[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 01/2014; 9(10):e109117. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Frontiers in Neural Circuits 01/2013; 7:109. · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Neuroscience 12/2012; 32(49):17646-17657. · 6.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Neuroscience 09/2012; 32(39):13488-500. · 6.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Neurophysiology 05/2012; 108(3):925-34. · 3.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 01/2012; 7(10):e47940. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Neurophysiology 01/2012; 107(1):103-13. · 3.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Disease Models and Mechanisms 11/2011; 5(2):248-58. · 4.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Neurophysiology 09/2008; 100(4):1716-23. · 3.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Neuroscience 05/2007; 27(17):4507-18. · 6.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Larval zebrafish provide a unique model for investigating the mechanisms involved in generating rhythmic patterns of behavior, such as swimming, due to the array of techniques available including genetics, optical imaging, and conventional electrophysiology. Because electrophysiological and imaging studies of rhythmic motor behaviors in paralyzed preparations depend on the ability to monitor the central motor pattern, we developed a fictive preparation in which the activity of axial motor neurons was monitored using extracellular recordings from peripheral nerves. We examined spontaneous and light induced fictive motor patterns in wild type and mutant larval zebrafish (4-6 days post-fertilization) paralyzed with curare. All spontaneous and light-induced preparations produced alternation of motor activity from side-to-side (mean contralateral phase = 50.7 +/- 7.0%; mean burst frequency = 35.6 +/- 4.7 Hz) and a progression of activity from head-to-tail (mean ipsilateral rostrocaudal delay = 0.8 +/- 0.5 ms per segment), consistent with lateral undulation and forward propulsion during swimming, respectively. The basic properties of the motor pattern were similar in spontaneous and light-induced swimming. This fictive preparation can be used in combination with conventional electrophysiological and imaging methods to investigate normal circuit function as well as to elucidate functional deficits in mutant lines. Toward this end, we show that two accordion class mutants, accordion and bandoneon, have alternating activity on opposite sides of the body, contradicting the hypothesis that their deficit results from the absence of the reciprocal glycinergic inhibition that is typically found in the spinal cord of swimming vertebrates.
Journal of Neurophysiology 07/2005; 93(6):3177-88. · 3.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Studies in chicks and mice have suggested that transcription factors mark functional subtypes of interneurons in the developing spinal cord. We used genetic, morphological, and physiological studies to test this proposed association in zebrafish. We found that Engrailed-1 expression uniquely marks a class of ascending interneurons, called circumferential ascending (CiA) interneurons, with ipsilateral axonal projections in both motor and sensory regions of spinal cord. These cells express the glycine transporter 2 gene and are the only known ipsilateral interneurons positive for this marker of inhibitory transmission. Patch recordings show that the CiA neurons are rhythmically active during swimming. Pairwise recordings from the CiA interneurons and postsynaptic cells reveal that the Engrailed-1 neurons produce monosynaptic, strychnine-sensitive inhibition of dorsal sensory interneurons and also inhibit more ventral neurons, including motoneurons and descending interneurons. We conclude that Engrailed-1 expression marks a class of inhibitory interneuron that seems to provide all of the ipsilateral glycinergic inhibition in the spinal cord of embryonic and larval fish. Individual Engrailed-1-positive cells are multifunctional, playing roles in both sensory gating and motor pattern generation. This primitive cell type may have given rise to several, more specialized glycinergic inhibitory interneurons in birds and mammals. Our data support the view that the subdivision of spinal cord into different regions by transcription factors defines a primitive functional organization of spinal interneurons that formed a developmental and evolutionary foundation on which more complex systems were built.
Journal of Neuroscience 07/2004; 24(25):5827-39. · 6.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genetically encoded calcium indicators, such as cameleon, have offered the promise of noninvasively monitoring activity of neurons, but no one has demonstrated whether these indicators can report calcium transients in neurons of behaving vertebrates. We show that cameleon can be expressed at high levels in sensory and spinal cord neurons in zebrafish by using neural-specific promoters in both transient expression experiments and in a stable transgenic line. Using standard confocal microscopy, calcium transients in identified motoneurons and spinal interneurons could be detected during escape behaviors produced by a touch on the head of the fish. Small movements of the restrained fish during the behavior did not represent a major problem for analyzing the calcium responses because of the ratiometric nature of cameleon. We conclude that cameleon can be used to noninvasively study the activity of neurons in an intact, behaving vertebrate. The ability to introduce an indicator genetically allows for studies of the functional roles of local interneurons that cannot easily be monitored with other approaches. Transgenic lines such as the one we generated can also be crossed into mutant lines of fish to study both structural and functional consequences of the mutations.
Journal of Neurophysiology 01/2004; 90(6):3986-97. · 3.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 10 11 The classic 'size principle' of motor control describes how increasingly forceful 12 movements arise by the recruitment of motoneurons of progressively larger size and force 13 output into the active pool. Here, we explore the activity of pools of spinal interneurons in 14 larval zebrafish and find that increases in swimming speed are not associated with the 15 simple addition of cells to the active pool. Instead, the recruitment of interneurons at 16 faster speeds is accompanied by the silencing of those driving movements at slower 17 speeds. This silencing occurs both between and within classes of rhythmically-active 18