been viewed as the 'final common pathway', serving as passive recipients merely conveying to the muscles the final motor program generated by upstream interneuron circuits. Here we reveal an unforeseen role of motor neurons in controlling the locomotor circuit function via gap junctions in zebrafish. These gap junctions mediate a retrograde analogue propagation of voltage fluctuations from motor neurons to control the synaptic release and recruitment of the upstream V2a interneurons that drive locomotion. Selective inhibition of motor neurons during ongoing locomotion de-recruits V2a interneurons and strongly influences locomotor circuit function. Rather than acting as separate units, gap junctions unite motor neurons and V2a interneurons into functional ensembles endowed with a retrograde analogue computation essential for locomotor rhythm generation. These results show that motor neurons are not a passive recipient of motor commands but an integral component of the neural circuits responsible for motor behaviour.

Delineating the Diversity of Spinal Interneurons in Locomotor Circuits.

Locomotion is common to all animals and is essential for survival. Neural circuits located in the spinal cord have been shown to be necessary and sufficient for the generation and control of the basic locomotor rhythm by activating muscles on either side of the body in a specific sequence. Activity in these neural circuits determines the speed, gait pattern, and direction of movement, so the specific locomotor pattern generated relies on the diversity of the neurons within spinal locomotor circuits. Here, we review findings demonstrating that developmental genetics can be used to identify populations of neurons that comprise these circuits and focus on recent work indicating that many of these populations can be further subdivided into distinct subtypes, with each likely to play complementary functions during locomotion. Finally, we discuss data describing the manner in which these populations interact with each other to produce efficient, task-dependent locomotion.

Neural progenitors organize in small-world networks to promote cell proliferation.

Coherent network activity among assemblies of interconnected cells is essential for diverse functions in the adult brain. However, cellular networks before formations of chemical synapses are poorly understood. Here, embryonic stem cell-derived neural progenitors were found to form networks exhibiting synchronous calcium ion (Ca(2+)) activity that stimulated cell proliferation. Immature neural cells established circuits that propagated electrical signals between neighboring cells, thereby activating voltage-gated Ca(2+) channels that triggered Ca(2+) oscillations. These network circuits were dependent on gap junctions, because blocking prevented electrotonic transmission both in vitro and in vivo. Inhibiting connexin 43 gap junctions abolished network activity, suppressed proliferation, and affected embryonic cortical layer formation. Cross-correlation analysis revealed highly correlated Ca(2+) activities in small-world networks that followed a scale-free topology. Graph theory predicts that such network designs are effective for biological systems. Taken together, these results demonstrate that immature cells in the developing brain organize in small-world networks that critically regulate neural progenitor proliferation.

Optogenetic activation of excitatory premotor interneurons is sufficient to generate coordinated locomotor activity in larval zebrafish.

Neural networks in the spinal cord can generate locomotion in the absence of rhythmic input from higher brain structures or sensory feedback because they contain an intrinsic source of excitation. However, the molecular identity of the spinal interneurons underlying the excitatory drive within the locomotor circuit has remained unclear. Using optogenetics, we show that activation of a molecularly defined class of ipsilateral premotor interneurons elicits locomotion. These interneurons represent the excitatory module of the locomotor networks and are sufficient to produce a coordinated swimming pattern in zebrafish. They correspond to the V2a interneuron class and express the transcription factor Chx10. They produce sufficient excitatory drive within the spinal networks to generate coordinated locomotor activity. Therefore, our results define the V2a interneurons as the excitatory module within the spinal locomotor networks that is sufficient to initiate and maintain locomotor activity.

Decoding the rules of recruitment of excitatory interneurons in the adult zebrafish locomotor network.

Neural networks in the spinal cord transform signals from the brain into coordinated locomotor movements. An optimal adjustment of the speed of locomotion entails a precise order of recruitment of interneurons underlying excitation within these networks. However, the mechanisms encoding the recruitment threshold of excitatory interneurons have remained unclear. Here we show, using a juvenile/adult zebrafish preparation, that excitatory V2a interneurons are incrementally recruited with increased swimming frequency. The order of recruitment is not imprinted by the topography or the input resistance of the V2a interneurons. Rather, it is determined by scaling the effect of excitatory synaptic currents by the input resistance. We also show that the locomotor networks are composed of multiple microcircuits encompassing subsets of V2a interneurons and motoneurons that are recruited in a continuum with increased swimming speeds. Thus, our results provide insights into the organization and mechanisms determining the recruitment of spinal microcircuits to ensure optimal execution of locomotor movements.

