Teresa Nicolson, PhD

All Publications

• Differential expression of mechanotransduction complex genes in auditory/vestibular hair cells in zebrafish. Frontiers in molecular neuroscience Smith, E. T., Sun, P., Yu, S. K., Raible, D. W., Nicolson, T. 2023; 16: 1274822

  Abstract

  Ciliated sensory cells such as photo- and olfactory receptors employ multiple types of opsins or hundreds of unique olfactory G-protein coupled receptors to respond to various wavelengths of light or odorants. With respect to hearing and balance, the mechanotransduction machinery involves fewer variants; however, emerging evidence suggests that specialization occurs at the molecular level. To address how the mechanotransduction complex varies in the inner ear, we characterized the expression of paralogous genes that encode components required for mechanotransduction in zebrafish hair cells using RNA-FISH and bioinformatic analysis. Our data indicate striking zonal differences in the expression of two components of the mechanotransduction complex which are known to physically interact, the transmembrane channel-like 1 and 2 (tmc1/2) family members and the calcium and integrin binding 2 and 3 (cib2/3) paralogues. tmc1, tmc2b, and cib3 are largely expressed in peripheral or extrastriolar hair cells, whereas tmc2a and cib2 are enriched in central or striolar hair cells. In addition, a gene implicated in deaf-blindness, ush1c, is highly enriched in a subset of extrastriolar hair cells. These results indicate that specific combinations of these components may optimize responses to mechanical stimuli in subtypes of sensory receptors within the inner ear.

  View details for DOI 10.3389/fnmol.2023.1274822

  View details for PubMedID 38035267

  View details for PubMedCentralID PMC10682102

• Transmembrane channel-like (Tmc) subunits contribute to frequency sensitivity in the zebrafish utricle. The Journal of neuroscience : the official journal of the Society for Neuroscience Sun, P., Smith, E., Nicolson, T. 2023

  Abstract

  Information about dynamic head motion is conveyed by a central 'striolar' zone of vestibular hair cells and afferent neurons in the inner ear. How vestibular hair cells are tuned to transduce dynamic stimuli at the molecular level is not well understood. Here we take advantage of the differential expression pattern of tmc1, tmc2a and tmc2b, which encode subunits of the mechanotransduction complex in zebrafish vestibular hair cells. To test the role of various combinations of Tmc subunits in transducing dynamic head movements, we measured reflexive eye movements induced by high frequency stimuli in single versus double tmc mutants. We found that tmc2a correlates with the broadest range of frequency sensitivity, whereas tmc2b mainly contributes to lower frequency responses. tmc1, which is excluded from the striolar zone, plays a minor role in sensing lower frequency stimuli. Our study suggests that the Tmc subunits impart functional differences to mechanotransduction of dynamic stimuli.Significance Statement Information about dynamic head movements is transmitted by sensory receptors, known as hair cells, in the labyrinth of the inner ear. The sensitivity of hair cells to fast or slow movements of the head differs according to cell type. Whether the mechanotransduction complex that converts mechanical stimuli into electrical signals in hair cells participates in conveying frequency information is not clear. Here we find that the transmembrane channel like 1/2 genes, which encode a central component of the complex, are differentially expressed in the utricle and contribute to frequency sensitivity in zebrafish.

  View details for DOI 10.1523/JNEUROSCI.1298-23.2023

  View details for PubMedID 37952940

• Sensory deficit screen identifies nsf mutation that differentially affects SNARE recycling and quality control. Cell reports Gao, Y., Khan, Y. A., Mo, W., White, K. I., Perkins, M., Pfuetzner, R. A., Trapani, J. G., Brunger, A. T., Nicolson, T. 2023; 42 (4): 112345

  Abstract

  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. Invitro 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.

  View details for DOI 10.1016/j.celrep.2023.112345

  View details for PubMedID 37027300

• Putting the Pieces Together: the Hair Cell Transduction Complex. Journal of the Association for Research in Otolaryngology : JARO Holt, J. R., Tobin, M., Elferich, J., Gouaux, E., Ballesteros, A., Yan, Z., Ahmed, Z. M., Nicolson, T. 2021

  Abstract

  Identification of the components of the mechanosensory transduction complex in hair cells has been a major research interest for many auditory and vestibular scientists and has attracted attention from outside the field. The past two decades have witnessed a number of significant advances with emergence of compelling evidence implicating at least a dozen distinct molecular components of the transduction machinery. Yet, how the pieces of this ensemble fit together and function in harmony to enable the senses of hearing and balance has not been clarified. The goal of this review is to summarize a 2021 symposium presented at the annual mid-winter meeting of the Association for Research in Otolaryngology. The symposium brought together the latest insights from within and beyond the field to examine individual components of the transduction complex and how these elements interact at molecular, structural, and biophysical levels to gate mechanosensitive channels and initiate sensory transduction in the inner ear. The review includes a brief historical background to set the stage for topics to follow that focus on structure, properties, and interactions of proteins such as CDH23, PCDH15, LHFPL5, TMIE, TMC1/2, and CIB2/3. We aim to present the diversity of ideas in this field and highlight emerging theories and concepts. This review will not only provide readers with a deeper appreciation of the components of the transduction apparatus and how they function together, but also bring to light areas of broad agreement, areas of scientific controversy, and opportunities for future scientific discovery.

  View details for DOI 10.1007/s10162-021-00808-0

  View details for PubMedID 34617206

• Navigating Hereditary Hearing Loss: Pathology of the Inner Ear. Frontiers in cellular neuroscience Nicolson, T. 2021; 15: 660812

  Abstract

  Inherited forms of deafness account for a sizable portion of hearing loss among children and adult populations. Many patients with sensorineural deficits have pathological manifestations in the peripheral auditory system, the inner ear. Within the hearing organ, the cochlea, most of the genetic forms of hearing loss involve defects in sensory detection and to some extent, signaling to the brain via the auditory cranial nerve. This review focuses on peripheral forms of hereditary hearing loss and how these impairments can be studied in diverse animal models or patient-derived cells with the ultimate goal of using the knowledge gained to understand the underlying biology and treat hearing loss.

  View details for DOI 10.3389/fncel.2021.660812

  View details for PubMedID 34093131

  View details for PubMedCentralID PMC8172992

• Disruption of tmc1/2a/2b genes in zebrafish reveals subunit requirements in subtypes of inner ear hair cells. The Journal of neuroscience : the official journal of the Society for Neuroscience Smith, E. T., Pacentine, I., Shipman, A., Hill, M., Nicolson, T. 2020

  Abstract

  Detection of sound and head movement requires mechanoelectrical transduction (MET) channels at tips of hair-cell stereocilia. In vertebrates, the transmembrane channel-like (TMC) proteins TMC1 and TMC2 fulfill critical roles in MET and substantial evidence implicates these TMCs as subunits of the MET channel. To identify developmental and functional roles of this Tmc subfamily in the zebrafish inner ear, we tested the effects of truncating mutations in tmc1, tmc2a, and tmc2b on in vivo mechanosensation at the onset of hearing and balance, before gender differentiation. We find that tmc1/2a/2b triple-mutant larvae cannot detect sound or orient with respect to gravity. They lack acoustic-evoked behavioral responses (AEBR), vestibular-induced eye movements (VIEM), and hair-cell activity as assessed with FM dye labeling and microphonic potentials. Despite complete loss of hair-cell function, tmc triple-mutant larvae retain normal gross morphology of hair bundles and proper trafficking of known MET components Protocadherin 15a (Pcdh15a), Lipoma HMGIC fusion partner-like 5 (Lhfpl5), and Transmembrane inner ear protein (Tmie). Transgenic, hair cell-specific expression of Tmc2b-mEGFP rescues the behavioral and physiological deficits in tmc triple mutants. Results from tmc single- and double- mutants evince a principle role for Tmc2a and Tmc2b in hearing and balance, respectively, whereas Tmc1 has lower overall impact. Our experiments reveal that in developing cristae, hair cells stratify into an upper, Tmc2a-dependent layer of teardrop shaped cells and a lower, Tmc1/2b-dependent tier of gourd shaped cells. Collectively our genetic evidence indicates that auditory/vestibular end organs and subsets of hair cells therein rely on distinct combinations of Tmc1/2a/2b.Significance StatementWe assessed the effects of tmc1/2a/2b truncation mutations on mechanoelectrical transduction (MET) in the inner-ear hair cells of larval zebrafish. tmc triple mutants lacked behavioral responses to sound and head movements, while further assays demonstrated no observable mechanosensitivity in the tmc1/2a/2b triple mutant inner ear. Examination of tmc double mutants revealed major contributions from Tmc2a and Tmc2b to macular function; however, Tmc1 had less overall impact. FM labeling of lateral cristae in tmc double mutants revealed the presence of two distinct cell types, an upper layer of teardrop shaped cells that rely on Tmc2a, and a lower layer of gourd shaped cells that rely on Tmc1/2b.

