A sensation for inflation: initial swim bladder inflation in larval zebrafish is mediated by the mechanosensory lateral line.

Larval zebrafish achieve neutral buoyancy by swimming up to the surface and taking in air through their mouths to inflate their swim bladders. We define this behavior as 'surfacing'. Little is known about the sensory basis for this underappreciated behavior of larval fish. A strong candidate is the mechanosensory lateral line, a hair cell-based sensory system that detects hydrodynamic information from sources such as water currents, predators, prey and surface waves. However, a role for the lateral line in mediating initial inflation of the swim bladder has not been reported. To explore the connection between the lateral line and surfacing, we used a genetic mutant (lhfpl5b-/-) that renders the zebrafish lateral line insensitive to mechanical stimuli. We observed that approximately half of these lateral line mutants over-inflate their swim bladders during initial inflation and become positively buoyant. Thus, we hypothesized that larval zebrafish use their lateral line to moderate interactions with the air-water interface during surfacing to regulate swim bladder inflation. To test the hypothesis that lateral line defects are responsible for swim bladder over-inflation, we showed that exogenous air is required for the hyperinflation phenotype and transgenic rescue of hair cell function restores normal inflation. We also found that chemical ablation of anterior lateral line hair cells in wild-type larvae causes hyperinflation. Furthermore, we show that manipulation of lateral line sensory information results in abnormal inflation. Finally, we report spatial and temporal differences in the surfacing behavior between wild-type and lateral line mutant larvae. In summary, we propose a novel sensory basis for achieving neutral buoyancy where larval zebrafish use their lateral line to sense the air-water interface and regulate initial swim bladder inflation.

Sensory deficit screen identifies nsf mutation that differentially affects SNARE recycling and quality control.

The AAA+ NSF complex is responsible for SNARE complex disassembly both before and after membrane fusion. Loss of NSF function results in pronounced developmental and degenerative defects. In a genetic screen for sensory deficits in zebrafish, we identified a mutation in nsf, I209N, that impairs hearing and balance in a dosage-dependent manner without accompanying defects in motility, myelination, and innervation. In vitro experiments demonstrate that while the I209N NSF protein recognizes SNARE complexes, the effects on disassembly are dependent upon the type of SNARE complex and I209N concentration. Higher levels of I209N protein produce a modest decrease in binary (syntaxin-SNAP-25) SNARE complex disassembly and residual ternary (syntaxin-1A-SNAP-25-synaptobrevin-2) disassembly, whereas at lower concentrations binary disassembly activity is strongly reduced and ternary disassembly activity is absent. Our study suggests that the differential effect on disassembly of SNARE complexes leads to selective effects on NSF-mediated membrane trafficking and auditory/vestibular function.

A sensation for inflation: initial swim bladder inflation in larval zebrafish is mediated by the mechanosensory lateral line.

Larval zebrafish achieve neutral buoyancy by swimming up to the surface and taking in air through their mouths to inflate their swim bladders. We define this behavior as 'surfacing'. Little is known about the sensory basis for this underappreciated behavior of larval fish. A strong candidate is the mechanosensory lateral line, a hair cell-based sensory system that detects hydrodynamic information from sources like water currents, predators, prey, and surface waves. However, a role for the lateral line in mediating initial inflation of the swim bladder has not been reported. To explore the connection between the lateral line and surfacing, we utilized a genetic mutant ( lhfpl5b -/- ) that renders the zebrafish lateral line insensitive to mechanical stimuli. We observe that approximately half of these lateral line mutants over-inflate their swim bladders during initial inflation and become positively buoyant. Thus, we hypothesize that larval zebrafish use their lateral line to moderate interactions with the air-water interface during surfacing to regulate swim bladder inflation. To test the hypothesis that lateral line defects are responsible for swim bladder over-inflation, we show exogenous air is required for the hyperinflation phenotype and transgenic rescue of hair cell function restores normal inflation. We also find that chemical ablation of anterior lateral line hair cells in wild type larvae causes hyperinflation. Furthermore, we show that manipulation of lateral line sensory information results in abnormal inflation. Finally, we report spatial and temporal differences in the surfacing behavior between wild type and lateral line mutant larvae. In summary, we propose a novel sensory basis for achieving neutral buoyancy where larval zebrafish use their lateral line to sense the air-water interface and regulate initial swim bladder inflation.

