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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC 2011 January 1.
Published in final edited form as:
PMCID: PMC2921932
NIHMSID: NIHMS221267

touché is required for touch evoked generator potentials within vertebrate sensory neurons

Abstract

The process by which light-touch in vertebrates is transformed into an electrical response in cutaneous mechanosensitive neurons is a largely unresolved question. To address this question we undertook a forward genetic screen in zebrafish (Danio rerio) to identify mutants exhibiting abnormal touch-evoked behaviors, despite the presence of sensory neurons and peripheral neurites. One family, subsequently named touché, was found to harbor a recessive mutation which produced offspring that were unresponsive to light-touch, but responded to a variety of other sensory stimuli. The optogenetic activation of motor behaviors by touché mutant sensory neurons expressing ChannelRhodopsin-2 suggested that the synaptic output of sensory neurons was intact, consistent with a defect in sensory neuron activation.

To explore sensory neuron activation we developed an in vivo preparation permitting the precise placement of a combined electrical and tactile stimulating probe upon eGFP positive peripheral neurites. In wild type larva electrical and tactile stimulation of peripheral neurites produced action potentials detectable within the cell body. In a subset of these sensory neurons an underlying generator potential could be observed in response to subthreshold tactile stimuli. A closer examination revealed that the amplitude of the generator potential was proportional to the stimulus amplitude. When assayed touché mutant sensory neurons also responded to electrical stimulation of peripheral neurites similar to wild type larvae, however tactile stimulation of these neurites failed to uncover a subset of sensory neurons possessing generator potentials. These findings suggest that touché is required for generator potentials, and that generator potentials underlie responsiveness to light-touch in zebrafish.

Keywords: Somatosensory, Mechanosensory, Touch, Mutant, Nociception, Patch Clamp

Introduction

The identification of mechanosensitive proteins in vertebrates using traditional approaches, such as affinity purification and expression cloning, has been hampered by the lack of high-affinity ligands and the likelihood that the mechanosensitive proteins must be co-expressed with additional membrane and cytoskeletal elements to function properly. Thus many researchers have looked to organisms amenable to forward genetic screens, such as C. elegans and Drosophila, in the hopes of identifying mechanosensitive proteins conserved across evolution. However many of the candidate proteins identified from screens in these invertebrate organisms (Colbert and Bargmann, 1995; Walker et al., 2000; Liedtke et al., 2003; O’Hagan et al., 2005; Kindt et al., 2007) have been shown to be unessential for light-touch in vertebrates (Sidi et al., 2003; Suzuki et al., 2003; Drew et al., 2004; Bautista et al., 2006; Kwan et al., 2006). These results raise the possibility that the proteins that mediate mechanotransduction in touch sensitive neurons may not be conserved across phylogeny. Therefore we and others have turned to zebrafish (Granato et al., 1996; Haffter et al., 1996), a vertebrate model organism amenable to both genetic and in vivo electrophysiological manipulations.

Zebrafish embryos develop externally, possess a relatively simple nervous system, and respond to tactile stimuli within the first day of development (Saint-Amant and Drapeau, 1998). The neurons that activate touch-evoked behaviors are segregated into two groups: trigeminal ganglia neurons relay tactile stimuli delivered to the craniofacial region (Sneddon, 2003), and Rohon-Beard neurons (RBs) within the spinal cord that relay tactile stimuli to the trunk and tail regions (Clarke et al., 1984). Both groups of neurons are likely polymodal as they have been shown to exhibit differential expression of several receptors implicated in nociception (Cockayne et al., 2000; Kucenas et al., 2003; Cockayne et al., 2005), and ubiquitous expression of TRPA1b which is essential for responsiveness to the noxious compound mustard oil (Prober et al., 2008).

