V3 Interneurons Are Active and Recruit Spinal Motor Neurons during In Vivo Fictive Swimming in Larval Zebrafish.

Survival for vertebrate animals is dependent on the ability to successfully find food, locate a mate, and avoid predation. Each of these behaviors requires motor control, which is set by a combination of kinematic properties. For example, the frequency and amplitude of motor output combine in a multiplicative manner to determine features of locomotion such as distance traveled, speed, force (thrust), and vigor. Although there is a good understanding of how different populations of excitatory spinal interneurons establish locomotor frequency, there is a less thorough mechanistic understanding for how locomotor amplitude is established. Recent evidence indicates that locomotor amplitude is regulated in part by a subset of functionally and morphologically distinct V2a excitatory spinal interneurons (Type II, nonbursting) in larval and adult zebrafish. Here, we provide direct evidence that most V3 interneurons (V3-INs), which are a developmentally and genetically defined population of ventromedial glutamatergic spinal neurons, are active during fictive swimming. We also show that elimination of the spinal V3-IN population reduces the proportion of active motor neurons (MNs) during fictive swimming but does not alter the range of locomotor frequencies produced. These data are consistent with V3-INs providing excitatory drive to spinal MNs during swimming in larval zebrafish and may contribute to the production of locomotor amplitude independently of locomotor frequency.

Repetitive optogenetic stimulation of glutamatergic neurons: An alternative to NMDA treatment for generating locomotor activity in spinalized zebrafish larvae.

N-methyl-d-aspartate (NMDA) application has conventionally been used to activate spinal networks to induce locomotion in spinalized animals. We recently described an alternative approach in which application of continuous blue light activates channelrhodopsin-2 in vesicular glutamate transporter 2a (vglut2a)-expressing spinal neurons to produce organized, rhythmic locomotor activity in spinally-transected larval zebrafish. This technique arguably enhances research validity, because endogenous glutamate is released into existing synapses instead of activating only a subset of glutamatergic (NMDA) receptors with an exogenous compound. Here, we explored the viability of this approach in the context of using it for longer-term experiments. Fictive swimming was induced through repetitive application of 10-s blue light stimuli to spinalized preparations for up to 60 min at intervals of 1, 3, or 15 min. Locomotor activity was maintained throughout the experimental timecourse, demonstrating the robustness of the system. Although locomotor bursts remained organized into episodes of activity, the number of bursts elicited during each successive stimulus decreased. This was in contrast to NMDA bath application, in which bursts became less episodically organized while the overall number of bursts remained unchanged. The efficacy of the repetitive optogenetic stimulation paradigm was demonstrated through application of exogenous dopamine, which reversibly decreased the number of bursts produced per stimulus compared with untreated preparations. Finally, increasing the stimulus interval to 15 min lessened, but did not eliminate locomotor fatigue from repetitive activation. Altogether, we established repetitive optogenetic stimulation of vglut2a-expressing neurons as a viable alternative to NMDA application for activation of the zebrafish spinal locomotor network.

L-type voltage-gated calcium channel agonists mitigate hearing loss and modify ribbon synapse morphology in the zebrafish model of Usher syndrome type 1.

The mariner (myo7aa-/- ) mutant is a zebrafish model for Usher syndrome type 1 (USH1). To further characterize hair cell synaptic elements in myo7aa-/- mutants, we focused on the ribbon synapse and evaluated ultrastructure, number and distribution of immunolabeled ribbons, and postsynaptic densities. By transmission electron microscopy, we determined that myo7aa-/- zebrafish have fewer glutamatergic vesicles tethered to ribbon synapses, yet maintain a comparable ribbon area. In myo7aa-/- hair cells, immunolocalization of Ctbp2 showed fewer ribbon-containing cells in total and an altered distribution of Ctbp2 puncta compared to wild-type hair cells. myo7aa-/- mutants have fewer postsynaptic densities - as assessed by MAGUK immunolabeling - compared to wild-type zebrafish. We quantified the circular swimming behavior of myo7aa-/- mutant fish and measured a greater turning angle (absolute smooth orientation). It has previously been shown that L-type voltage-gated calcium channels are necessary for ribbon localization and occurrence of postsynaptic density; thus, we hypothesized and observed that L-type voltage-gated calcium channel agonists change behavioral and synaptic phenotypes in myo7aa-/- mutants in a drug-specific manner. Our results indicate that treatment with L-type voltage-gated calcium channel agonists alter hair cell synaptic elements and improve behavioral phenotypes of myo7aa-/- mutants. Our data support that L-type voltage-gated calcium channel agonists induce morphological changes at the ribbon synapse - in both the number of tethered vesicles and regarding the distribution of Ctbp2 puncta - shift swimming behavior and improve acoustic startle response.