  View details for DOI 10.1523/JNEUROSCI.0163-20.2020

  View details for PubMedID 32371604

• The lhfpl5 Ohnologs lhfpl5a and lhfpl5b Are Required for Mechanotransduction in Distinct Populations of Sensory Hair Cells in Zebrafish FRONTIERS IN MOLECULAR NEUROSCIENCE Erickson, T., Pacentine, I. V., Venuto, A., Clemens, R., Nicolson, T. 2020; 12: 320

  Abstract

  Hair cells sense and transmit auditory, vestibular, and hydrodynamic information by converting mechanical stimuli into electrical signals. This process of mechano-electrical transduction (MET) requires a mechanically gated channel localized in the apical stereocilia of hair cells. In mice, lipoma HMGIC fusion partner-like 5 (LHFPL5) acts as an auxiliary subunit of the MET channel whose primary role is to correctly localize PCDH15 and TMC1 to the mechanotransduction complex. Zebrafish have two lhfpl5 genes (lhfpl5a and lhfpl5b), but their individual contributions to MET channel assembly and function have not been analyzed. Here we show that the zebrafish lhfpl5 genes are expressed in discrete populations of hair cells: lhfpl5a expression is restricted to auditory and vestibular hair cells in the inner ear, while lhfpl5b expression is specific to hair cells of the lateral line organ. Consequently, lhfpl5a mutants exhibit defects in auditory and vestibular function, while disruption of lhfpl5b affects hair cells only in the lateral line neuromasts. In contrast to previous reports in mice, localization of Tmc1 does not depend upon Lhfpl5 function in either the inner ear or lateral line organ. In both lhfpl5a and lhfpl5b mutants, GFP-tagged Tmc1 and Tmc2b proteins still localize to the stereocilia of hair cells. Using a stably integrated GFP-Lhfpl5a transgene, we show that the tip link cadherins Pcdh15a and Cdh23, along with the Myo7aa motor protein, are required for correct Lhfpl5a localization at the tips of stereocilia. Our work corroborates the evolutionarily conserved co-dependence between Lhfpl5 and Pcdh15, but also reveals novel requirements for Cdh23 and Myo7aa to correctly localize Lhfpl5a. In addition, our data suggest that targeting of Tmc1 and Tmc2b proteins to stereocilia in zebrafish hair cells occurs independently of Lhfpl5 proteins.

  View details for DOI 10.3389/fnmol.2019.00320

  View details for Web of Science ID 000509937300001

  View details for PubMedID 32009898

  View details for PubMedCentralID PMC6974483

• Temporal Vestibular Deficits in synaptojanin 1 (synj1) Mutants. Frontiers in molecular neuroscience Gao, Y., Nicolson, T. 2020; 13: 604189

  Abstract

  The lipid phosphatase synaptojanin 1 (synj1) is required for the disassembly of clathrin coats on endocytic compartments. In neurons such activity is necessary for the recycling of endocytosed membrane into synaptic vesicles. Mutations in zebrafish synj1 have been shown to disrupt the activity of ribbon synapses in sensory hair cells. After prolonged mechanical stimulation of hair cells, both phase locking of afferent nerve activity and the recovery of spontaneous release of synaptic vesicles are diminished in synj1 mutants. Presumably as a behavioral consequence of these synaptic deficits, synj1 mutants are unable to maintain an upright posture. To probe vestibular function with respect to postural control in synj1 mutants, we developed a method for assessing the vestibulospinal reflex (VSR) in larvae. We elicited the VSR by rotating the head and recorded tail movements. As expected, the VSR is completely absent in pcdh15a and lhfpl5a mutants that lack inner ear function. Conversely, lhfpl5b mutants, which have a selective loss of function of the lateral line organ, have normal VSRs, suggesting that the hair cells of this organ do not contribute to this reflex. In contrast to mechanotransduction mutants, the synj1 mutant produces normal tail movements during the initial cycles of rotation of the head. Both the amplitude and temporal aspects of the response are unchanged. However, after several rotations, the VSR in synj1 mutants was strongly diminished or absent. Mutant synj1 larvae are able to recover, but the time required for the reappearance of the VSR after prolonged stimulation is dramatically increased in synj1 mutants. Collectively, the data demonstrate a behavioral correlate of the synaptic defects caused by the loss of synj1 function. Our results suggest that defects in synaptic vesicle recycling give rise to fatigue of ribbons synapses and possibly other synapses of the VS circuit, leading to the loss of postural control.

  View details for DOI 10.3389/fnmol.2020.604189

  View details for PubMedID 33584199

• Subunits of the mechano-electrical transduction channel, Tmc1/2b, require Tmie to localize in zebrafish sensory hair cells PLOS GENETICS Pacentine, I. V., Nicolson, T. 2019; 15 (2): e1007635

  Abstract

  Mutations in transmembrane inner ear (TMIE) cause deafness in humans; previous studies suggest involvement in the mechano-electrical transduction (MET) complex in sensory hair cells, but TMIE's precise role is unclear. In tmie zebrafish mutants, we observed that GFP-tagged Tmc1 and Tmc2b, which are subunits of the MET channel, fail to target to the hair bundle. In contrast, overexpression of Tmie strongly enhances the targeting of Tmc1-GFP and Tmc2b-GFP to stereocilia. To identify the motifs of Tmie underlying the regulation of the Tmcs, we systematically deleted or replaced peptide segments. We then assessed localization and functional rescue of each mutated/chimeric form of Tmie in tmie mutants. We determined that the first putative helix was dispensable and identified a novel critical region of Tmie, the extracellular region and transmembrane domain, which is required for both mechanosensitivity and Tmc2b-GFP expression in bundles. Collectively, our results suggest that Tmie's role in sensory hair cells is to target and stabilize Tmc channel subunits to the site of MET.

  View details for PubMedID 30726219

• Subunits of the mechano-electrical transduction channel, Tmc1/2b, require Tmie to localize in zebrafish sensory hair cells. PlosGenetics Pacentine, I. V., Nicolson, T. 2019
• Zebrafish: from genes and neurons to circuits, behavior and disease. Journal of neurogenetics Chandrasekhar, A., Guo, S., Masai, I., Nicolson, T., Wu, C. F. 2017; 31 (3): 59-60

  View details for DOI 10.1080/01677063.2017.1359589

  View details for PubMedID 28868983

• The genetics of hair-cell function in zebrafish. Journal of neurogenetics Nicolson, T. 2017; 31 (3): 102-112

  Abstract

  Our ears are remarkable sensory organs, providing the important senses of balance and hearing. The complex structure of the inner ear, or 'labyrinth', along with the assorted neuroepithelia, have evolved to detect head movements and sounds with impressive sensitivity. The rub is that the inner ear is highly vulnerable to genetic lesions and environmental insults. According to National Institute of Health estimates, hearing loss is one of the most commonly inherited or acquired sensorineural diseases. To understand the causes of deafness and balance disorders, it is imperative to understand the underlying biology of the inner ear, especially the inner workings of the sensory receptors. These receptors, which are termed hair cells, are particularly susceptible to genetic mutations - more than two dozen genes are associated with defects in this cell type in humans. Over the past decade, a substantial amount of progress has been made in working out the molecular basis of hair-cell function using vertebrate animal models. Given the transparency of the inner ear and the genetic tools that are available, zebrafish have become an increasingly popular animal model for the study of deafness and vestibular dysfunction. Mutagenesis screens for larval defects in hearing and balance have been fruitful in finding key components, many of which have been implicated in human deafness. This review will focus on the genes that are required for hair-cell function in zebrafish, with a particular emphasis on mechanotransduction. In addition, the generation of new tools available for the characterization of zebrafish hair-cell mutants will be discussed.