In a previous study, the membrane properties of RBs from several potential mechanosensitive mutants were chosen for detailed electrophysiological analysis (Ribera and Nusslein-Volhard, 1998). Results from this study revealed that most mutant sensory neurons possessed defects within their excitable properties, and therefore are not strong candidates for mutations affecting mechanosensation. Recognizing the need for additional touch-unresponsive mutants we undertook another forward genetic screen to identify novel mutants with abnormal touch-evoked behaviors. From our screen one family (mi173), subsequently named touché (toumi173), was found to harbor a recessive mutation which resulted in offspring that were selectively unresponsive to light-touch. Employing a novel in vivo recording technique we failed to uncover a subset of sensory neurons in touché mutants which responded to tactile stimulation with generator potentials. These findings indicate that touché is required for generator potentials, and that sensory neurons with generator potentials underlie responsiveness to light-touch in zebrafish.

Materials and Methods

Animal care and use

Zebrafish were bred and raised according to approved guidelines set forth by the Animal Experimentation Ethics Committee, Université de Montréal and the University Committee on Use and Care of Animals, University of Michigan. Staging of embryos was performed as described previously (Kimmel et al., 1995). The touché allele mi173 (toumi173) was identified in a screen conducted at the University of Michigan, Ann Arbor following previously published procedures (Haffter and Nusslein-Volhard, 1996).

Mapping

A mapping family for touché was established by crossing a touché male carrier (Michigan genetic background) with a wild type WIK female (Zebrafish Resource Center, Eugene, Oregon). Offspring from this mapping family were subjected to bulk segregate analysis (Postlethwait et al., 1994) according to the Zon lab protocol (http://zfrhmaps.tch.harvard.edu) using 20 wild type sibling and 20 touché mutants.

Behavioral analysis

All reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Embryos at ~24 hours post-fertilization (hpf) were dechorionated with pronase, staged and segregated according to their responsiveness to touch. Tactile and nociceptive stimuli were delivered by striking the tail of an embryo with a sideways motion up to 3 times with a pair of No.5 forceps and pinching of the tail, respectively. The speed of tactile stimuli was assessed offline by measuring tip displacement (Fig. 1A) as a function of time. Embryos were exposed to noxious stimuli individually in 24 well plates: allyl-isothiocyanate (Acros Organics, Geel, Belgium) at 500μM mustard oil in sham (1% DMSO, v/v in Evans), or acidified Evans (pH 4.5 adjusted with acetic acid, sham Evans pH 7.5).

Figure 1
Touché mutants fail to respond to light-touch, but respond to tail-pinching. (A) Time-lapse images of responses to tactile stimuli applied to the tail of a 26 hours post-fertilization (hpf) embryos. Scale bar here and below 500μm. (B) ...

Electrically-evoked bouts of swimming used in the stopping assay were elicited using a bipolar electrode and an A-M Systems (Sequim, WA) Model 2100 isolated pulse stimulator in larvae embedded in 1% (w/v) low-melting point agarose. To mimic head-first collisions a jet of water was applied to the head (20psi, 50ms) using a Picospritzer III (Parker Hannifin, Fairfield, NJ). The electrical stimulus was paired with an LED (Fig. 3A), and the timing of both was controlled by pClamp 8 software using a Digidata 1200 interface (Axon Instruments, Union City, CA).

Figure 3
Touché mutants lack a stopping response. (A) Time-lapse images of wild type and touché mutants at 48–52hpf during electrically-evoked swimming trials wherein the counter stimulus was applied. The electrical stimulus delivered by ...

Acoustic-vestibular responses were assayed between 2 and 4 days post-fertilization (dpf). At 2dpf the ability of lateral-line stimuli to activate motor behaviors was examined by placing larvae individually into the center of a 60mm dish in 3ml of fish water (Westerfield, 2000). To the side of the dish was added 1ml of fish water by pipette to cause passive displacement of the larvae. Larvae were scored as positive responders if motor activity was observed before the larvae stopped moving passively. The contribution of inner-ear hair cell function to body posture was assayed at 3dpf by counting the number of dorsal-up orientated larva. The ability to respond to acoustic stimuli was assayed by placing larvae into 60mm dishes in 4ml of fish water atop a metal plate. The plate was sharply struck with a metal rod from a constant height to cause a loud audible sound. Some experiments were performed on the mutants and siblings simultaneously to rule out subtle changes in threshold for detection.