GABAA α subunit control of hyperactive behavior in developing zebrafish.

GABAA receptors mediate rapid responses to the neurotransmitter gamma-aminobutyric acid and are robust regulators of the brain and spinal cord neural networks that control locomotor behaviors, such as walking and swimming. In developing zebrafish, gross pharmacological blockade of these receptors causes hyperactive swimming, which is also a feature of many zebrafish epilepsy models. Although GABAA receptors are important to control locomotor behavior, the large number of subunits and homeostatic compensatory mechanisms have challenged efforts to determine subunit-selective roles. To address this issue, we mutated each of the 8 zebrafish GABAA α subunit genes individually and in pairs using a CRISPR-Cas9 somatic inactivation approach and, then, we examined the swimming behavior of the mutants at 2 developmental stages, 48 and 96 h postfertilization. We found that disrupting the expression of specific pairs of subunits resulted in different abnormalities in swimming behavior at 48 h postfertilization. Mutation of α4 and α5 selectively resulted in longer duration swimming episodes, mutations in α3 and α4 selectively caused excess, large-amplitude body flexions (C-bends), and mutation of α3 and α5 resulted in increases in both of these measures of hyperactivity. At 96 h postfertilization, hyperactive phenotypes were nearly absent, suggesting that homeostatic compensation was able to overcome the disruption of even multiple subunits. Taken together, our results identify subunit-selective roles for GABAA α3, α4, and α5 in regulating locomotion. Given that these subunits exhibit spatially restricted expression patterns, these results provide a foundation to identify neurons and GABAergic networks that control discrete aspects of locomotor behavior.

Recording Channelrhodopsin-Evoked Field Potentials and Startle Responses from Larval Zebrafish.

Zebrafish are an excellent model organism to study many aspects of vertebrate sensory encoding and behavior. Their escape responses begin with a C-shaped body bend followed by several swimming bouts away from the potentially threatening stimulus. This highly stereotyped motor behavior provides a model for studying startle reflexes and the neural circuitry underlying multisensory encoding and locomotion. Channelrhodopsin (ChR2) can be expressed in the lateral line and ear hair cells of zebrafish and can be excited in vivo to elicit these rapid forms of escape. Here we review our methods for studying transgenic ChR2-expressing zebrafish larvae, including screening for positive expression of ChR2 and recording field potentials and high-speed videos of optically evoked escape responses. We also highlight important features of the acquired data and provide a brief review of other zebrafish research that utilizes or has the potential to benefit from ChR2 and optogenetics.

Mathematical Modeling and Analyses of Interspike-Intervals of Spontaneous Activity in Afferent Neurons of the Zebrafish Lateral Line.

Without stimuli, hair cells spontaneously release neurotransmitter leading to spontaneous generation of action potentials (spikes) in innervating afferent neurons. We analyzed spontaneous spike patterns recorded from the lateral line of zebrafish and found that distributions of interspike intervals (ISIs) either have an exponential shape or an "L" shape that is characterized by a sharp decay but wide tail. ISI data were fitted to renewal-process models that accounted for the neuron refractory periods and hair-cell synaptic release. Modeling the timing of synaptic release using a mixture of two exponential distributions yielded the best fit for our ISI data. Additionally, lateral line ISIs displayed positive serial correlation and appeared to exhibit switching between faster and slower modes of spike generation. This pattern contrasts with previous findings from the auditory system where ISIs tended to have negative serial correlation due to synaptic depletion. We propose that afferent neuron innervation with multiple and heterogenous hair-cells synapses, each influenced by changes in calcium domains, can serve as a mechanism for the random switching behavior. Overall, our analyses provide evidence of how physiological similarities and differences between synapses and innervation patterns in the auditory, vestibular, and lateral line systems can lead to variations in spontaneous activity.

Teaching Dose-response Relationships Through Aminoglycoside Block of Mechanotransduction Channels in Lateral Line Hair Cells of Larval Zebrafish.