Glutamate receptor subtypes differentially contribute to optogenetically activated swimming in spinally transected zebrafish larvae.

The spinal cord (SC) contains neural networks that are capable of producing organized locomotor activity autonomously from the brain. Locomotor activity can be induced in spinally transected (spinalized) animals by adding a source of tonic excitation to activate spinal networks. This is commonly accomplished by activating N-methyl-d-aspartate (NMDA) glutamate receptors through bath application of NMDA. More recently, optogenetic approaches have enabled both activation and inactivation of neuronal cell populations to control the activity of locomotor networks. Larval zebrafish are exceptionally amenable to optogenetic techniques due to their transparency, which permits noninvasive light delivery. In this study, we induced locomotor activity in spinalized transgenic zebrafish larvae that expressed channelrhodopsin-2 in all subtypes of spinal vesicular glutamate transporter 2a (vglut2a)-expressing neurons by applying 10 s of constant blue light to the preparations. The resultant locomotor activity possessed all of the characteristics of swimming: bilateral alternation, rostrocaudal progression, and organization into discrete swimming episodes. Spatially restricted light application revealed that illumination of the rostral SC produced more robust activity than illumination of the caudal SC. Moreover, illumination of only three body segments was sufficient to produce fictive swimming. Intriguingly, organized swimming activity persisted during NMDA receptor antagonism but was disrupted by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonism. Hence, AMPA receptor signaling is required for episodically-organized swimming, whereas NMDA receptor signaling is not necessary.NEW & NOTEWORTHY Spinal locomotor networks have the intrinsic capacity to transform unpatterned excitatory input into patterned output. Conventionally, spinally mediated fictive locomotor activity is experimentally elicited by N-methyl-d-aspartate (NMDA) application to bias the network toward activation. We present a novel experimental paradigm that permits spatially and temporally controllable activation of spinal vesicular glutamate transporter 2a-expressing neurons in larval zebrafish, eliciting patterned locomotor activity that is not dependent on NMDA receptor signaling.

Dietary Contaminants and Their Effects on Zebrafish Embryos.

Dietary contaminants are often an over-looked factor in the health of zebrafish. Typically, water is considered to be the source for most contaminants, especially within an aquatic environment. For this reason, source water for zebrafish recirculating systems is highly regulated and monitored daily. Most facilities use reverse osmosis or de-ionized water filtration systems to purify incoming water to ensure that contaminants, as well as pathogens, do not enter their zebrafish housing units. However, diets are rarely tested for contaminants and, in the case of manufactured zebrafish feeds, since the product is marketed for aquaculture or aquarium use it is assumed that the feed is acceptable for animals used for research. The following provides examples as to how contaminants could lead to negative effects on development and behavior of developing zebrafish.

Intraspinal serotonergic signaling suppresses locomotor activity in larval zebrafish.