  View details for DOI 10.1080/01677063.2017.1342246

  View details for PubMedID 28705044

  View details for PubMedCentralID PMC6080859

• Enlargement of Ribbons in Zebrafish Hair Cells Increases Calcium Currents But Disrupts Afferent Spontaneous Activity and Timing of Stimulus Onset. The Journal of neuroscience : the official journal of the Society for Neuroscience Sheets, L., He, X. J., Olt, J., Schreck, M., Petralia, R. S., Wang, Y. X., Zhang, Q., Beirl, A., Nicolson, T., Marcotti, W., Trapani, J. G., Kindt, K. S. 2017; 37 (26): 6299-6313

  Abstract

  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.

  View details for DOI 10.1523/JNEUROSCI.2878-16.2017

  View details for PubMedID 28546313

  View details for PubMedCentralID PMC5490065

• Integration of Tmc1/2 into the mechanotransduction complex in zebrafish hair cells is regulated by Transmembrane O-methyltransferase (Tomt). eLife Erickson, T., Morgan, C. P., Olt, J., Hardy, K., Busch-Nentwich, E., Maeda, R., Clemens, R., Krey, J. F., Nechiporuk, A., Barr-Gillespie, P. G., Marcotti, W., Nicolson, T. 2017; 6

  Abstract

  Transmembrane O-methyltransferase (TOMT/LRTOMT) is responsible for non-syndromic deafness DFNB63. However, the specific defects that lead to hearing loss have not been described. Using a zebrafish model of DFNB63, we show that the auditory and vestibular phenotypes are due to a lack of mechanotransduction (MET) in Tomt-deficient hair cells. GFP-tagged Tomt is enriched in the Golgi of hair cells, suggesting that Tomt might regulate the trafficking of other MET components to the hair bundle. We found that Tmc1/2 proteins are specifically excluded from the hair bundle in tomt mutants, whereas other MET complex proteins can still localize to the bundle. Furthermore, mouse TOMT and TMC1 can directly interact in HEK 293 cells, and this interaction is modulated by His183 in TOMT. Thus, we propose a model of MET complex assembly where Tomt and the Tmcs interact within the secretory pathway to traffic Tmc proteins to the hair bundle.

  View details for DOI 10.7554/eLife.28474

  View details for PubMedID 28534737

  View details for PubMedCentralID PMC5462536

• Functional Analysis of the Transmembrane and Cytoplasmic Domains of Pcdh15a in Zebrafish Hair Cells. The Journal of neuroscience : the official journal of the Society for Neuroscience Maeda, R., Pacentine, I. V., Erickson, T., Nicolson, T. 2017; 37 (12): 3231-3245

  Abstract

  Protocadherin 15 (PCDH15) is required for mechanotransduction in sensory hair cells as a component of the tip link. Isoforms of PCDH15 differ in their cytoplasmic domains (CD1, CD2, and CD3), but share the extracellular and transmembrane (TMD) domains, as well as an intracellular domain known as the common region (CR). In heterologous expression systems, both the TMD and CR of PCDH15 have been shown to interact with members of the mechanotransduction complex. The in vivo significance of these protein-protein interaction domains of PCDH15 in hair cells has not been determined. Here, we examined the localization and function of the two isoforms of zebrafish Pcdh15a (CD1 and CD3) in pcdh15a-null mutants by assessing Pcdh15a transgene-mediated rescue of auditory/vestibular behavior and hair cell morphology and activity. We found that either isoform alone was able to rescue the Pcdh15a-null phenotype and that the CD1- or CD3-specific regions were dispensable for hair bundle integrity and labeling of hair cells with FM4-64, which was used as a proxy for mechanotransduction. When either the CR or TMD domain was deleted, the mutated proteins localized to the stereocilial tips, but were unable to rescue FM4-64 labeling. Disrupting both domains led to a complete failure of Pcdh15a to localize to the hair bundle. Our findings demonstrate that the TMD and cytoplasmic CR domains are required for the in vivo function of Pcdh15a in zebrafish hair cells.SIGNIFICANCE STATEMENT Tip links transmit force to mechanotransduction channels at the tip of hair bundles in sensory hair cells. One component of tip links is Protocadherin 15 (PCDH15). Here, we demonstrate that, when transgenically expressed, either zebrafish Pcdh15a-cytodomain 1 (CD1) or Pcdh15a-CD3 can rescue the phenotype of a pcdh15a-null mutant. Even when lacking the specific regions for CD1 or CD3, truncated Pcdh15a that contains the so-called common region (CR) at the cytoplasmic/membrane interface still has the ability to rescue similar to full-length Pcdh15a. In contrast, Pcdh15a lacking the entire cytoplasmic domain is not functional. These results demonstrate that the CR plays a key role in the mechanotransduction complex in hair cells.

  View details for DOI 10.1523/JNEUROSCI.2216-16.2017

  View details for PubMedID 28219986

  View details for PubMedCentralID PMC5373116

• Cell type-specific transcriptomic analysis by thiouracil tagging in zebrafish. Methods in cell biology Erickson, T., Nicolson, T. 2016; 135: 309-28

  Abstract

  Transcriptomic studies are important tools for understanding the development and function of the different cell types that make up complex tissues. Zebrafish (Danio rerio) is a valuable organism for modeling key aspects of vertebrate development, cell biology, and human disease. However, the small size of individual larvae and relative scarcity of certain cell types in zebrafish can hamper efforts to collect enough pure material for cell type-specific transcriptomic studies. Thus, there is a need in the zebrafish field for spatially and temporally resolved gene expression assays. This chapter will discuss the general principles behind the TU-tagging method to isolate cell type-specific RNAs and provide guidance in designing and executing TU-tagging experiments in zebrafish.

  View details for DOI 10.1016/bs.mcb.2016.04.009

  View details for PubMedID 27443933

• Dopamine Modulates the Activity of Sensory Hair Cells. The Journal of neuroscience : the official journal of the Society for Neuroscience Toro, C., Trapani, J. G., Pacentine, I., Maeda, R., Sheets, L., Mo, W., Nicolson, T. 2015; 35 (50): 16494-503

  Abstract

  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.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.

  View details for DOI 10.1523/JNEUROSCI.1691-15.2015

  View details for PubMedID 26674873

  View details for PubMedCentralID PMC4679827

• Ribbon synapses in zebrafish hair cells. Hearing research Nicolson, T. 2015; 330 (Pt B): 170-7

  Abstract

  The basic architecture and functionality of ribbon synapses of mechanosensitive hair cells are well conserved among vertebrates. Forward and reverse genetic methods in zebrafish (Danio rerio) have identified components that are critical for the development and function of ribbon synapses. This review will focus on the findings of these genetic approaches, and discuss some emergent concepts on the role of the ribbon body and calcium in synapse development, and how perturbations in synaptic vesicles lead to a loss of temporal fidelity at ribbon synapses. This article is part of a Special Issue entitled .