Here we introduce a novel set of laboratory exercises for teaching about hair cell structure and function and dose-response relationships via fluorescence microscopy. Through fluorescent labeling of lateral line hair cells, students assay aminoglycoside block of mechanoelectrical transduction (MET) channels in larval zebrafish. Students acquire and quantify images of hair cells fluorescently labeled with FM 1-43, which enters the hair cell through MET channels. Blocking FM 1-43 uptake with different concentrations of dihydrostreptomycin (DHS) results in dose-dependent reduction in hair-cell fluorescence. This method allows students to generate dose-response curves for the percent fluorescence reduction at different concentrations of DHS, which are then visualized to examine the blocking behavior of DHS using the Hill equation. Finally, students present their findings in lab reports structured as scientific papers. Together these laboratory exercises give students the opportunity to learn about hair cell mechanotransduction, pharmacological block of ion channels, and dose-dependent relationships including the Hill equation, while also exposing students to the zebrafish model organism, fluorescent labeling and microscopy, acquisition and analysis of images, and the presentation of experimental findings. These simple yet comprehensive techniques are appropriate for an undergraduate biology or neuroscience classroom laboratory.

Enlargement of Ribbons in Zebrafish Hair Cells Increases Calcium Currents But Disrupts Afferent Spontaneous Activity and Timing of Stimulus Onset.

In sensory hair cells of auditory and vestibular organs, the ribbon synapse is required for the precise encoding of a wide range of complex stimuli. Hair cells have a unique presynaptic structure, the synaptic ribbon, which organizes both synaptic vesicles and calcium channels at the active zone. Previous work has shown that hair-cell ribbon size is correlated with differences in postsynaptic activity. However, additional variability in postsynapse size presents a challenge to determining the specific role of ribbon size in sensory encoding. To selectively assess the impact of ribbon size on synapse function, we examined hair cells in transgenic zebrafish that have enlarged ribbons, without postsynaptic alterations. Morphologically, we found that enlarged ribbons had more associated vesicles and reduced presynaptic calcium-channel clustering. Functionally, hair cells with enlarged ribbons had larger global and ribbon-localized calcium currents. Afferent neuron recordings revealed that hair cells with enlarged ribbons resulted in reduced spontaneous spike rates. Additionally, despite larger presynaptic calcium signals, we observed fewer evoked spikes with longer latencies from stimulus onset. Together, our work indicates that hair-cell ribbon size influences the spontaneous spiking and the precise encoding of stimulus onset in afferent neurons.SIGNIFICANCE STATEMENT Numerous studies support that hair-cell ribbon size corresponds with functional sensitivity differences in afferent neurons and, in the case of inner hair cells of the cochlea, vulnerability to damage from noise trauma. Yet it is unclear whether ribbon size directly influences sensory encoding. Our study reveals that ribbon enlargement results in increased ribbon-localized calcium signals, yet reduces afferent spontaneous activity and disrupts the timing of stimulus onset, a distinct aspect of auditory and vestibular encoding. These observations suggest that varying ribbon size alone can influence sensory encoding, and give further insight into how hair cells transduce signals that cover a wide dynamic range of stimuli.

Intensity-dependent timing and precision of startle response latency in larval zebrafish.

KEY POINTS: Using high-speed videos time-locked with whole-animal electrical recordings, simultaneous measurement of behavioural kinematics and field potential parameters of C-start startle responses allowed for discrimination between short-latency and long-latency C-starts (SLCs vs. LLCs) in larval zebrafish. Apart from their latencies, SLC kinematics and SLC field potential parameters were intensity independent. Increasing stimulus intensity increased the probability of evoking an SLC and decreased mean SLC latencies while increasing their precision; subtraction of field potential latencies from SLC latencies revealed a fixed time delay between the two measurements that was intensity independent. The latency and the precision in the latency of the SLC field potentials were linearly correlated to the latencies and precision of the first evoked action potentials (spikes) in hair-cell afferent neurons of the lateral line. Together, these findings indicate that first spike latency (FSL) is a fast encoding mechanism that can serve to precisely initiate startle responses when speed is critical for survival. ABSTRACT: Vertebrates rely on fast sensory encoding for rapid and precise initiation of startle responses. In afferent sensory neurons, trains of action potentials (spikes) encode stimulus intensity within the onset time of the first evoked spike (first spike latency; FSL) and the number of evoked spikes. For speed of initiation of startle responses, FSL would be the more advantageous mechanism to encode the intensity of a threat. However, the intensity dependence of FSL and spike number and whether either determines the precision of startle response initiation is not known. Here, we examined short-latency startle responses (SLCs) in larval zebrafish and tested the hypothesis that first spike latencies and their precision (jitter) determine the onset time and precision of SLCs. We evoked startle responses via activation of Channelrhodopsin (ChR2) expressed in ear and lateral line hair cells and acquired high-speed videos of head-fixed larvae while simultaneously recording underlying field potentials. This method allowed for discrimination between primary SLCs and less frequent, long-latency startle responses (LLCs). Quantification of SLC kinematics and field potential parameters revealed that, apart from their latencies, they were intensity independent. We found that increasing stimulus intensity decreased SLC latencies while increasing their precision, which was significantly correlated with corresponding changes in field potential latencies and their precision. Single afferent neuron recordings from the lateral line revealed a similar intensity-dependent decrease in first spike latencies and their jitter, which could account for the intensity-dependent changes in timing and precision of startle response latencies.

