Projects in my section aim at clarifying the mechanisms of synapse formation and function, with an emphasis on the nicotinic synapses. Nicotinic receptors are found widely in the nervous system, and play important roles in various aspects of behavior and pathological conditions. Specifically, their involvement has been strongly implicated in alcoholism. We use the nervous system of zebrafish as the experimental model. In this fiscal year, two papers resulting from a genetic screening conducted in my section and two papers examining the physiology of nervous system were published. A manuscript entitled Identification and modulation of voltage-gated Ca2+ current in zebrafish Rohon-Beard neurons was published in the Journal of Neurophysiology. The abstract is as follows. Electrically excitable cells have voltage-dependent ion channels on the plasma membrane that regulate membrane permeability to specific ions. Voltage-gated Ca2+ channels (VGCCs) are especially important as Ca2+ serves as both a charge carrier and second messenger. Zebrafish (Danio rerio) are an important model vertebrate for studies of neuronal excitability, circuits, and behavior. However, electrophysiological properties of zebrafish VGCCs remain largely unexplored because a suitable preparation for whole cell voltage-clamp studies is lacking. Rohon-Beard (R-B) sensory neurons represent an attractive candidate for this purpose because of their relatively large somata and functional homology to mammalian dorsal root ganglia (DRG) neurons. Transgenic zebrafish expressing green fluorescent protein in R-B neurons, (Isl2b:EGFP)ZC7, were used to identify dissociated neurons suitable for whole cell patch-clamp experiments. Based on biophysical and pharmacological properties, zebrafish R-B neurons express both high- and low-voltage-gated Ca2+ current (HVA- and LVA-ICa, respectively). Ni+-sensitive LVA-ICa occur in the minority of R-B neurons (30%) and ω-conotoxin GVIA-sensitive CaV2.2 (N-type) Ca2+ channels underlie the vast majority (90%) of HVA-ICa. To identify G protein coupled receptors (GPCRs) that modulate HVA-ICa, a panel of neurotransmitters was screened. Application of GABA/baclofen or serotonin produced a voltage-dependent inhibition while application of the mu-opioid agonist DAMGO resulted in a voltage-independent inhibition. Unlike in mammalian neurons, GPCR-mediated voltage-dependent modulation of ICa appears to be transduced primarily via a cholera toxin-sensitive Gαsubunit. These results provide the basis for using the zebrafish model system to understanding Ca2+ channel function, and in turn, how Ca2+ channels contribute to mechanosensory function. A manuscript entitled Identification of adult renal progenitor cells capable of nephron formation and regeneration in zebrafish was published in Nature. The abstact is as follows. Loss of kidney function underlies many renal diseases. Mammals can partly repair their nephrons (the functional units of the kidney), but cannot form new ones. By contrast, fish add nephrons throughout their lifespan and regenerate nephrons de novo after injury, providing a model for understanding how mammalian renal regeneration may be therapeutically activated. Here we trace the source of new nephrons in the adult zebrafish to small cellular aggregates containing nephron progenitors. Transplantation of single aggregates comprising 10-30 cells is sufficient to engraft adults and generate multiple nephrons. Serial transplantation experiments to test self-renewal revealed that nephron progenitors are long-lived and possess significant replicative potential, consistent with stem-cell activity. Transplantation of mixed nephron progenitors tagged with either green or red fluorescent proteins yielded some mosaic nephrons, indicating that multiple nephron progenitors contribute to a single nephron. Consistent with this, live imaging of nephron formation in transparent larvae showed that nephrogenic aggregates form by the coalescence of multiple cells and then differentiate into nephrons. Taken together, these data demonstrate that the zebrafish kidney probably contains self-renewing nephron stem/progenitor cells. The identification of these cells paves the way to isolating or engineering the equivalent cells in mammals and developing novel renal regenerative therapies. A manuscript entitled Formation of the spinal network in zebrafish determined by domain-specific Pax genes was published in the Journal of Comparative Neurology. The abstract is as follows. In the formation of the spinal network, various transcription factors interact to develop specific cell types. By using a gene trap technique, we established a stable line of zebrafish in which the red fluorescent protein (RFP) was inserted into the pax8 gene. RFP insertion marked putative pax8-lineage cells with fluorescence and inhibited pax8 expression in homozygous embryos. Pax8 homozygous embryos displayed defects in the otic vesicle, as previously reported in studies with morpholinos. The pax8 homozygous embryos survived to adulthood, in contrast to mammalian counterparts that die prematurely. RFP is expressed in the dorsal spinal cord. Examination of the axon morphology revealed that RFP+ neurons include commissural bifurcating longitudinal (CoBL) interneurons, but other inhibitory neurons such as commissural local (CoLo) interneurons and circumferential ascending (CiA) interneurons do not express RFP. We examined the effect of inhibiting pax2a/pax8 expression on interneuron development. In pax8 homozygous fish, the RFP+ cells underwent differentiation similar to that of pax8 heterozygous fish, and the swimming behavior remained intact. In contrast, the RFP+ cells of pax2a/pax8 double mutants displayed altered cell fates. CoBLs were not observed. Instead, RFP+ cells exhibited axons descending ipsilaterally, a morphology resembling that of V2a/V2b interneurons. A manuscript entitled An acetylcholine receptor lacking both gamma and epsilon subunits mediates transmission in zebrafish slow muscle synapses was published in the Journal of General Physiology. The abstact is as follows. Fast and slow skeletal muscle types in larval zebrafish can be distinguished by a fivefold difference in the time course of their synaptic decay. Single-channel recordings indicate that this difference is conferred through kinetically distinct nicotinic acetylcholine receptor (AChR) isoforms. The underlying basis for this distinction was explored by cloning zebrafish muscle AChR subunit cDNAs and expressing them in Xenopus laevis oocytes. Measurements of single-channel conductance and mean open burst duration assigned alpha2;beta;delta;epsilon to fast muscle synaptic current. Contrary to expectations, receptors composed of only alpha;beta;delta subunits (presumed to be alpha2;beta;delta2 receptors) recapitulated the kinetics and conductance of slow muscle single-channel currents. Additional evidence in support of gamma/epsilon-less receptors as mediators of slow muscle synapses was reflected in the inward current rectification of heterologously expressed alpha2;beta;delta2 receptors, a property normally associated with neuronal-type nicotinic receptors. Similar rectification was reflected in both single-channel and synaptic currents in slow muscle, distinguishing them from fast muscle. The final evidence for alpha2;beta;delta2 receptors in slow muscle was provided by our ability to convert fast muscle synaptic currents to those of slow muscle by knocking down epsilon subunit expression in vivo. Thus, for the first time, muscle synaptic function can be ascribed to a receptor isoform that is composed of only three different subunits. The unique functional features offered by the alpha2;beta;delta2 receptor likely play a central role in mediating the persistent contractions characteristic to this muscle type.
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