Light-evoked (480nm) activation of motor behaviors was performed by injecting 50pg of plasmid containing Channelrhodopsin-2 coupled to Enhanced Yellow Fluorescent Protein (ChR2-eYFP) under the control of the Isl1-SS-enhancer:Gal4-VP16:UAS-E1b promoter (Douglass et al., 2008) diluted in 5nl of water containing 0.01% phenol red into freshly fertilized embryos from touché carrier incrosses at the 1–2 cell stage using a Nanoinject II system (Drummond Scientific, Broomall, PA). Embryos were screened at ~24–27hpf for expression eYFP and touch responsiveness using an Olympus dissecting microscope (SZX7) fitted with epifluorescence. Light-evoked motor behaviors were triggered manually by opening a shutter on individual embryos in 24 well plates.

Behaviors were recorded at 30 and 200Hz using FleaR2 (FL2-20S4M-C) and Grasshopper (GRAS-03K2M-C) cameras from Point Grey (Richmond, BC). Images were captured with PRG Flycap, and analyzed off-line using ImageJ (http://rsbweb.nih.gov/ij/).

FM1-43 dye uptake and immunohistochemistry

For dye uptake FM1-43 (Invitrogen, Carlsbad, CA), a dye thought to enter hair cells through the mechanotransduction channel, was applied at 3 μM in Evans (see below) for 30s. Thereafter larvae were washed in Evans containing the local anesthetic tricaine (0.02% w/v) five times (5X) for 10s each. Dye uptake was assessed on a dissecting microscope fitted with a Qicam FAST 1394 camera from Qimaging (Surrey, BC).

For labeling eGFP and ChR2-eYFP larvae were fixed in 4% paraformaldehyde for 30min and then washed 5X for 10min in washing buffer (PBS containing 0.1% Triton X-100), and 2X for 10min in blocking buffer (wash buffer containing 2mg/ml bovine serum albumin). Rabbit anti- EGFP (Torrey Pines Biolabs, East Orange, NJ) primary was diluted 1/1000 in blocking buffer and bound overnight. Thereafter larvae were washed 5X for 10min in washing buffer, 2X for 10min in blocking buffer. Secondary antibody (anti-rabbit Alexa-488, Molecular Probes) diluted 1/1000 in blocking buffer was bound for 4h. Larvae were then washed 5X for 10min with washing buffer and then mounted in 70% glycerol in PBS. Images were captured with a spinning disk confocal (Quorum) microscope (Olympus, BX-51). Three segments above the anus were analyzed for Rohon-Beard (RBs) cell counts. For trigeminal neuron cell counts the average diameter of a trigeminal neuron was first determined to be ~8μm. Optical stacks of trigeminal ganglia were then divided into 8μm optical sections, starting 4μm into the ganglion, and neurons with discernable cell bodies were counted under double blind conditions. Innervation of the skin by peripheral neurites was determined by thresholding all neurite projections in the skin for the 3 somites centered on the anal somite under double blind conditions. Thereafter the percentage of the total thresholded area relative to total area was determined using ImageJ.

Recording methods

Electrophysiological recording from zebrafish were obtained from neurons at room temperature using methods similar to those previously described (Hamill et al., 1981; Ribera and Nusslein-Volhard, 1998; Drapeau et al., 1999). In brief larvae were anesthetized in Evans recording solution (in mM): 134 NaCl, 2.9 KCl,2.1 CaCl2, 1.2 MgCl2, 10 glucose, 10 HEPES, pH 7.5 with NaOH containing 0.02% (w/v) tricaine. The skin of a larva pinned to a 35mm SylgardR coated dish was removed with a pair of No.5 forceps. The solution was exchanged with Evans containing 15μM curare flowing throughout the recording session at ~1ml/min. To gain access to the spinal cord the bath solution was replaced with recording solution containing 4mg/ml collagenase Type XI and incubated until the muscle started to separate (~10min). Thereafter the muscle was peeled away using suction applied to a broken pipette (~50μm). The internal recording solution contained (in mM): 116 K-gluconate, 16 KCl, 2 MgCl2, 10 HEPES, 10 EGTA, at pH 7.2 with KOH and 0.1% SulforhodamineB for cell type identification. Borosilicate glass electrodes had resistances of 5–8MΩ when filled with internal recording solution. Recordings were made with an Axopatch 200B amplifier (Axon Instruments, Union City, CA) low passed filtered at 1–5kHz and sampled at 1–10kHz. Electrical and tactile stimuli were delivered through the use of a bipolar stimulating probe (A-M Systems Model 2100) controlled by a piezoelectric stimulator (Moffatt and Hume, 2007). Probes were placed upon eGFP positive neurites ~100μm from the cell body. Generator potential amplitudes were normalized for RB input resistance, and scaled to zero which represents the displacement preceding the first observable membrane depolarization as described previously (Drew et al., 2002). Data acquisition was controlled by pClamp 10 software using a Digidata 1440A interface. The initial data analysis was done with Clampfit 10, and figures were prepared using Sigma Plot 11.0.