  View details for DOI 10.1016/j.heares.2015.04.003

  View details for PubMedID 25916266

  View details for PubMedCentralID PMC4620066

• Identification of sensory hair-cell transcripts by thiouracil-tagging in zebrafish. BMC genomics Erickson, T., Nicolson, T. 2015; 16: 842

  Abstract

  Sensory hair cells are exquisitely sensitive to mechanical stimuli and as such, are prone to damage and apoptosis during dissections or in vitro manipulations. Thiouracil (TU)-tagging is a noninvasive method to label cell type-specific transcripts in an intact organism, thereby meeting the challenge of how to analyze gene expression in hair cells without the need to sort cells. We adapted TU-tagging to zebrafish to identify novel transcripts expressed in the sensory hair cells of the developing acoustico-lateralis organs.We created a transgenic line of zebrafish expressing the T.gondii uracil phospho-ribosyltransferase (UPRT) enzyme specifically in the hair cells of the inner ear and lateral line organ. RNA was labeled by exposing 3 days post-fertilization (dpf) UPRT transgenic larvae to 2.5 mM 4-thiouracil (4TU) for 15 hours. Following total RNA isolation, poly(A) mRNA enrichment, and purification of TU-tagged RNA, deep sequencing was performed on the input and TU-tagged RNA samples.Analysis of the RNA sequencing data revealed the expression of 28 transcripts that were significantly enriched (adjusted p-value < 0.05) in the UPRT TU-tagged RNA relative to the input sample. Of the 25 TU-tagged transcripts with mammalian homologs, the expression of 18 had not been previously demonstrated in zebrafish hair cells. The hair cell-restricted expression for 17 of these transcripts was confirmed by whole mount mRNA in situ hybridization in 3 dpf larvae.The hair cell-restricted pattern of expression of these genes offers insight into the biology of this receptor cell type and may serve as useful markers to study the development and function of sensory hair cells. In addition, our study demonstrates the utility of TU-tagging to study nascent transcripts in specific cell types that are relatively rare in the context of the whole zebrafish larvae.

  View details for DOI 10.1186/s12864-015-2072-5

  View details for PubMedID 26494580

  View details for PubMedCentralID PMC4619078

• Characterization of Ribeye subunits in zebrafish hair cells reveals that exogenous Ribeye B-domain and CtBP1 localize to the basal ends of synaptic ribbons. PloS one Sheets, L., Hagen, M. W., Nicolson, T. 2014; 9 (9): e107256

  Abstract

  Synaptic ribbons are presynaptic structures formed by the self-association of RIBEYE-the main structural component of ribbon synapses. RIBEYE consists of two domains: a unique N-terminal A-domain and a C-terminal B-domain that is identical to the transcription co-repressor C-terminal binding protein 2 (CtBP2). Previous studies in cell lines have shown that RIBEYE A-domain alone is sufficient to form ribbon-like aggregates and that both A- and B- domains form homo-and heterotypic interactions. As these interactions are likely the basis for synaptic-ribbon assembly and structural plasticity, we wanted to examine how zebrafish Ribeye A- and B- domains interact with synaptic ribbons in vivo. To that end, we characterized the localization of exogenously expressed Ribeye A- and B- domains and the closely related protein, CtBP1, in the hair cells of transgenic zebrafish larvae. Unexpectedly, exogenously expressed Ribeye A-domain showed variable patterns of localization in hair cells; one zebrafish paralog of A-domain failed to self-associate or localize to synaptic ribbons, while the other self-assembled but sometimes failed to localize to synaptic ribbons. By contrast, Ribeye B-domain/CtBP2 was robustly localized to synaptic ribbons. Moreover, both exogenously expressed B-domain/CtBP2 and CtBP1 were preferentially localized to the basal end of ribbons adjacent to the postsynaptic density. Overexpression of B-domain/CtBP2 also appeared to affect synaptic-ribbon composition; endogenous levels of ribbon-localized Ribeye were significantly reduced as hair cells matured in B-domain/CtBP2 transgenic larvae compared to wild-type. These results reveal how exogenously expressed Ribeye domains interact with synaptic ribbons, and suggest a potential organization of elements within the ribbon body.

  View details for DOI 10.1371/journal.pone.0107256

  View details for PubMedID 25208216

  View details for PubMedCentralID PMC4160224

• Tip-link protein protocadherin 15 interacts with transmembrane channel-like proteins TMC1 and TMC2. Proceedings of the National Academy of Sciences of the United States of America Maeda, R., Kindt, K. S., Mo, W., Morgan, C. P., Erickson, T., Zhao, H., Clemens-Grisham, R., Barr-Gillespie, P. G., Nicolson, T. 2014; 111 (35): 12907-12

  Abstract

  The tip link protein protocadherin 15 (PCDH15) is a central component of the mechanotransduction complex in auditory and vestibular hair cells. PCDH15 is hypothesized to relay external forces to the mechanically gated channel located near its cytoplasmic C terminus. How PCDH15 is coupled to the transduction machinery is not clear. Using a membrane-based two-hybrid screen to identify proteins that bind to PCDH15, we detected an interaction between zebrafish Pcdh15a and an N-terminal fragment of transmembrane channel-like 2a (Tmc2a). Tmc2a is an ortholog of mammalian TMC2, which along with TMC1 has been implicated in mechanotransduction in mammalian hair cells. Using the above-mentioned two-hybrid assay, we found that zebrafish Tmc1 and Tmc2a can interact with the CD1 or CD3 cytoplasmic domain isoforms of Pcdh15a, and this interaction depends on the common region shared between the two Pcdh15 isoforms. Moreover, an interaction between mouse PCDH15-CD3 and TMC1 or TMC2 was observed in both yeast two-hybrid assays and coimmunoprecipitation experiments. To determine whether the Pcdh15-Tmc interaction is relevant to mechanotransduction in vivo, we overexpressed N-terminal fragments of Tmc2a in zebrafish hair cells. Overexpression of the Tmc2a N terminus results in mislocalization of Pcdh15a within hair bundles, together with a significant decrease in mechanosensitive responses, suggesting that a Pcdh15a-Tmc complex is critical for mechanotransduction. Together, these results identify an evolutionarily conserved association between the fish and mouse orthologs of PCDH15 and TMC1 and TMC2, supporting the notion that TMCs are key components of the transduction complex in hair cells.

  View details for DOI 10.1073/pnas.1402152111

  View details for PubMedID 25114259

  View details for PubMedCentralID PMC4156717

• Mutations in ap1b1 cause mistargeting of the Na(+)/K(+)-ATPase pump in sensory hair cells. PloS one Clemens Grisham, R., Kindt, K., Finger-Baier, K., Schmid, B., Nicolson, T. 2013; 8 (4): e60866

  Abstract

  The hair cells of the inner ear are polarized epithelial cells with a specialized structure at the apical surface, the mechanosensitive hair bundle. Mechanotransduction occurs within the hair bundle, whereas synaptic transmission takes place at the basolateral membrane. The molecular basis of the development and maintenance of the apical and basal compartments in sensory hair cells is poorly understood. Here we describe auditory/vestibular mutants isolated from forward genetic screens in zebrafish with lesions in the adaptor protein 1 beta subunit 1 (ap1b1) gene. Ap1b1 is a subunit of the adaptor complex AP-1, which has been implicated in the targeting of basolateral membrane proteins. In ap1b1 mutants we observed that although the overall development of the inner ear and lateral-line organ appeared normal, the sensory epithelium showed progressive signs of degeneration. Mechanically-evoked calcium transients were reduced in mutant hair cells, indicating that mechanotransduction was also compromised. To gain insight into the cellular and molecular defects in ap1b1 mutants, we examined the localization of basolateral membrane proteins in hair cells. We observed that the Na(+)/K(+)-ATPase pump (NKA) was less abundant in the basolateral membrane and was mislocalized to apical bundles in ap1b1 mutant hair cells. Accordingly, intracellular Na(+) levels were increased in ap1b1 mutant hair cells. Our results suggest that Ap1b1 is essential for maintaining integrity and ion homeostasis in hair cells.