Serotonin (5HT) is a modulator of many vital processes in the spinal cord (SC), such as production of locomotion. In the larval zebrafish, intraspinal serotonergic neurons (ISNs) are a source of spinal 5HT that, despite the availability of numerous genetic and optical tools, has not yet been directly shown to affect the spinal locomotor network. In order to better understand the functions of ISNs, we used a combination of strategies to investigate ISN development, morphology, and function. ISNs were optically isolated from one another by photoconverting Kaede fluorescent protein in individual cells, permitting morphometric analysis as they developed in vivo. ISN neurite lengths and projection distances exhibited the greatest amount of change between 3 and 4 days post-fertilization (dpf) and appeared to stabilize by 5 dpf. Overall ISN innervation patterns were similar between cells and between SC regions. ISNs possessed rostrally-extending neurites resembling dendrites and a caudally-extending neurite resembling an axon, which terminated with an enlarged growth cone-like structure. Interestingly, these enlargements remained even after neurite extension had ceased. Functionally, application of exogenous 5HT reduced spinally-produced motor nerve bursting. A selective 5HT reuptake inhibitor and ISN activation with channelrhodopsin-2 each produced similar effects to 5HT, indicating that spinally-intrinsic 5HT originating from the ISNs has an inhibitory effect on the spinal locomotor network. Taken together this suggests that the ISNs are morphologically mature by 5 dpf and supports their involvement in modulating the activity of the spinal locomotor network. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018.

An Adult Zebrafish Diet Contaminated with Chromium Reduces the Viability of Progeny.

The lack of standardized diet for laboratory animals can have profound effects on animal health and lead to less reproducible research outcomes. Live diets are commonly used in zebrafish culture and, although they are a more natural feed than flake or pellet food, are also a potential source of pathogens and toxic compounds. Heavy metals are a group of such compounds, which can accumulate in fish leading to developmental abnormalities, reduced growth, and increased rates of mortality. Two to three weeks after feeding adult zebrafish a new lot of nonhatching decapsulated brine shrimp cysts (Decaps), embryos at the University of Minnesota Zebrafish Core Facility (ZCF) and the University of Utah Centralized Zebrafish Animal Resource (CZAR) began to exhibit an orange color in the yolk, and larval health began to decline. The concentration of chromium in the Decaps (69.6 mg/kg) was more than 30 times that of other zebrafish diets tested (up to 2.1 mg/kg) and is thought to be the cause of the observed symptoms. Within 3 weeks of removing the Decaps from the feeding regimen, the orange coloration in the yolks began to diminish, the morphological abnormalities began to subside, and larval survival rates began to increase. Thus, implementation of standardized zebrafish diets and regular feed-quality testing may help to prevent the introduction of contaminants to zebrafish research facilities.

Intraspinal serotonergic neurons consist of two, temporally distinct populations in developing zebrafish.

Zebrafish intraspinal serotonergic neuron (ISN) morphology and distribution have been examined in detail at different ages; however, some aspects of the development of these cells remain unclear. Although antibodies to serotonin (5-HT) have detected ISNs in the ventral spinal cord of embryos, larvae, and adults, the only tryptophan hydroxylase (tph) transcript that has been described in the spinal cord is tph1a. Paradoxically, spinal tph1a is only expressed transiently in embryos, which brings the source of 5-HT in the ISNs of larvae and adults into question. Because the pet1 and tph2 promoters drive transgene expression in the spinal cord, we hypothesized that tph2 is expressed in spinal cords of zebrafish larvae. We confirmed this hypothesis through in situ hybridization. Next, we used 5-HT antibody labeling and transgenic markers of tph2-expressing neurons to identify a transient population of ISNs in embryos that was distinct from ISNs that appeared later in development. The existence of separate ISN populations may not have been recognized previously due to their shared location in the ventral spinal cord. Finally, we used transgenic markers and immunohistochemical labeling to identify the transient ISN population as GABAergic Kolmer-Agduhr double-prime (KA″) neurons. Altogether, this study revealed a novel developmental paradigm in which KA″ neurons are transiently serotonergic before the appearance of a stable population of tph2-expressing ISNs.

Coordination of fictive motor activity in the larval zebrafish is generated by non-segmental mechanisms.