Origin of excitation underlying locomotion in the spinal circuit of zebrafish.

Neural circuits in the spinal cord transform instructive signals from the brain into well-coordinated locomotor movements by virtue of rhythm-generating components. Although evidence suggests that excitatory interneurons are the essence of locomotor rhythm generation, their molecular identity and the assessment of their necessity have remained unclear. Here we show, using larval zebrafish, that V2a interneurons represent an intrinsic source of excitation necessary for the normal expression of the locomotor rhythm. Acute and selective ablation of these interneurons increases the threshold of induction of swimming activity, decreases the burst frequency, and alters the coordination of the rostro-caudal propagation of activity. Thus, our results argue that V2a interneurons represent a source of excitation that endows the spinal circuit with the capacity to generate locomotion.

Pattern of innervation and recruitment of different classes of motoneurons in adult zebrafish.

In vertebrates, spinal circuits drive rhythmic firing in motoneurons in the appropriate sequence to produce locomotor movements. These circuits become active early during development and mature gradually to acquire the flexibility necessary to accommodate the increased behavioral repertoire of adult animals. The focus here is to elucidate how different pools of motoneurons are organized and recruited and how membrane properties contribute to their mode of operation. For this purpose, we have used the in vitro preparation of adult zebrafish. We show that different motoneuron pools are organized in a somatotopic fashion in the motor column related to the type of muscle fibers (slow, intermediate, fast) they innervate. During swimming, the different motoneuron pools are recruited in a stepwise manner from slow, to intermediate, to fast to cover the full range of locomotor frequencies seen in intact animals. The spike threshold, filtering properties, and firing patterns of the different motoneuron pools are graded in a manner that relates to their order of recruitment. Our results thus show that motoneurons in adult zebrafish are organized into distinct modules, each with defined locations, properties, and recruitment patterns tuned to precisely match the muscle properties and hence produce swimming of different speeds and modalities.

Histone H2AX-dependent GABA(A) receptor regulation of stem cell proliferation.

Stem cell self-renewal implies proliferation under continued maintenance of multipotency. Small changes in numbers of stem cells may lead to large differences in differentiated cell numbers, resulting in significant physiological consequences. Proliferation is typically regulated in the G1 phase, which is associated with differentiation and cell cycle arrest. However, embryonic stem (ES) cells may lack a G1 checkpoint. Regulation of proliferation in the 'DNA damage' S/G2 cell cycle checkpoint pathway is known for its role in the maintenance of chromatin structural integrity. Here we show that autocrine/paracrine gamma-aminobutyric acid (GABA) signalling by means of GABA(A) receptors negatively controls ES cell and peripheral neural crest stem (NCS) cell proliferation, preimplantation embryonic growth and proliferation in the boundary-cap stem cell niche, resulting in an attenuation of neuronal progenies from this stem cell niche. Activation of GABA(A) receptors leads to hyperpolarization, increased cell volume and accumulation of stem cells in S phase, thereby causing a rapid decrease in cell proliferation. GABA(A) receptors signal through S-phase checkpoint kinases of the phosphatidylinositol-3-OH kinase-related kinase family and the histone variant H2AX. This signalling pathway critically regulates proliferation independently of differentiation, apoptosis and overt damage to DNA. These results indicate the presence of a fundamentally different mechanism of proliferation control in these stem cells, in comparison with most somatic cells, involving proteins in the DNA damage checkpoint pathway.

Molecular blueprints for spinal circuit modules controlling locomotor speed in zebrafish.

The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules, yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Here we use adult zebrafish to link the molecular diversity of motoneurons (MNs) and the rhythm-generating V2a interneurons (INs) with the modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of MNs and V2a INs reflects their functional segregation into slow, intermediate or fast subtypes. Furthermore, we reveal shared molecular signatures between V2a INs and MNs of the three speed circuit modules. Overall, by characterizing how the molecular diversity of MNs and V2a INs relates to their function, connectivity and behavior, our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general.

Gating the polarity of endocannabinoid-mediated synaptic plasticity by nitric oxide in the spinal locomotor network.