  View details for DOI 10.1371/journal.pone.0060866

  View details for PubMedID 23593334

  View details for PubMedCentralID PMC3625210

• Towards a Comprehensive Catalog of Zebrafish Behavior 1.0 and Beyond ZEBRAFISH Kalueff, A. V., Gebhardt, M., Stewart, A. M., Cachat, J. M., Brimmer, M., Chawla, J. S., Craddock, C., Kyzar, E. J., Roth, A., Landsman, S., Gaikwad, S., Robinson, K., Baatrup, E., Tierney, K., Shamchuk, A., Norton, W., Miller, N., Nicolson, T., Braubach, O., Gilman, C. P., Pittman, J., Rosemberg, D. B., Gerlai, R., Echevarria, D., Lamb, E., Neuhauss, S. C., Weng, W., Bally-Cuif, L., Schneider, H. 2013; 10 (1): 70-86

  Abstract

  Zebrafish (Danio rerio) are rapidly gaining popularity in translational neuroscience and behavioral research. Physiological similarity to mammals, ease of genetic manipulations, sensitivity to pharmacological and genetic factors, robust behavior, low cost, and potential for high-throughput screening contribute to the growing utility of zebrafish models in this field. Understanding zebrafish behavioral phenotypes provides important insights into neural pathways, physiological biomarkers, and genetic underpinnings of normal and pathological brain function. Novel zebrafish paradigms continue to appear with an encouraging pace, thus necessitating a consistent terminology and improved understanding of the behavioral repertoire. What can zebrafish 'do', and how does their altered brain function translate into behavioral actions? To help address these questions, we have developed a detailed catalog of zebrafish behaviors (Zebrafish Behavior Catalog, ZBC) that covers both larval and adult models. Representing a beginning of creating a more comprehensive ethogram of zebrafish behavior, this effort will improve interpretation of published findings, foster cross-species behavioral modeling, and encourage new groups to apply zebrafish neurobehavioral paradigms in their research. In addition, this glossary creates a framework for developing a zebrafish neurobehavioral ontology, ultimately to become part of a unified animal neurobehavioral ontology, which collectively will contribute to better integration of biological data within and across species.

  View details for DOI 10.1089/zeb.2012.0861

  View details for Web of Science ID 000317736900009

  View details for PubMedID 23590400

  View details for PubMedCentralID PMC3629777

• Presynaptic CaV1.3 channels regulate synaptic ribbon size and are required for synaptic maintenance in sensory hair cells. The Journal of neuroscience : the official journal of the Society for Neuroscience Sheets, L., Kindt, K. S., Nicolson, T. 2012; 32 (48): 17273-86

  Abstract

  L-type calcium channels (Ca(V)1) are involved in diverse processes, such as neurotransmission, hormone secretion, muscle contraction, and gene expression. In this study, we uncover a role for Ca(V)1.3a in regulating the architecture of a cellular structure, the ribbon synapse, in developing zebrafish sensory hair cells. By combining in vivo calcium imaging with confocal and super-resolution structured illumination microscopy, we found that genetic disruption or acute block of Ca(V)1.3a channels led to enlargement of synaptic ribbons in hair cells. Conversely, activating channels reduced both synaptic-ribbon size and the number of intact synapses. Along with enlarged presynaptic ribbons in ca(V)1.3a mutants, we observed a profound loss of juxtaposition between presynaptic and postsynaptic components. These synaptic defects are not attributable to loss of neurotransmission, because vglut3 mutants lacking neurotransmitter release develop relatively normal hair-cell synapses. Moreover, regulation of synaptic-ribbon size by Ca(2+) influx may be used by other cell types, because we observed similar pharmacological effects on pinealocyte synaptic ribbons. Our results indicate that Ca(2+) influx through Ca(V)1.3 fine tunes synaptic ribbon size during hair-cell maturation and that Ca(V)1.3 is required for synaptic maintenance.

  View details for DOI 10.1523/JNEUROSCI.3005-12.2012

  View details for PubMedID 23197719

  View details for PubMedCentralID PMC3718275

• Rapid positional cloning of zebrafish mutations by linkage and homozygosity mapping using whole-genome sequencing. Development (Cambridge, England) Obholzer, N., Swinburne, I. A., Schwab, E., Nechiporuk, A. V., Nicolson, T., Megason, S. G. 2012; 139 (22): 4280-90

  Abstract

  Forward genetic screens in zebrafish have identified >9000 mutants, many of which are potential disease models. Most mutants remain molecularly uncharacterized because of the high cost, time and labor investment required for positional cloning. These costs limit the benefit of previous genetic screens and discourage future screens. Drastic improvements in DNA sequencing technology could dramatically improve the efficiency of positional cloning in zebrafish and other model organisms, but the best strategy for cloning by sequencing has yet to be established. Using four zebrafish inner ear mutants, we developed and compared two approaches for 'cloning by sequencing': one based on bulk segregant linkage (BSFseq) and one based on homozygosity mapping (HMFseq). Using BSFseq we discovered that mutations in lmx1b and jagged1b cause abnormal ear morphogenesis. With HMFseq we validated that the disruption of cdh23 abolishes the ear's sensory functions and identified a candidate lesion in lhfpl5a predicted to cause nonsyndromic deafness. The success of HMFseq shows that the high intrastrain polymorphism rate in zebrafish eliminates the need for time-consuming map crosses. Additionally, we analyzed diversity in zebrafish laboratory strains to find areas of elevated diversity and areas of fixed homozygosity, reinforcing recent findings that genome diversity is clustered. We present a database of >15 million sequence variants that provides much of this approach's power. In our four test cases, only a single candidate single nucleotide polymorphism (SNP) remained after subtracting all database SNPs from a mutant's critical region. The saturation of the common SNP database and our open source analysis pipeline MegaMapper will improve the pace at which the zebrafish community makes unique discoveries relevant to human health.

  View details for DOI 10.1242/dev.083931

  View details for PubMedID 23052906

  View details for PubMedCentralID PMC3478692

• The Usher gene cadherin 23 is expressed in the zebrafish brain and a subset of retinal amacrine cells. Molecular vision Glover, G., Mueller, K. P., Söllner, C., Neuhauss, S. C., Nicolson, T. 2012; 18: 2309-22

  Abstract

  To characterize the expression pattern of cadherin 23 (cdh23) in the zebrafish visual system, and to determine whether zebrafish cdh23 mutants have retinal defects similar to those present in the human disease Usher syndrome 1D.In situ hybridization and immunohistochemistry were used to characterize cdh23 expression in the zebrafish, and to evaluate cdh23 mutants for retinal degeneration. Visual function was assessed by measurement of the optokinetic response in cdh23 siblings and mutants.We detected cdh23 mRNA expression in multiple nuclei of both the developing and adult central nervous system. In the retina, cdh23 mRNA was expressed in a small subset of amacrine cells, beginning at 70 h postfertilization and continuing through adulthood. No expression was detected in photoreceptors. The cdh23-positive population of amacrine cells was GABAergic. Examination of homozygous larvae expressing two different mutant alleles of cdh23-cdh23(tc317e) or cdh23(tj264a)-revealed no detectable morphological retinal defects or degeneration. In addition, the optokinetic response to moving gratings of varied contrast or spatial frequency was normal in both mutants.Unlike in other vertebrates, cdh23 is not detectable in zebrafish photoreceptors. Instead, cdh23 is expressed by a small subset of GABAergic amacrine cells. Moreover, larvae with mutations in cdh23 do not exhibit any signs of gross retinal degeneration or dysfunction. The role played by cdh23 in human retinal function is likely performed by either a different gene or an unidentified cdh23 splice variant in the retina that is not affected by the above mutations.