The cellular and network basis for most vertebrate locomotor central pattern generators (CPGs) is incompletely characterized, but organizational models based on known CPG architectures have been proposed. Segmental models propose that each spinal segment contains a circuit that controls local coordination and sends longer projections to coordinate activity between segments. Unsegmented/continuous models propose that patterned motor output is driven by gradients of neurons and synapses that do not have segmental boundaries. We tested these ideas in the larval zebrafish, an animal that swims in discrete episodes, each of which is composed of coordinated motor bursts that progress rostrocaudally and alternate from side to side. We perturbed the spinal cord using spinal transections or strychnine application and measured the effect on fictive motor output. Spinal transections eliminated episode structure, and reduced both rostrocaudal and side-to-side coordination. Preparations with fewer intact segments were more severely affected, and preparations consisting of midbody and caudal segments were more severely affected than those consisting of rostral segments. In reduced preparations with the same number of intact spinal segments, side-to-side coordination was more severely disrupted than rostrocaudal coordination. Reducing glycine receptor signaling with strychnine reversibly disrupted both rostrocaudal and side-to-side coordination in spinalized larvae without disrupting episodic structure. Both spinal transection and strychnine decreased the stability of the motor rhythm, but this effect was not causal in reducing coordination. These results are inconsistent with a segmented model of the spinal cord and are better explained by a continuous model in which motor neuron coordination is controlled by segment-spanning microcircuits.

Abnormal differentiation of dopaminergic neurons in zebrafish trpm7 mutant larvae impairs development of the motor pattern.

Transient receptor potential, melastatin-like 7 (Trpm7) is a combined ion channel and kinase implicated in the differentiation or function of many cell types. Early lethality in mice and frogs depleted of the corresponding gene impedes investigation of the functions of this protein particularly during later stages of development. By contrast, zebrafish trpm7 mutant larvae undergo early morphogenesis normally and thus do not have this limitation. The mutant larvae are characterized by multiple defects including melanocyte cell death, transient paralysis, and an ion imbalance that leads to the development of kidney stones. Here we report a requirement for Trpm7 in differentiation or function of dopaminergic neurons in vivo. First, trpm7 mutant larvae are hypomotile and fail to make a dopamine-dependent developmental transition in swim-bout length. Both of these deficits are partially rescued by the application of levodopa or dopamine. Second, histological analysis reveals that in trpm7 mutants a significant fraction of dopaminergic neurons lack expression of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Third, trpm7 mutants are unusually sensitive to the neurotoxin 1-methyl-4-phenylpyridinium, an oxidative stressor, and their motility is partially rescued by application of the iron chelator deferoxamine, an anti-oxidant. Finally, in SH-SY5Y cells, which model aspects of human dopaminergic neurons, forced expression of a channel-dead variant of TRPM7 causes cell death. In summary, a forward genetic screen in zebrafish has revealed that both melanocytes and dopaminergic neurons depend on the ion channel Trpm7. The mechanistic underpinning of this dependence requires further investigation.

Quantification of locomotor activity in larval zebrafish: considerations for the design of high-throughput behavioral studies.

High-throughput behavioral studies using larval zebrafish often assess locomotor activity to determine the effects of experimental perturbations. However, the results reported by different groups are difficult to compare because there is not a standardized experimental paradigm or measure of locomotor activity. To address this, we investigated the effects that several factors, including the stage of larval development and the physical dimensions (depth and diameter) of the behavioral arena, have on the locomotor activity produced by larval zebrafish. We provide evidence for differences in locomotor activity between larvae at different stages and when recorded in wells of different depths, but not in wells of different diameters. We also show that the variability for most properties of locomotor activity is less for older than younger larvae, which is consistent with previous reports. Finally, we show that conflicting interpretations of activity level can occur when activity is assessed with a single measure of locomotor activity. Thus, we conclude that although a combination of factors should be considered when designing behavioral experiments, the use of older larvae in deep wells will reduce the variability of locomotor activity, and that multiple properties of locomotor activity should be measured to determine activity level.

Necessary, sufficient and permissive: a single locomotor command neuron important for intersegmental coordination.