Natural Bizbenzoquinoline Derivatives Protect Zebrafish Lateral Line Sensory Hair Cells from Aminoglycoside Toxicity.

Moderate to severe hearing loss affects 360 million people worldwide and most often results from damage to sensory hair cells. Hair cell damage can result from aging, genetic mutations, excess noise exposure, and certain medications including aminoglycoside antibiotics. Aminoglycosides are effective at treating infections associated with cystic fibrosis and other life-threatening conditions such as sepsis, but cause hearing loss in 20-30% of patients. It is therefore imperative to develop new therapies to combat hearing loss and allow safe use of these potent antibiotics. We approach this drug discovery question using the larval zebrafish lateral line because zebrafish hair cells are structurally and functionally similar to mammalian inner ear hair cells and respond similarly to toxins. We screened a library of 502 natural compounds in order to identify novel hair cell protectants. Our screen identified four bisbenzylisoquinoline derivatives: berbamine, E6 berbamine, hernandezine, and isotetrandrine, each of which robustly protected hair cells from aminoglycoside-induced damage. Using fluorescence microscopy and electrophysiology, we demonstrated that the natural compounds confer protection by reducing antibiotic uptake into hair cells and showed that hair cells remain functional during and after incubation in E6 berbamine. We also determined that these natural compounds do not reduce antibiotic efficacy. Together, these natural compounds represent a novel source of possible otoprotective drugs that may offer therapeutic options for patients receiving aminoglycoside treatment.

Dopamine Modulates the Activity of Sensory Hair Cells.

The senses of hearing and balance are subject to modulation by efferent signaling, including the release of dopamine (DA). How DA influences the activity of the auditory and vestibular systems and its site of action are not well understood. Here we show that dopaminergic efferent fibers innervate the acousticolateralis epithelium of the zebrafish during development but do not directly form synapses with hair cells. However, a member of the D1-like receptor family, D1b, tightly localizes to ribbon synapses in inner ear and lateral-line hair cells. To assess modulation of hair-cell activity, we reversibly activated or inhibited D1-like receptors (D1Rs) in lateral-line hair cells. In extracellular recordings from hair cells, we observed that D1R agonist SKF-38393 increased microphonic potentials, whereas D1R antagonist SCH-23390 decreased microphonic potentials. Using ratiometric calcium imaging, we found that increased D1R activity resulted in larger calcium transients in hair cells. The increase of intracellular calcium requires Cav1.3a channels, as a Cav1 calcium channel antagonist, isradipine, blocked the increase in calcium transients elicited by the agonist SKF-38393. Collectively, our results suggest that DA is released in a paracrine fashion and acts at ribbon synapses, likely enhancing the activity of presynaptic Cav1.3a channels and thereby increasing neurotransmission. SIGNIFICANCE STATEMENT: The neurotransmitter dopamine acts in a paracrine fashion (diffusion over a short distance) in several tissues and bodily organs, influencing and regulating their activity. The cellular target and mechanism of the action of dopamine in mechanosensory organs, such as the inner ear and lateral-line organ, is not clearly understood. Here we demonstrate that dopamine receptors are present in sensory hair cells at synaptic sites that are required for signaling to the brain. When nearby neurons release dopamine, activation of the dopamine receptors increases the activity of these mechanosensitive cells. The mechanism of dopamine activation requires voltage-gated calcium channels that are also present at hair-cell synapses.