  View details for PubMedID 22977299

  View details for PubMedCentralID PMC3441156

• Kinocilia mediate mechanosensitivity in developing zebrafish hair cells. Developmental cell Kindt, K. S., Finch, G., Nicolson, T. 2012; 23 (2): 329-41

  Abstract

  Mechanosensitive cilia are vital to signaling and development across many species. In sensory hair cells, sound and movement are transduced by apical hair bundles. Each bundle is comprised of a single primary cilium (kinocilium) flanked by multiple rows of actin-filled projections (stereocilia). Extracellular tip links that interconnect stereocilia are thought to gate mechanosensitive channels. In contrast to stereocilia, kinocilia are not critical for hair-cell mechanotransduction. However, by sequentially imaging the structure of hair bundles and mechanosensitivity of individual lateral-line hair cells in vivo, we uncovered a central role for kinocilia in mechanosensation during development. Our data demonstrate that nascent hair cells require kinocilia and kinocilial links for mechanosensitivity. Although nascent hair bundles have correct planar polarity, the polarity of their responses to mechanical stimuli is initially reversed. Later in development, a switch to correctly polarized mechanosensitivity coincides with the formation of tip links and the onset of tip-link-dependent mechanotransduction.

  View details for DOI 10.1016/j.devcel.2012.05.022

  View details for PubMedID 22898777

  View details for PubMedCentralID PMC3426295

• Both pre- and postsynaptic activity of Nsf prevents degeneration of hair-cell synapses. PloS one Mo, W., Nicolson, T. 2011; 6 (11): e27146

  Abstract

  Vesicle fusion contributes to the maintenance of synapses in the nervous system by mediating synaptic transmission, release of neurotrophic factors, and trafficking of membrane receptors. N-ethylmaleimide-sensitive factor (NSF) is indispensible for dissociation of the SNARE-complex following vesicle fusion. Although NSF function has been characterized extensively in vitro, the in vivo role of NSF in vertebrate synaptogenesis is relatively unexplored. Zebrafish possess two nsf genes, nsf and nsfb. Here, we examine the function of either Nsf or Nsfb in the pre- and postsynaptic cells of the zebrafish lateral line organ and demonstrate that Nsf, but not Nsfb, is required for maintenance of afferent synapses in hair cells. In addition to peripheral defects in nsf mutants, neurodegeneration of glutamatergic synapses in the central nervous system also occurs in the absence of Nsf function. Expression of an nsf transgene in a null background indicates that stabilization of synapses requires Nsf function in both hair cells and afferent neurons. To identify potential targets of Nsf-mediated fusion, we examined the expression of genes implicated in stabilizing synapses and found that transcripts for multiple genes including brain-derived neurotrophic factor (bdnf) were significantly reduced in nsf mutants. With regard to trafficking of BDNF, we observed a striking accumulation of BDNF in the neurites of nsf mutant afferent neurons. In addition, injection of recombinant BDNF protein partially rescued the degeneration of afferent synapses in nsf mutants. These results establish a role for Nsf in the maintenance of synaptic contacts between hair cells and afferent neurons, mediated in part via the secretion of trophic signaling factors.

  View details for DOI 10.1371/journal.pone.0027146

  View details for PubMedID 22073277

  View details for PubMedCentralID PMC3207842

• Ribeye is required for presynaptic Ca(V)1.3a channel localization and afferent innervation of sensory hair cells. Development (Cambridge, England) Sheets, L., Trapani, J. G., Mo, W., Obholzer, N., Nicolson, T. 2011; 138 (7): 1309-19

  Abstract

  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.

  View details for DOI 10.1242/dev.059451

  View details for PubMedID 21350006

  View details for PubMedCentralID PMC3050663

• Mechanism of spontaneous activity in afferent neurons of the zebrafish lateral-line organ. The Journal of neuroscience : the official journal of the Society for Neuroscience Trapani, J. G., Nicolson, T. 2011; 31 (5): 1614-23

  Abstract

  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).

  View details for DOI 10.1523/JNEUROSCI.3369-10.2011

  View details for PubMedID 21289170

  View details for PubMedCentralID PMC3065326

• Physiological recordings from zebrafish lateral-line hair cells and afferent neurons. Methods in cell biology Trapani, J. G., Nicolson, T. 2010; 100: 219-31

  Abstract

  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.

  View details for DOI 10.1016/B978-0-12-384892-5.00008-6

  View details for PubMedID 21111219

• Quantification of vestibular-induced eye movements in zebrafish larvae. BMC neuroscience Mo, W., Chen, F., Nechiporuk, A., Nicolson, T. 2010; 11: 110

  Abstract

  Vestibular reflexes coordinate movements or sensory input with changes in body or head position. Vestibular-evoked responses that involve the extraocular muscles include the vestibulo-ocular reflex (VOR), a compensatory eye movement to stabilize retinal images. Although an angular VOR attributable to semicircular canal stimulation was reported to be absent in free-swimming zebrafish larvae, recent studies reveal that vestibular-induced eye movements can be evoked in zebrafish larvae by both static tilts and dynamic rotations that tilt the head with respect to gravity.We have determined herein the basis of sensitivity of the larval eye movements with respect to vestibular stimulus, developmental stage, and sensory receptors of the inner ear. For our experiments, video recordings of larvae rotated sinusoidally at 0.25 Hz were analyzed to quantitate eye movements under infrared illumination. We observed a robust response that appeared as early as 72 hours post fertilization (hpf), which increased in amplitude over time. Unlike rotation about an earth horizontal axis, rotation about an earth vertical axis at 0.25 Hz did not evoke eye movements. Moreover, vestibular-induced responses were absent in mutant cdh23 larvae and larvae lacking anterior otoliths.Our results provide evidence for a functional vestibulo-oculomotor circuit in 72 hpf zebrafish larvae that relies upon sensory input from anterior/utricular otolith organs.

  View details for DOI 10.1186/1471-2202-11-110

  View details for PubMedID 20815905

  View details for PubMedCentralID PMC2941499

• In vivo evidence for transdifferentiation of peripheral neurons. Development (Cambridge, England) Wright, M. A., Mo, W., Nicolson, T., Ribera, A. B. 2010; 137 (18): 3047-56

  Abstract

  It is commonly thought that differentiated neurons do not give rise to new cells, severely limiting the potential for regeneration and repair of the mature nervous system. However, we have identified cells in zebrafish larvae that first differentiate into dorsal root ganglia sensory neurons but later acquire a sympathetic neuron phenotype. These transdifferentiating neurons are present in wild-type zebrafish. However, they are increased in number in larvae that have a mutant voltage-gated sodium channel gene, scn8aa. Sodium channel knock-down promotes migration of differentiated sensory neurons away from the ganglia. Once in a new environment, sensory neurons transdifferentiate regardless of sodium channel expression. These findings reveal an unsuspected plasticity in differentiated neurons that points to new strategies for treatment of nervous system disease.

  View details for DOI 10.1242/dev.052696

  View details for PubMedID 20685733

  View details for PubMedCentralID PMC2926955

• Synaptojanin1 is required for temporal fidelity of synaptic transmission in hair cells. PLoS genetics Trapani, J. G., Obholzer, N., Mo, W., Brockerhoff, S. E., Nicolson, T. 2009; 5 (5): e1000480

  Abstract

  To faithfully encode mechanosensory information, auditory/vestibular hair cells utilize graded synaptic vesicle (SV) release at specialized ribbon synapses. The molecular basis of SV release and consequent recycling of membrane in hair cells has not been fully explored. Here, we report that comet, a gene identified in an ENU mutagenesis screen for zebrafish larvae with vestibular defects, encodes the lipid phosphatase Synaptojanin 1 (Synj1). Examination of mutant synj1 hair cells revealed basal blebbing near ribbons that was dependent on Cav1.3 calcium channel activity but not mechanotransduction. Synaptojanin has been previously implicated in SV recycling; therefore, we tested synaptic transmission at hair-cell synapses. Recordings of post-synaptic activity in synj1 mutants showed relatively normal spike rates when hair cells were mechanically stimulated for a short period of time at 20 Hz. In contrast, a sharp decline in the rate of firing occurred during prolonged stimulation at 20 Hz or stimulation at a higher frequency of 60 Hz. The decline in spike rate suggested that fewer vesicles were available for release. Consistent with this result, we observed that stimulated mutant hair cells had decreased numbers of tethered and reserve-pool vesicles in comparison to wild-type hair cells. Furthermore, stimulation at 60 Hz impaired phase locking of the postsynaptic activity to the mechanical stimulus. Following prolonged stimulation at 60 Hz, we also found that mutant synj1 hair cells displayed a striking delay in the recovery of spontaneous activity. Collectively, the data suggest that Synj1 is critical for retrieval of membrane in order to maintain the quantity, timing of fusion, and spontaneous release properties of SVs at hair-cell ribbon synapses.