In this report we posed the overarching question: What multiple contributions can a single neuron have on controlling the behavior of an animal, especially within a given context? To address this timely question, we studied the neuron R3b-1 in the medicinal leech. This bilaterally paired neuron descends from the cephalic ganglion and projects uninterrupted through the segmental ganglia comprising the nerve cord; its terminal arbors invade each hemi-ganglion. We discovered that a single R3b-1 neuron functions as a command neuron in the strictest sense, as it was both necessary and sufficient for fictive crawling behavior. Aside from these command-related properties, we determined that R3b-1 modulates the cycle period of crawl motor activity. R3b-1 has previously been shown to activate swimming behavior, but when the CNS was exposed to dopamine (DA), crawling became the exclusive locomotor pattern produced by R3b-1. DA exposure also led to bursting in R3b-1 that matched periods observed during fictive crawling, even when potential ascending inputs from crawl oscillators were removed. Although the above attributes render R3b-1 an intriguing cell, it is its ability to permit the coordination of the segmentally distributed crawl oscillators that makes this multifunctional neuron so notable. To our knowledge, this cell provides the first biological example of a single command neuron that is also vital for the intersegmental coordination of a locomotor behavior. Furthermore, our study highlights the importance of DA as an internal contextual cue that can integrate functional layers of the nervous system for adaptive behavior.

TTX-resistant NMDA receptor-mediated membrane potential oscillations in neonatal mouse Hb9 interneurons.

Conditional neuronal membrane potential oscillations have been identified as a potential mechanism to help support or generate rhythmogenesis in neural circuits. A genetically identified population of ventromedial interneurons, called Hb9, in the mouse spinal cord has been shown to generate TTX-resistant membrane potential oscillations in the presence of NMDA, serotonin and dopamine, but these oscillatory properties are not well characterized. Hb9 interneurons are rhythmically active during fictive locomotor-like behavior. In this study, we report that exogenous N-Methyl-D-Aspartic acid (NMDA) application is sufficient to produce membrane potential oscillations in Hb9 interneurons. In contrast, exogenous serotonin and dopamine application, alone or in combination, are not sufficient. The properties of NMDA-induced oscillations vary among the Hb9 interneuron population; their frequency and amplitude increase with increasing NMDA concentration. NMDA does not modulate the T-type calcium current (I(Ca(T))), which is thought to be important in generating locomotor-like activity, in Hb9 neurons. These results suggest that NMDA receptor activation is sufficient for the generation of TTX-resistant NMDA-induced membrane potential oscillations in Hb9 interneurons.

The conserved dopaminergic diencephalospinal tract mediates vertebrate locomotor development in zebrafish larvae.

The most conserved part of the vertebrate dopaminergic system is the orthopedia (otp)-expressing diencephalic neuronal population that constitutes the dopaminergic diencephalospinal tract (DDT). Although studies in the neonatal murine spinal cord in vitro suggest an early locomotor role of the DDT, the function of the DDT in developing vertebrates in vivo remains unknown. Here, we investigated the role of the DDT in the locomotor development of zebrafish larvae. To assess the development of the behavioral and neural locomotor pattern, we used high-throughput video tracking in combination with peripheral nerve recordings. We found a behavioral and neural correspondence in the developmental switch from an immature to mature locomotor pattern. Blocking endogenous dopamine receptor 4 (D(4)R) signaling in vivo either before or after the developmental switch prevented or reversed the switch, respectively. Spinal transections of post-switch larvae reestablished the immature locomotor pattern, which was rescued to a mature-like pattern via spinal D(4)R agonism. Selective chemogenetic ablation of otp b (otpb) neurons that contribute to the DDT perpetuated the immature locomotor pattern in vivo. This phenotype was recapitulated by diencephalic transections that removed the dopaminergic otpb population and was rescued to a mature-like locomotor pattern by D(4)R agonism. We conclude that the dopaminergic otpb population, via the DDT, is responsible for spinal D(4)R signaling to mediate the developmental switch to the mature locomotor pattern of zebrafish. These results, integrated with the mammalian literature, suggest that the DDT represents an evolutionarily conserved neuromodulatory system that is necessary for normal vertebrate locomotor development.