The final motor output underlying behavior arises from an appropriate balance between excitation and inhibition within neural networks. Retrograde signaling by endocannabinoids adapts synaptic strengths and the global activity of neural networks. In the spinal cord, endocannabinoids are mobilized postsynaptically from network neurons and act retrogradely on presynaptic cannabinoid receptors to potentiate the locomotor frequency. However, it is still unclear whether mechanisms exist within the locomotor networks that determine the sign of the modulation by cannabinoid receptors to differentially regulate excitation and inhibition. In this study, using the lamprey spinal cord in vitro, we first report that 2-AG (2-arachidonyl glycerol) is mobilized by network neurons and underlies a form of modulation that is embedded within the locomotor networks. We then show that the polarity of the endocannabinoid modulation is gated by nitric oxide to enable simultaneously potentiation of excitation and depression of inhibition within the spinal locomotor networks. Our results suggest that endocannabinoid and nitric oxide systems interact to mediate inversion of the polarity of synaptic plasticity within the locomotor networks. Thus, endocannabinoid and nitric oxide shift in the excitation-inhibition balance to set the excitability of the spinal locomotor network.

Differential regulation of synaptic transmission by pre- and postsynaptic SK channels in the spinal locomotor network.

The generation of activity in the central nervous system requires precise tuning of cellular properties and synaptic transmission. Neural networks in the spinal cord produce coordinated locomotor movements. Synapses in these networks need to be equipped with multiple mechanisms that regulate their operation over varying regimes to produce locomotor activity at different frequencies. Using the in vitro lamprey spinal cord, we explored whether Ca(2+) influx via different routes in postsynaptic soma and dendrites and in presynaptic terminals can activate apamin-sensitive Ca(2+)-activated K(+) (SK) channels and thereby shape synaptic transmission. We show that postsynaptic SK channels are tightly coupled to Ca(2+) influx via NMDA receptors. Activation of these channels by synaptically induced NMDA-dependent Ca(2+) transients restrains the time course of the synaptic current and the amplitude of the synaptic potential. In addition, presynaptic SK channels are activated by Ca(2+) influx via voltage-gated channels and control the waveform of the action potential and the resulting Ca(2+) dynamics in the axon terminals. The coupling of SK channels to different Ca(2+) sources, pre- and postsynaptically, acts as a negative feedback mechanism to shape synaptic transmission. Thus SK channels can play a pivotal role in setting the dynamic range of synapses and enabling short-term plasticity in the spinal locomotor network.

Modular circuit organization for speed control of locomotor movements.

Our movements and actions stem from complex processes in the central nervous system. Precise adaptation of locomotor movements is essential for effectively interacting with the environment. To understand the mechanisms underlying these movements, it is crucial to determine the organization of spinal circuits at the level of individual neurons and synapses. This review highlights the insights gained from studying spinal circuits in adult zebrafish and discusses their broader implications for our understanding of locomotor control across species.

Brainstem circuits encoding start, speed, and duration of swimming in adult zebrafish.

The flexibility of locomotor movements requires an accurate control of their start, duration, and speed. How brainstem circuits encode and convey these locomotor parameters remains unclear. Here, we have combined in vivo calcium imaging, electrophysiology, anatomy, and behavior in adult zebrafish to address these questions. We reveal that the detailed parameters of locomotor movements are encoded by two molecularly, topographically, and functionally segregated glutamatergic neuron subpopulations within the nucleus of the medial longitudinal fasciculus. The start, duration, and changes of locomotion speed are encoded by vGlut2+ neurons, whereas vGlut1+ neurons encode sudden changes to high speed/high amplitude movements. Ablation of vGlut2+ neurons compromised slow-explorative swimming, whereas vGlut1+ neuron ablation impaired fast swimming. Our results provide mechanistic insights into how separate brainstem subpopulations implement flexible locomotor commands. These two brainstem command subpopulations are suitably organized to integrate environmental cues and hence generate flexible swimming movements to match the animal's behavioral needs.

Initiation of locomotion in adult zebrafish.