  View details for DOI 10.1371/journal.pgen.1000480

  View details for PubMedID 19424431

  View details for PubMedCentralID PMC2673039

• Vesicular glutamate transporter 3 is required for synaptic transmission in zebrafish hair cells. The Journal of neuroscience : the official journal of the Society for Neuroscience Obholzer, N., Wolfson, S., Trapani, J. G., Mo, W., Nechiporuk, A., Busch-Nentwich, E., Seiler, C., Sidi, S., Söllner, C., Duncan, R. N., Boehland, A., Nicolson, T. 2008; 28 (9): 2110-8

  Abstract

  Hair cells detect sound and movement and transmit this information via specialized ribbon synapses. Here we report that asteroid, a gene identified in an ethylnitrosourea mutagenesis screen of zebrafish larvae for auditory/vestibular mutants, encodes vesicular glutamate transporter 3 (Vglut3). A splice site mutation in exon 2 of vglut3 results in a severe truncation of the predicted protein product and morpholinos directed against the vglut3 ATG start site or the affected splice junction replicate the asteroid phenotype. In situ hybridization shows that vglut3 is exclusively expressed in hair cells of the ear and lateral line organ. A second transporter gene, vglut1, is also expressed in zebrafish hair cells, but the level of vglut1 mRNA is not increased in the absence of Vglut3. Antibodies against Vglut3 label the basal end of hair cells and labeling is not present in asteroid/vglut3 mutants. Based on the localization of Vglut3 in hair cells, we suspected that the lack of vestibulo-ocular and acoustic startle reflexes in asteroid/vglut3 mutants was attributable to a defect in synaptic transmission in hair cells. In support of this notion, action currents in postsynaptic acousticolateralis neurons are absent in asteroid/vglut3 mutants. At the ultrastructural level, mutant asteroid/vglut3 hair cells show a decrease in the number of ribbon-associated synaptic vesicles, indicating a role for Vglut3 in synaptic vesicle biogenesis and/or tethering to the ribbon body. Lack of postsynaptic action currents in the mutants suggests that the remaining hair-cell synaptic vesicles contain insufficient levels of glutamate for generation of action potentials in first-order neurons.

  View details for DOI 10.1523/JNEUROSCI.5230-07.2008

  View details for PubMedID 18305245

• The genetics of hearing and balance in zebrafish. Annual review of genetics Nicolson, T. 2005; 39: 9-22

  Abstract

  The zebrafish is an excellent model system for studying the molecular basis of inner ear development and function. The eggs develop ex utero and the ear is transparent for the first few weeks of life. Forward genetic screens and antisense technology have helped to elucidate the signaling pathways and molecules required for inner ear development and function. This review addresses the most recent advances in our understanding of how the ear forms and discusses the molecules in hair cells that are essential for sensing sound and movement in the zebrafish.

  View details for DOI 10.1146/annurev.genet.39.073003.105049

  View details for PubMedID 16285850

• Mutated otopetrin 1 affects the genesis of otoliths and the localization of Starmaker in zebrafish. Development genes and evolution Söllner, C., Schwarz, H., Geisler, R., Nicolson, T. 2004; 214 (12): 582-90

  Abstract

  Otoliths in bony fishes and otoconia in mammals are composite crystals consisting of calcium carbonate and proteins. These biominerals are part of the gravity and linear acceleration detection system of the inner ear. Mutations in otopetrin 1 have been shown to result in lack of otoconia in tilted and mergulhador mutant mice. The molecular function of Otopetrin 1, a novel protein that contains ten predicted transmembrane domains, however, has remained elusive. Here we show that a mutation in the orthologous gene in zebrafish is responsible for the complete absence of otoliths in backstroke mutants. We examined the localization of Starmaker, a secreted protein that is highly abundant in otoliths in backstroke mutants. Starmaker protein accumulated within cells of the otic epithelium, indicating a possible defect in secretion. Our data suggest that Otopetrin 1 in zebrafish may be involved in the protein trafficking of components required for formation of biominerals in the ear.

  View details for DOI 10.1007/s00427-004-0440-2

  View details for PubMedID 15480759

• Molecules and mechanisms of mechanotransduction 34th Annual Meeting of the Society-for-Neuroscience Goodman, M. B., Lumpkin, E. A., Ricci, A., Tracey, W. D., Kernan, M., Nicolson, T. SOC NEUROSCIENCE. 2004: 9220–22

  View details for DOI 10.1523/JNEUROSCI.3342-04.2004

  View details for PubMedID 15496654

• Myosin VI is required for structural integrity of the apical surface of sensory hair cells in zebrafish. Developmental biology Seiler, C., Ben-David, O., Sidi, S., Hendrich, O., Rusch, A., Burnside, B., Avraham, K. B., Nicolson, T. 2004; 272 (2): 328-38

  Abstract

  Unconventional myosins have been associated with hearing loss in humans, mice, and zebrafish. Mutations in myosin VI cause both recessive and dominant forms of nonsyndromic deafness in humans and deafness in Snell's waltzer mice associated with abnormal fusion of hair cell stereocilia. Although myosin VI has been implicated in diverse cellular processes such as vesicle trafficking and epithelial morphogenesis, the role of this protein in the sensory hair cells remains unclear. To investigate the function of myosin VI in zebrafish, we cloned and examined the expression pattern of myosin VI, which is duplicated in the zebrafish genome. One duplicate, myo6a, is expressed in a ubiquitous pattern during early development and at later stages, and is highly expressed in the brain, gut, and kidney. myo6b, on the other hand, is predominantly expressed in the sensory epithelium of the ear and lateral line at all developmental stages examined. Both molecules have different splice variants expressed in these tissues. Using a candidate gene approach, we show that myo6b is satellite, a gene responsible for auditory/vestibular defects in zebrafish larvae. Examination of hair cells in satellite mutants revealed that stereociliary bundles are irregular and disorganized. At the ultrastructural level, we observed that the apical surface of satellite mutant hair cells abnormally protrudes above the epithelium and the membrane near the base of the stereocilia is raised. At later stages, stereocilia fused together. We conclude that zebrafish myo6b is required for maintaining the integrity of the apical surface of hair cells, suggesting a conserved role for myosin VI in regulation of actin-based interactions with the plasma membrane.

  View details for DOI 10.1016/j.ydbio.2004.05.004

  View details for PubMedID 15282151

• Mutations in cadherin 23 affect tip links in zebrafish sensory hair cells. Nature Söllner, C., Rauch, G. J., Siemens, J., Geisler, R., Schuster, S. C., Müller, U., Nicolson, T. 2004; 428 (6986): 955-9

  Abstract

  Hair cells have highly organized bundles of apical projections, or stereocilia, that are deflected by sound and movement. Displacement of stereocilia stretches linkages at the tips of stereocilia that are thought to gate mechanosensory channels. To identify the molecular machinery that mediates mechanotransduction in hair cells, zebrafish mutants were identified with defects in balance and hearing. In sputnik mutants, stereociliary bundles are splayed to various degrees, with individuals displaying reduced or absent mechanotransduction. Here we show that the defects in sputnik mutants are caused by mutations in cadherin 23 (cdh23). Mutations in Cdh23 also cause deafness and vestibular defects in mice and humans, and the protein is present in hair bundles. We show that zebrafish Cdh23 protein is concentrated near the tips of hair bundles, and that tip links are absent in homozygous sputnik(tc317e) larvae. Moreover, tip links are absent in larvae carrying weak alleles of cdh23 that affect mechanotransduction but not hair bundle integrity. We conclude that Cdh23 is an essential tip link component required for hair-cell mechanotransduction.