Episodic swimming in the larval zebrafish is generated by a spatially distributed spinal network with modular functional organization.

Despite the diverse methods vertebrates use for locomotion, there is evidence that components of the locomotor central pattern generator (CPG) are conserved across species. When zebrafish begin swimming early in development, they perform short episodes of activity separated by periods of inactivity. Within these episodes, the trunk flexes with side-to-side alternation and the traveling body wave progresses rostrocaudally. To characterize the distribution of the swimming CPG along the rostrocaudal axis, we performed transections of the larval zebrafish spinal cord and induced fictive swimming using N-methyl-d-aspartate (NMDA). In both intact and spinalized larvae, bursting is found throughout the rostrocaudal extent of the spinal cord, and the properties of fictive swimming observed were dependent on the concentration of NMDA. We isolated series of contiguous spinal segments by performing multiple spinal transections on the same larvae. Although series from all regions of the spinal cord have the capacity to produce bursts, the capacity to produce organized episodes of fictive swimming has a rostral bias: in the rostral spinal cord, only 12 contiguous body segments are necessary, whereas 23 contiguous body segments are necessary in the caudal spinal cord. Shorter series of segments were often active but produced either continuous rhythmic bursting or sporadic, nonrhythmic bursting. Both episodic and continuous bursting alternated between the left and right sides of the body and showed rostrocaudal progression, demonstrating the functional dissociation of the circuits responsible for episodic structure and fine burst timing. These findings parallel results in mammalian locomotion, and we propose a hierarchical model of the larval zebrafish swimming CPG.

Low-threshold calcium currents contribute to locomotor-like activity in neonatal mice.

In this study, we examined the contribution of a low-threshold calcium current [I(Ca(T))] to locomotor-related activity in the neonatal mouse. Specifically, the role of I(Ca(T)) was studied during chemically induced, locomotor-like activity in the isolated whole cord and in a genetically distinct population of ventromedial spinal interneurons marked by the homeobox gene Hb9. In isolated whole spinal cords, cycle frequency was decreased in the presence of low-threshold calcium channel blockers, which suggests a role for I(Ca(T)) in the network that produces rhythmic, locomotor-like activity. Additionally, we used Hb9 interneurons as a model to study the cellular responses to application of low-threshold calcium channel blockers. In transverse slice preparations from transgenic Hb9::enhanced green fluorescent protein neonatal mice, N-methyl-d-aspartate-induced membrane potential oscillations in identified Hb9 interneurons also slowed in frequency with application of nickel when fast, spike-mediated, synaptic transmission was blocked with TTX. Voltage-clamp and immunolabeling experiments confirmed expression of I(Ca(T)) and channels, respectively, in Hb9 interneurons located in the ventromedial spinal cord. Taken together, these results provide support that T-type calcium currents play an important role in network-wide rhythm generation during chemically evoked, fictive locomotor activity.

Mutation of zebrafish dihydrolipoamide branched-chain transacylase E2 results in motor dysfunction and models maple syrup urine disease.

Analysis of zebrafish mutants that demonstrate abnormal locomotive behavior can elucidate the molecular requirements for neural network function and provide new models of human disease. Here, we show that zebrafish quetschkommode (que) mutant larvae exhibit a progressive locomotor defect that culminates in unusual nose-to-tail compressions and an inability to swim. Correspondingly, extracellular peripheral nerve recordings show that que mutants demonstrate abnormal locomotor output to the axial muscles used for swimming. Using positional cloning and candidate gene analysis, we reveal that a point mutation disrupts the gene encoding dihydrolipoamide branched-chain transacylase E2 (Dbt), a component of a mitochondrial enzyme complex, to generate the que phenotype. In humans, mutation of the DBT gene causes maple syrup urine disease (MSUD), a disorder of branched-chain amino acid metabolism that can result in mental retardation, severe dystonia, profound neurological damage and death. que mutants harbor abnormal amino acid levels, similar to MSUD patients and consistent with an error in branched-chain amino acid metabolism. que mutants also contain markedly reduced levels of the neurotransmitter glutamate within the brain and spinal cord, which probably contributes to their abnormal spinal cord locomotor output and aberrant motility behavior, a trait that probably represents severe dystonia in larval zebrafish. Taken together, these data illustrate how defects in branched-chain amino acid metabolism can disrupt nervous system development and/or function, and establish zebrafish que mutants as a model to better understand MSUD.