Converging axons collectively initiate and maintain synaptic selectivity in a constantly remodeling sensory organ.

Sensory receptors are the functional link between the environment and the brain. The repair of sensory organs enables animals to continuously detect environmental stimuli. However, receptor cell turnover can affect sensory acuity by changing neural connectivity patterns. In zebrafish, two to four postsynaptic lateralis afferent axons converge into individual peripheral mechanosensory organs called neuromasts, which contain hair cell receptors of opposing planar polarity. Yet, each axon exclusively synapses with hair cells of identical polarity during development and regeneration to transmit unidirectional mechanical signals to the brain. The mechanism that governs this exceptionally accurate and resilient synaptic selectivity remains unknown. We show here that converging axons are mutually dependent for polarity-selective connectivity. If rendered solitary, these axons establish simultaneous functional synapses with hair cells of opposing polarities to transmit bidirectional mechanical signals. Remarkably, nonselectivity by solitary axons can be corrected upon the reintroduction of additional axons. Collectively, our results suggest that lateralis synaptogenesis is intrinsically nonselective and that interaxonal interactions continuously rectify mismatched synapses. This dynamic organization of neural connectivity may represent a general solution to maintain coherent synaptic transmission from sensory organs undergoing frequent variations in the number and spatial distribution of receptor cells.

Optical stimulation of zebrafish hair cells expressing channelrhodopsin-2.

Vertebrate hair cells are responsible for the high fidelity encoding of mechanical stimuli into trains of action potentials (spikes) in afferent neurons. Here, we generated a transgenic zebrafish line expressing Channelrhodopsin-2 (ChR2) under the control of the hair-cell specific myo6b promoter, in order to examine the role of the mechanoelectrical transduction (MET) channel in sensory encoding in afferent neurons. We performed in vivo recordings from afferent neurons of the zebrafish lateral line while activating hair cells with either mechanical stimuli from a waterjet or optical stimuli from flashes of ∼470-nm light. Comparison of the patterns of encoded spikes during 100-ms stimuli revealed no difference in mean first spike latency between the two modes of activation. However, there was a significant increase in the variability of first spike latency during optical stimulation as well as an increase in the mean number of spikes per stimulus. Next, we compared encoding of spikes during hair-cell stimulation at 10, 20, and 40-Hz. Consistent with the increased variability of first spike latency, we saw a significant decrease in the vector strength of phase-locked spiking during optical stimulation. These in vivo results support a physiological role for the MET channel in the high fidelity of first spike latency seen during encoding of mechanical sensory stimuli. Finally, we examined whether remote activation of hair cells via ChR2 activation was sufficient to elicit escape responses in free-swimming larvae. In transgenic larvae, 100-ms flashes of ∼470-nm light resulted in escape responses that occurred concomitantly with field recordings indicating Mauthner cell activity. Altogether, the myo6b:ChR2 transgenic line provides a platform to investigate hair-cell function and sensory encoding, hair-cell sensory input to the Mauthner cell, and the ability to remotely evoke behavior in free-swimming zebrafish.

Quantification of exocytosis kinetics by DIC image analysis of cortical lawns.

Cortical lawns prepared from sea urchin eggs have offered a robust in vitro system for study of regulated exocytosis and membrane fusion events since their introduction by Vacquier almost 40 years ago (Vacquier in Dev Biol 43:62-74, 1975). Lawns have been imaged by phase contrast, darkfield, differential interference contrast, and electron microscopy. Quantification of exocytosis kinetics has been achieved primarily by light scattering assays. We present simple differential interference contrast image analysis procedures for quantifying the kinetics and extent of exocytosis in cortical lawns using an open vessel that allows rapid solvent equilibration and modification. These preparations maintain the architecture of the original cortices, allow for cytological and immunocytochemical analyses, and permit quantification of variation within and between lawns. When combined, these methods can shed light on factors controlling the rate of secretion in a spatially relevant cellular context. We additionally provide a subroutine for IGOR Pro® that converts raw data from line scans of cortical lawns into kinetic profiles of exocytosis. Rapid image acquisition reveals spatial variations in time of initiation of individual granule fusion events with the plasma membrane not previously reported.

