Development of Motor Networks in Zebrafish Embryos

General mechanisms of motor network development have often been examined in the spinal cord because of its relative simplicity when compared to higher parts of the brain. Indeed, most of our current understanding of motor pattern generation comes from work in the lower vertebrate spinal cord. Nevertheless, very little is known about the initial stages of motor network formation and the interplay between genes and electrical activity. Recent research has led to the establishment of the zebrafish as a key model system to study the genetics of neural development. The spinal cord of zebrafish is amenable to optical and electrophysiological analysis of neuronal activity even at the earliest embryonic stages when the network is immature. The combination of physiology and genetics in the same animal model should lead to insights into the basic mechanisms of motor circuit formation. This paper reviews recent work on the development of zebrafish motor activity and discusses them in the context of the current knowledge of embryonic and larval zebrafish spinal cord morphology and physiology.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63184/1/zeb.2006.3.173.pd

Whole-cell patch-clamp recordings from identified spinal neurons in the zebrafish embryo

We describe a preparation for obtaining patch-clamp recordings from identified embryonic spinal cord interneurons, motoneurons and sensory neurons in an in vivo zebrafish preparation. This preparation is used to study the spatial and temporal patterns of spontaneous and touch-evoked electrical activity during the initial development of circuitry in the spinal cord. The combination of these physiological techniques with the powerful genetic and molecular tools available in the zebrafish has the potential to increase our understanding of the complex interactions between genes and electrical activity during the development of the vertebrate nervous system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43239/1/11022_2004_Article_5149581.pd

Na V 1.6a is required for normal activation of motor circuits normally excited by tactile stimulation

A screen for zebrafish motor mutants identified two noncomplementing alleles of a recessive mutation that were named non-active ( nav mi89 and nav mi130 ). nav embryos displayed diminished spontaneous and touch-evoked escape behaviors during the first 3 days of development. Genetic mapping identified the gene encoding Na V 1.6a ( scn8aa ) as a potential candidate for nav . Subsequent cloning of scn8aa from the two alleles of nav uncovered two missense mutations in Na V 1.6a that eliminated channel activity when assayed heterologously. Furthermore, the injection of RNA encoding wild-type scn8aa rescued the nav mutant phenotype indicating that scn8aa was the causative gene of nav . In-vivo electrophysiological analysis of the touch-evoked escape circuit indicated that voltage-dependent inward current was decreased in mechanosensory neurons in mutants, but they were able to fire action potentials. Furthermore, tactile stimulation of mutants activated some neurons downstream of mechanosensory neurons but failed to activate the swim locomotor circuit in accord with the behavioral response of initial escape contractions but no swimming. Thus, mutant mechanosensory neurons appeared to respond to tactile stimulation but failed to initiate swimming. Interestingly fictive swimming could be initiated pharmacologically suggesting that a swim circuit was present in mutants. These results suggested that Na V 1.6a was required for touch-induced activation of the swim locomotor network. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70:508–522, 2010Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75774/1/20791_ftp.pd

Stac3 is a component of the excitation–contraction coupling machinery and mutated in Native American myopathy

Excitation-contraction coupling, the process that regulates contractions by skeletal muscles, transduces changes in membrane voltage by activating release of Ca2+ from internal stores to initiate muscle contraction. Defects in EC coupling are associated with muscle diseases. Here we identify Stac3 as a novel component of the EC coupling machinery. Using a zebrafish genetic screen, we generate a locomotor mutation that is mapped to stac3. We provide electrophysiological, Ca2+ imaging, immunocytochemical and biochemical evidence that Stac3 participates in excitation-contraction coupling in muscles. Furthermore, we reveal that a mutation in human STAC3 as the genetic basis of the debilitating Native American myopathy (NAM). Analysis of NAM stac3 in zebrafish shows that the NAM mutation decreases excitation-contraction coupling. These findings enhance our understanding of both excitation-contraction coupling and the pathology of myopathies

Development of motor behaviors and activity patterns of spinal neurons in the zebrafish embryo