Continuous shifts in the active set of spinal interneurons during changes in locomotor speed.

The classic 'size principle' of motor control describes how increasingly forceful movements arise by the recruitment of motoneurons of progressively larger size and force output into the active pool. We explored the activity of pools of spinal interneurons in larval zebrafish and found that increases in swimming speed were not associated with the simple addition of cells to the active pool. Instead, the recruitment of interneurons at faster speeds was accompanied by the silencing of those driving movements at slower speeds. This silencing occurred both between and within classes of rhythmically active premotor excitatory interneurons. Thus, unlike motoneurons, there is a continuous shift in the set of cells driving the behavior, even though changes in the speed of the movements and the frequency of the motor pattern appear to be smoothly graded. We conclude that fundamentally different principles may underlie the recruitment of motoneuron and interneuron pools.

Synaptic homeostasis in a zebrafish glial glycine transporter mutant.

Truncated escape responses characteristic of the zebrafish shocked mutant result from a defective glial glycine transporter (GlyT1). In homozygous GlyT1 mutants, irrigating brain ventricles with glycine-free solution rescues normal swimming. Conversely, elevating brain glycine levels restores motility defects. These experiments are consistent with previous studies that demonstrate regulation of global glycine levels in the CNS as a primary function of GlyT1. As GlyT1 mutants mature, their ability to mount an escape response naturally recovers. To understand the basis of this recovery, we assay synaptic transmission in primary spinal motor neurons by measuring stimulus-evoked postsynaptic potentials. At the peak of the motility defect, inhibitory synaptic potentials are both significantly larger and more prolonged indicating a prominent role for GlyT1 in shaping fast synaptic transmission. However, as GlyT1 mutants naturally regain their ability to swim, the amplitude of inhibitory potentials decreases to below wild-type levels. In parallel with diminishing synaptic potentials, the glycine concentration required to evoke the mutant motility defect increases 61-fold during behavioral recovery. Behavioral recovery is also mirrored by a reduction in the levels of both glycine receptor protein and transcript. These results suggest that increased CNS glycine tolerance and reduced glycine receptor expression in GlyT1 mutants reflect compensatory mechanisms for functional recovery from excess nervous system inhibition.

Persistent sodium currents participate in fictive locomotion generation in neonatal mouse spinal cord.

The persistent sodium current (I(Na(P))) has been implicated in the regulation of synaptic integration, intrinsic membrane properties, and rhythm generation in many types of neurons. We characterized I(Na(P)) in commissural interneurons (CINs) in the neonatal (postnatal days 0-3) mouse spinal cord; it is activated at subthreshold potentials, inactivates slowly, and can be blocked by low concentrations of riluzole. The role of I(Na(P)) in locomotor pattern generation was examined by applying riluzole during fictive locomotion induced by NMDA, serotonin, and dopamine or by stimulation of the cauda equina. Blockade of I(Na(P)) has marginal effects on the locomotion frequency but progressively weakens the rhythmic firing and locomotor-related membrane oscillation of CINs and motoneurons (MNs) and the locomotor-like bursts in ventral roots, until the motor pattern ceases. Riluzole directly affects the intrinsic firing properties of CINs and MNs, reducing their ability to fire repetitively during tonic depolarizations and raising their spike threshold. At the same time, riluzole has little effects on the strength of spike-evoked synaptic transmission onto CINs and MNs. Our results suggest that I(Na(P)) is essential for the generation of the locomotor pattern and acts in part by regulating the frequency of interneuron firing in the central pattern generator for locomotion.