  View details for DOI 10.1038/nature02484

  View details for PubMedID 15057246

• gemini encodes a zebrafish L-type calcium channel that localizes at sensory hair cell ribbon synapses. The Journal of neuroscience : the official journal of the Society for Neuroscience Sidi, S., Busch-Nentwich, E., Friedrich, R., Schoenberger, U., Nicolson, T. 2004; 24 (17): 4213-23

  Abstract

  L-type Ca2+ channels (LTCCs) drive the bulk of voltage-gated Ca2+ entry in vertebrate inner ear hair cells (HCs) and are essential for mammalian auditory processing. LTCC currents have been implicated in neurotransmitter release at the HC afferent active zone, the ribbon synapse. It is likely that LTCCs play a direct role in vesicle fusion; however, the subcellular localization of the channels in HCs has not been fully resolved. Via positional cloning, we show that mutations in a zebrafish LTCC encoding gene, cav1.3a, underlie the auditory-vestibular defects of gemini (gem) circler mutants. gem homozygous receptor mutant HCs display normal cell viability, afferent synaptogenesis, and peripheral innervation, yet exhibit strongly reduced extracellular potentials (approximately 50% of wild-type potentials). Apical FM1-43 uptake, however, is unaffected in gem mutant HCs, suggesting that mechanotransduction channels are functional. Using a Gem-specific antibody, we show that the bulk of Gem/Ca(v)1.3a immunoreactivity in HCs is restricted to basally located focal spots. The number and location of focal spots relative to nerve terminals, and their remarkable ring-shaped structure, which is reminiscent of synaptic dense bodies, are consistent with Gem/Ca(v)1.3a channels clustering at HC ribbon synapses.

  View details for DOI 10.1523/JNEUROSCI.0223-04.2004

  View details for PubMedID 15115817

• Control of crystal size and lattice formation by starmaker in otolith biomineralization. Science (New York, N.Y.) Söllner, C., Burghammer, M., Busch-Nentwich, E., Berger, J., Schwarz, H., Riekel, C., Nicolson, T. 2003; 302 (5643): 282-6

  Abstract

  The stone-like otoliths from the ears of teleost fishes are involved in balance and hearing and consist of calcium carbonate crystallites embedded in a protein framework. We report that a previously unknown gene, starmaker, is required in zebrafish for otolith morphogenesis. Reduction of starmaker activity by injection of modified antisense oligonucleotides causes a change in the crystal lattice structure and thus a change in otolith morphology. The expression pattern of starmaker, along with the presence of the protein on the growing otolith, suggest that the expression levels of starmaker control the shape of the otoliths.

  View details for DOI 10.1126/science.1088443

  View details for PubMedID 14551434

• NompC TRP channel required for vertebrate sensory hair cell mechanotransduction. Science (New York, N.Y.) Sidi, S., Friedrich, R. W., Nicolson, T. 2003; 301 (5629): 96-9

  Abstract

  The senses of hearing and balance in vertebrates rely on the sensory hair cells (HCs) of the inner ear. The central element of the HC's transduction apparatus is a mechanically gated ion channel of unknown identity. Here we report that the zebrafish ortholog of Drosophila no mechanoreceptor potential C (nompC), which encodes a transient receptor potential (TRP) channel, is critical for HC mechanotransduction. In zebrafish larvae, nompC is selectively expressed in sensory HCs. Morpholino-mediated removal of nompC function eliminated transduction-dependent endocytosis and electrical responses in HCs, resulting in larval deafness and imbalance. These observations indicate that nompC encodes a vertebrate HC mechanotransduction channel.

  View details for DOI 10.1126/science.1084370

  View details for PubMedID 12805553

• Mariner is defective in myosin VIIA: a zebrafish model for human hereditary deafness. Human molecular genetics Ernest, S., Rauch, G. J., Haffter, P., Geisler, R., Petit, C., Nicolson, T. 2000; 9 (14): 2189-96

  Abstract

  The zebrafish (Danio rerio) possesses two mechanosensory organs believed to be homologous to each other: the inner ear, which is responsible for the senses of audition and equilibrium, and the lateral line organ, which is involved in the detection of water movements. Eight zebrafish circler or auditory/vestibular mutants appear to have defects specific to sensory hair cell function. The circler genes may therefore encode components of the mechanotransduction apparatus and/or be the orthologous counterparts of the genes underlying human hereditary deafness. In this report, we show that the phenotype of the circler mutant, mariner, is due to mutations in the gene encoding Myosin VIIA, an unconventional myosin which is expressed in sensory hair cells and is responsible for various types of hearing disorder in humans, namely Usher 1B syndrome, DFNB2 and DFNA11. Our analysis of the fine structure of hair bundles in the mariner mutants suggests that a missense mutation within the C-terminal FERM domain of the tail of Myosin VIIA has the potential to dissociate the two different functions of the protein in hair bundle integrity and apical endocytosis. Notably, mariner sensory hair cells display morphological and functional defects that are similar to those present in mouse shaker-1 hair cells which are defective in Myosin VIIA. Thus, this study demonstrates the striking conservation of the function of Myosin VIIA throughout vertebrate evolution and establishes mariner as the first fish model for human hereditary deafness.

  View details for PubMedID 10958658

• Defective calmodulin-dependent rapid apical endocytosis in zebrafish sensory hair cell mutants. Journal of neurobiology Seiler, C., Nicolson, T. 1999; 41 (3): 424-34

  Abstract

  Vertebrate mechanosensory hair cells contain a narrow "pericuticular" zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1-43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function.

  View details for PubMedID 10526320

• A radiation hybrid map of the zebrafish genome NATURE GENETICS Geisler, R., Rauch, G. J., Baier, H., van Bebber, F., Bross, L., Dekens, M. P., Finger, K., Fricke, C., GATES, M. A., Geiger, H., Geiger-Rudolph, S., Gilmour, D., Glaser, S., Gnugge, L., Habeck, H., Hingst, K., Holley, S., Keenan, J., Kirn, A., Knaut, H., Lashkari, D., Maderspacher, F., Martyn, U., Neuhauss, S., Neumann, C., Nicolson, T., Pelegri, F., Ray, R., Rick, J. M., Roehl, H., Roeser, T., Schauerte, H. E., Schier, A. F., Schonberger, U., Schonthaler, H. B., Schulte-Merker, S., Seydler, C., Talbot, W. S., Weiler, C., Nusslein-Volhard, C., Haffter, P. 1999; 23 (1): 86-89

  Abstract

  Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

  View details for Web of Science ID 000082337300022

  View details for PubMedID 10471505

• Genetic analysis of vertebrate sensory hair cell mechanosensation: the zebrafish circler mutants. Neuron Nicolson, T., Rüsch, A., Friedrich, R. W., Granato, M., Ruppersberg, J. P., Nüsslein-Volhard, C. 1998; 20 (2): 271-83

  Abstract

  The molecular basis of sensory hair cell mechanotransduction is largely unknown. In order to identify genes that are essential for mechanosensory hair cell function, we characterized a group of recently isolated zebrafish motility mutants. These mutants are defective in balance and swim in circles but have no obvious morphological defects. We examined the mutants using calcium imaging of acoustic-vibrational and tactile escape responses, high resolution microscopy of sensory neuroepithelia in live larvae, and recordings of extracellular hair cell potentials (microphonics). Based on the analyses, we have identified several classes of genes. Mutations in sputnik and mariner affect hair bundle integrity. Mutant astronaut and cosmonaut hair cells have relatively normal microphonics and thus appear to affect events downstream of mechanotransduction. Mutant orbiter, mercury, and gemini larvae have normal hair cell morphology and yet do not respond to acoustic-vibrational stimuli. The microphonics of lateral line hair cells of orbiter, mercury, and gemini larvae are absent or strongly reduced. Therefore, these genes may encode components of the transduction apparatus.

  View details for PubMedID 9491988