The development of spinal circuits underlying motor behaviors was examined in zebrafish. Zebrafish embryos showed three sequential, stereotyped behaviors: a transient period of spontaneous coiling contractions, followed by touch-evoked rapid coils, and swimming. Lesioning the hindbrain eliminated swimming and touch responses, but not the spontaneous contractions.The first (spontaneous) behavior was chosen for further analysis in order to characterize the underlying circuit. In vivo patch clamp recordings were obtained from identified spinal neurons. These neurons showed periodic depolarizations that triggered rhythmic bursts of action potentials with a frequency and duration that were consistent with those of the spontaneous contractions. As with the behavior, transecting the spinal cord at the hindbrain border did not affect the rhythmic activity patterns of the neurons. Surprisingly the contractions and the periodic depolarizations were insensitive to both general and specific blockade of synaptic transmission. The periodic depolarizations were suppressed by heptanol and by intracellular acidification treatments that are known to uncouple gap junctions, indicating that electrotonic synapses could underlie network synchronization during the earliest motor behavior.Paired recordings were obtained from identified spinal neurons. These showed that active ipsilateral neurons were electrically coupled in a simple network consisting initially of motoneurons and only three types of interneurons. Therefore, this early spinal circuit consists of rhythmically active and electrically coupled neurons. Furthermore, this circuit is also initially independent of the main neurotransmitter systems, sensory inputs, and descending hindbrain projections. The descending projections are required later in development for the onset of touch responses and swimming

Touch Responsiveness in Zebrafish Requires Voltage-Gated Calcium Channel 2.1b

The molecular and physiological basis of the touch-unresponsive zebrafish mutant fakir has remained elusive. Here we report that the fakir phenotype is caused by a missense mutation in the gene encoding voltage-gated calcium channel 2.1b (CACNA1Ab). Injection of RNA encoding wild-type CaV2.1 restores touch responsiveness in fakir mutants, whereas knockdown of CACNA1Ab via morpholino oligonucleotides recapitulates the fakir mutant phenotype. Fakir mutants display normal current-evoked synaptic communication at the neuromuscular junction but have attenuated touch-evoked activation of motor neurons. NMDA-evoked fictive swimming is not affected by the loss of CaV2.1b, suggesting that this channel is not required for motor pattern generation. These results, coupled with the expression of CACNA1Ab by sensory neurons, suggest that CaV2.1b channel activity is necessary for touch-evoked activation of the locomotor network in zebrafish

Touch responsiveness in zebrafish requires voltage-gated calcium channel 2.1b

The molecular and physiological basis of the touch-unresponsive zebrafish mutant fakir has remained elusive. Here we report that the fakir phenotype is caused by a missense mutation in the gene encoding voltage-gated calcium channel 2.1b (CACNA1Ab). Injection of RNA encoding wild-type CaV2.1 restores touch responsiveness in fakir mutants, whereas knockdown of CACNA1Ab via morpholino oligonucleotides recapitulates the fakir mutant phenotype. Fakir mutants display normal current-evoked synaptic communication at the neuromuscular junction but have attenuated touch-evoked activation of motor neurons. NMDA-evoked fictive swimming is not affected by the loss of CaV2.1b, suggesting that this channel is not required for motor pattern generation. These results, coupled with the expression of CACNA1Ab by sensory neurons, suggest that CaV2.1b channel activity is necessary for touch-evoked activation of the locomotor network in zebrafish

Zebrafish bandoneon mutants display behavioral defects due to a mutation in the glycine receptor β-subunit

Bilateral alternation of muscle contractions requires reciprocal inhibition between the two sides of the hindbrain and spinal cord, and disruption of this inhibition should lead to simultaneous activation of bilateral muscles. At 1 day after fertilization, wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. Similar simultaneous contractions are observed in wild-type embryos treated with strychnine, a blocker of the inhibitory glycine receptor (GlyR). This result suggests that glycinergic synaptic transmission is defective in beo mutants. Muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the CNS. Recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. Furthermore, immunostaining with an antibody against GlyR showed clusters in wild-type neurons but not in beo neurons. These data suggest that the failure of GlyRs to aggregate at synaptic sites causes impairment of glycinergic transmission and abnormal behavior in beo mutants. Indeed, mutations in the GlyR β-subunit, which are thought to be required for proper localization of GlyRs, were identified as the basis for the beo mutation. These data demonstrate that GlyRβ is essential for physiologically relevant clustering of GlyRs in vivo. Because GlyR mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, the zebrafish beo mutant should be a useful animal model for this condition