Recording field potentials from zebrafish larvae during escape responses.

Among vertebrates, startle responses are a ubiquitous method for alerting, and avoiding or escaping from alarming or dangerous stimuli. In zebrafish larvae, fast escape behavior is easily evoked through either acoustic or tactile stimuli. For example, a light touch to the head will excite trigeminal neurons that in turn excite a large reticulospinal neuron in the hindbrain called the Mauthner cell (M-cell). The M-cell action potential then travels down the contralateral trunk of the larva exciting motoneurons, which subsequently excite the entire axial musculature, producing a large amplitude body bend away from the source of the stimulus. This body conformation is known as the "C-bend" due to the shape of the larva during the behavior. As a result of the semi-synchronized activation of the M-cell, the population of motor neurons, and the axial trunk muscles, a large field potential is generated and can be recorded from free-swimming or fixed-position larvae. Undergraduate laboratories that record field potentials during escape responses in larval zebrafish are relatively simple to setup and allow students to observe and study the escape reflex circuit. Furthermore, by testing hypotheses, analyzing data and writing journal-style laboratory reports, students have multiple opportunities to learn about many neuroscience topics including vertebrate reflexes; sensory transduction; synaptic-, neuro-, and muscle-physiology; the M-cell mediated escape response; and the zebrafish as a model organism. Here, we detail the equipment, software, and recording setup necessary to observe field potentials in an undergraduate teaching lab. Additionally, we discuss potential advanced laboratory exercises and pedagogical outcomes. Finally, we note possible low-cost alternatives for recording field potentials.

Tools, methods, and applications for optophysiology in neuroscience.

The advent of optogenetics and genetically encoded photosensors has provided neuroscience researchers with a wealth of new tools and methods for examining and manipulating neuronal function in vivo. There exists now a wide range of experimentally validated protein tools capable of modifying cellular function, including light-gated ion channels, recombinant light-gated G protein-coupled receptors, and even neurotransmitter receptors modified with tethered photo-switchable ligands. A large number of genetically encoded protein sensors have also been developed to optically track cellular activity in real time, including membrane-voltage-sensitive fluorophores and fluorescent calcium and pH indicators. The development of techniques for controlled expression of these proteins has also increased their utility by allowing the study of specific populations of cells. Additionally, recent advances in optics technology have enabled both activation and observation of target proteins with high spatiotemporal fidelity. In combination, these methods have great potential in the study of neural circuits and networks, behavior, animal models of disease, as well as in high-throughput ex vivo studies. This review collects some of these new tools and methods and surveys several current and future applications of the evolving field of optophysiology.

Rabconnectin3α promotes stable activity of the H+ pump on synaptic vesicles in hair cells.

Acidification of synaptic vesicles relies on the vacuolar-type ATPase (V-ATPase) and provides the electrochemical driving force for neurotransmitter exchange. The regulatory mechanisms that ensure assembly of the V-ATPase holoenzyme on synaptic vesicles are unknown. Rabconnectin3α (Rbc3α) is a potential candidate for regulation of V-ATPase activity because of its association with synaptic vesicles and its requirement for acidification of intracellular compartments. Here, we provide the first evidence for a role of Rbc3α in synaptic vesicle acidification and neurotransmission. In this study, we characterized mutant alleles of rbc3α isolated from a large-scale screen for zebrafish with auditory/vestibular defects. We show that Rbc3α is localized to basal regions of hair cells in which synaptic vesicles are present. To determine whether Rbc3α regulates V-ATPase activity, we examined the acidification of synaptic vesicles and localization of the V-ATPase in hair cells. In contrast to wild-type hair cells, we observed that synaptic vesicles had elevated pH, and a cytosolic subunit of the V-ATPase was no longer enriched in synaptic regions of mutant hair cells. As a consequence of defective acidification of synaptic vesicles, afferent neurons in rbc3α mutants had reduced firing rates and reduced accuracy of phase-locked action potentials in response to mechanical stimulation of hair cells. Collectively, our data suggest that Rbc3α modulates synaptic transmission in hair cells by promoting V-ATPase activity in synaptic vesicles.