Results

Touché is a novel light-touch unresponsive mutant

In contrast to other organisms a clear difference between light-touch and nociceptive stimuli, and their related behavioral responses, has not been defined for zebrafish. In an attempt to address this deficit we first examined the behavioral responsiveness of zebrafish embryos to increasing forces delivered by calibrated animal hairs and commercially available von Frey filaments. However the lowest hairs and filaments obtainable (~5 – 8mg) consistently resulted in the activation of motor behaviors in zebrafish embryos. As work from other organisms predicted that zebrafish embryos might display a graded responsiveness to increasing forces we reasoned that ~5mg was greater than the minimal force detectable by zebrafish embryos. Therefore we explored whether varying the speed at which a tactile stimulus was delivered might uncover speeds, and related forces at which zebrafish embryos exhibited a graded responsiveness to tactile stimuli. In response to stimuli below 5mm/s all zebrafish embryos failed to respond to touch (Fig. 1A–B). Increasing the speed of delivery resulted in a corresponding increase in the percentage of responsive embryos, until at 20mm/s when all embryos examined were responsive. Closer examination also revealed that the maximum number of coils performed by zebrafish embryos in response to light-touch was three.

To elicit a more nociceptive response the tips from a pair of forceps were closed upon the caudal fin, herein referred to as tail-pinching (Fig. 1C). In response to tail-pinching zebrafish embryos were found to consistently perform 3 or more coils, with a range of 3 – 12 coils (Fig. 1D). Thus zebrafish embryos exhibit at least two different levels of responses to increasing forces, with light-touch stimuli evoking ≤ 3 coils and nociceptive stimuli evoking ≥ 3 coils.

In a screen for novel zebrafish mutants exhibiting abnormal touch-evoked motor behaviors one family (mi173), subsequently named touché (toumi173), was found to harbor a recessive mutation which resulted in offspring that were unresponsive to light-touch (Fig. 1A–B), but retained the ability to respond to tail-pinching (Fig. 1C–D). To determine whether touché represented a new touch-unresponsive mutant we rough mapped the touché locus to chromosome 2. Of the previously identified touch-unresponsive mutants (Granato et al., 1996) only macho (maott261) has been mapped to chromosome 2 (www.zfin.org), and therefore complementation analysis was performed with macho. Pair wise crosses between macho and touché complemented the mutant phenotypes indicating that touché is not a new allele of macho. Collectively these findings indicate that touché represents a new touch-unresponsive mutant, deficient in the ability to respond light-touch.

Touché mutants are responsive to multiple noxious stimuli

The finding that touché mutants were unresponsive to light-touch, but responded to tail-pinching prompted an investigation into the responsiveness of touché mutants to other modes of sensory stimuli. During the first few days of development, zebrafish are responsive to the compound commonly known as mustard oil (allyl-isothiocyanate) and to low pH. The behavioral responsiveness of zebrafish to mustard oil was shown to require TRPA1b, a member of the TRP superfamily of cation channels expressed by zebrafish sensory neurons (Prober et al., 2008). To examine the responsiveness of wild type and touché mutants to mustard oil, embryos were transferred into petri dishes preloaded with mustard oil. Under control conditions (Evan’s containing 1% DMSO) wild type and touché mutants were found to be predominantly inactive (Fig. 2A–B). However when exposed to mustard oil wild type and touché mutants were both significantly more active. Comparisons failed to uncover a significant difference in the time active in mustard oil between wild type and touché mutants.