Motor behavior is generated by specific neural circuits. Those producing locomotion are located in the spinal cord, and their activation depends on descending inputs from the brain or on sensory inputs. In this study, we have used an in vitro brainstem-spinal cord preparation from adult zebrafish to localize a region where stimulation of descending inputs can induce sustained locomotor activity. We show that a brief stimulation of descending inputs at the junction between the brainstem and spinal cord induces long-lasting swimming activity. The swimming frequencies induced are remarkably similar to those observed in freely moving adult fish, arguing that the induced locomotor episode is highly physiological. The motor pattern is mediated by activation of ionotropic glutamate and glycine receptors in the spinal cord and is not the result of synaptic interactions between neurons at the site of the stimulation in the brainstem. We also compared the activity of motoneurons during locomotor activity induced by electrical stimulation of descending inputs and by exogenously applied NMDA. Prolonged NMDA application changes the shape of the synaptic drive and action potentials in motoneurons. When escape activity occurs, the swimming activity in the intact zebrafish was interrupted and some of the motoneurons involved became inhibited in vitro. Thus, the descending inputs seem to act as a switch to turn on the activity of the spinal locomotor network in the caudal spinal cord. We propose that recurrent synaptic activity within the spinal locomotor circuits can transform a brief input into a well coordinated and long-lasting swimming pattern.

Na,K-ATPase signal transduction triggers CREB activation and dendritic growth.

Dendritic growth is pivotal in the neurogenesis of cortical neurons. The sodium pump, or Na,K-ATPase, is an evolutionarily conserved protein that, in addition to its central role in establishing the electrochemical gradient, has recently been reported to function as a receptor and signaling mediator. Although a large body of evidence points toward a dual function for the Na,K-ATPase, few biological implications of this signaling pathway have been described. Here we report that Na,K-ATPase signal transduction triggers dendritic growth as well as a transcriptional program dependent on cAMP response element binding protein (CREB) and cAMP response element (CRE)-mediated gene expression, primarily regulated via Ca(2+)/calmodulin-dependent protein (CaM) kinases. The signaling cascade mediating dendritic arbor growth also involves intracellular Ca(2+) oscillations and sustained phosphorylation of mitogen-activated protein (MAP) kinases. Thus, our results suggest a novel role for the Na,K-ATPase as a modulator of dendritic growth in developing neurons.

Efficient production of mesencephalic dopamine neurons by Lmx1a expression in embryonic stem cells.

Signaling factors involved in CNS development have been used to control the differentiation of embryonic stem cells (ESCs) into mesencephalic dopamine (mesDA) neurons, but tend to generate a limited yield of desired cell type. Here we show that forced expression of Lmx1a, a transcription factor functioning as a determinant of mesDA neurons during embryogenesis, effectively can promote the generation of mesDA neurons from mouse and human ESCs. Under permissive culture conditions, 75%-95% of mouse ESC-derived neurons express molecular and physiological properties characteristic of bona fide mesDA neurons. Similar to primary mesDA neurons, these cells integrate and innervate the striatum of 6-hydroxy dopamine lesioned neonatal rats. Thus, the enriched generation of functional mesDA neurons by forced expression of Lmx1a may be of future importance in cell replacement therapy of Parkinson disease.

Protocol to visualize distinct motoneuron pools in adult zebrafish via injection of retrograde tracers.

In adult zebrafish, slow, intermediate, and fast muscle fibers occupy distinct regions of the axial muscle, allowing the use of retrograde tracers for selective targeting of the motoneurons (MNs) innervating them. Here, we describe a protocol to label distinct MN pools and tissue processing for visualization with confocal laser microscopy. We outline the different steps for selective labeling of primary and secondary MNs together with spinal cord fixation, isolation, mounting, and imaging. For complete details on the use and execution of this protocol, please refer to Pallucchi et al. (2022)1 and Ampatzis et al. (2013).2.

Developmental switch in the function of inhibitory commissural V0d interneurons in zebrafish.

During development, all animals undergo major adaptations to accommodate behavioral flexibility and diversity. How these adaptations are reflected in the changes in the motor circuits controlling our behaviors remains poorly understood. Here, we show, using a combination of techniques applied at larval and adult zebrafish stages, that the pattern-generating V0d inhibitory interneurons within the locomotor circuit undergo a developmental switch in their role. In larvae, we show that V0d interneurons have a primary function in high-speed motor behavior yet are redundant for explorative swimming. By contrast, adult V0d interneurons have diversified into speed-dependent subclasses, with an overrepresentation of those active at the slowest speeds. The ablation of V0d interneurons in adults disrupts slow explorative swimming, which is associated with a loss of mid-cycle inhibition onto target motoneurons. Thus, we reveal a developmental switch in V0d interneuron function from a role in high-speed motor behavior to a function in timing and thus coordinating slow explorative locomotion. Our study suggests that early motor circuit composition is not predictive of the adult system but instead undergoes major functional transformations during development.