Mechanism of spontaneous activity in afferent neurons of the zebrafish lateral-line organ.

Many auditory, vestibular, and lateral-line afferent neurons display spontaneous action potentials. This spontaneous spiking is thought to result from hair-cell glutamate release in the absence of stimuli. Spontaneous release at hair-cell resting potentials presumably results from Ca(V)1.3 L-type calcium channel activity. Here, using intact zebrafish larvae, we recorded robust spontaneous spiking from lateral-line afferent neurons in the absence of external stimuli. Consistent with the above assumptions, spiking was absent in mutants that lacked either Vesicular glutamate transporter 3 (Vglut3) or Ca(V)1.3. We then tested the hypothesis that spontaneous spiking resulted from sustained Ca(V)1.3 activity due to depolarizing currents that are active at rest. Mechanotransduction currents (I(MET)) provide a depolarizing influence to the resting potential. However, following block of I(MET), spontaneous spiking persisted and was characterized by longer interspike intervals and increased periods of inactivity. These results suggest that an additional depolarizing influence maintains the resting potential within the activation range of Ca(V)1.3. To test whether the hyperpolarization-activated cation current, I(h) participates in setting the resting potential, we applied I(h) antagonists. Both ZD7288 and DK-AH 269 reduced spontaneous activity. Finally, concomitant block of I(MET) and I(h) essentially abolished spontaneous activity, ostensibly by hyperpolarization outside of the activation range for Ca(V)1.3. Together, our data support a mechanism for spontaneous spiking that results from Ca(2+)-dependent neurotransmitter release at hair-cell resting potentials that are maintained within the activation range of Ca(V)1.3 channels through active I(MET) and I(h).

Ribeye is required for presynaptic Ca(V)1.3a channel localization and afferent innervation of sensory hair cells.

Ribbon synapses of the ear, eye and pineal gland contain a unique protein component: Ribeye. Ribeye consists of a novel aggregation domain spliced to the transcription factor CtBP2 and is one of the most abundant proteins in synaptic ribbon bodies. Although the importance of Ribeye for the function and physical integrity of ribbon synapses has been shown, a specific role in synaptogenesis has not been described. Here, we have modulated Ribeye expression in zebrafish hair cells and have examined the role of Ribeye in synapse development. Knockdown of ribeye resulted in fewer stimulus-evoked action potentials from afferent neurons and loss of presynaptic Ca(V)1.3a calcium channel clusters in hair cells. Additionally, afferent innervation of hair cells was reduced in ribeye morphants, and the reduction was correlated with depletion of Ribeye punctae. By contrast, transgenic overexpression of Ribeye resulted in Ca(V)1.3a channels colocalized with ectopic aggregates of Ribeye protein. Overexpression of Ribeye, however, was not sufficient to create ectopic synapses. These findings reveal two distinct functions of Ribeye in ribbon synapse formation--clustering Ca(V)1.3a channels at the presynapse and stabilizing contacts with afferent neurons--and suggest that Ribeye plays an organizing role in synaptogenesis.

Physiological recordings from zebrafish lateral-line hair cells and afferent neurons.

Sensory signal transduction, the process by which the features of external stimuli are encoded into action potentials, is a complex process that is not fully understood. In fish and amphibia, the lateral-line organ detects water movement and vibration and is critical for schooling behavior and the detection of predators and prey. The lateral-line system in zebrafish serves as an ideal platform to examine encoding of stimuli by sensory hair cells. Here, we describe methods for recording hair-cell microphonics and activity of afferent neurons using intact zebrafish larvae. The recordings are performed by immobilizing and mounting larvae for optimal stimulation of lateral-line hair cells. Hair cells are stimulated with a pressure-controlled water jet and a recording electrode is positioned next to the site of mechanotransduction in order to record microphonics--extracellular voltage changes due to currents through hair-cell mechanotransduction channels. Another readout of the hair-cell activity is obtained by recording action currents from single afferent neurons in response to water-jet stimulation of innervated hair cells. When combined, these techniques make it possible to probe the function of the lateral-line sensory system in an intact zebrafish using controlled, repeatable, physiological stimuli.