Figure 2
Touché mutants respond normally to noxious stimuli. (A) Time-lapse images of wild type and touché mutants in the presence of mustard oil (500μM) respond with increased motor activity at 26hpf. Scale bar 500μm. (B) Analysis ...

In contrast to mustard oil, the process by which low pH is perceived by zebrafish and transformed into a behavioral response is unknown. However it is likely to involve member(s) of the Acid Sensing Ion Channel (ASIC) family and/or TRPV1, which are known to be expressed by a subset of sensory neurons in zebrafish (Paukert et al., 2004; Caron et al., 2008). To determine whether touché mutants were capable of perceiving and responding to low pH we exposed larvae to acidified Evan’s recording solution (pH 4.5). When compared to normal Evan’s (pH 7.5) wild type and touché mutants were both found to be significantly more active (Fig. 2B). Comparisons of time active between wild type and touché mutants again failed to uncover a significant difference. Thus touché is not required for the behavioral responsiveness to these noxious stimuli.

Touché mutants lack a stopping response

After the onset of swimming it was noted that touché mutants often failed to stop swimming upon head-first collisions with the sides of dishes. This “stopping response” has been shown to require feedback from mechanosensitive trigeminal neurons in Xenopus laevis embryos (Boothby and Roberts, 1992a, b). To examine this finding in detail, larvae were restrained in agar, with the tail and head regions exposed to allow for the delivery of stimuli and swimming. Larvae were then induced to swim by passing a current across the caudal tail using a bipolar stimulating electrode (Fig. 3A), which has been shown to activate zebrafish sensory neurons in vivo (Higashijima et al., 2003). In an equal number of trials a puff of water referred to as the counter stimulus (cs) was delivered to the head 500ms after an electrical stimulus (es). In wild type larvae the counter stimulus resulted in a significant reduction in the duration of swimming (Fig. 3B). In contrast, no significant reduction in the duration of swimming was observed in touché mutants following the counter stimulus. Therefore touché mutants lack two sensory evoked behaviors known to require input from mechanosensitive neurons.

Touché mutants respond to acoustic-vestibular stimuli

To rule out a wholesale disruption of mechanosensitive processes in touché mutants, we assayed behaviors dependent upon input from mechanosensitive hair cells of the inner ear and the lateral-line. Mechanotransduction within these hair cells allow zebrafish to respond to water displacement, acoustic stimuli (tapping), and contribute to a dorsal-up body posture. As a first level of characterization of mechanotransduction within these hair cells, we exposed embryos to the dye FM1-43, which is thought to enter hair cells through the mechanotransduction channel (Seiler and Nicolson, 1999). Following incubation in FM1-43, wild type and touché mutant hair cells both exhibited uptake of the fluorescent dye (Fig 4A).

Figure 4
Touché mutants respond to acoustic-vestibular stimuli. (A) FM1-43 dye uptake by hair cells of the lateral-line (the location of hairs cells within a few neuromasts are indicated arrows) is present in both wild type and touché mutants 48–52hpf. ...

Behavioral defects mediated by mutations of proteins essential to the function of the lateral-line and inner ear hair cells become obvious between the second and fourth days of development (Nicolson et al., 1998). When assayed, no differences in the responsiveness to water displacement (Fig. 4B–C), or tapping (Fig. 4D–E) were found between wild type and touché mutants. Similarly wild type and touché mutants were both found to be dorsally orientated on day four (Fig. 4B and 4D). Thus mechanotransduction within touché mutant hair cells appears present, indicating that the touché mutation does not affect all mechanically sensitive cells.

Sensory neuron anatomy in touché mutants

The behavioral results thus far suggest a role for touché within touch-sensitive neurons. This role could be functional, such as in the relay of tactile stimuli to second order neurons. Alternatively touché could be required developmentally for the differentiation and/or retention of touch sensitive neurons. As a first step in distinguishing between these two possibilities, the presence and morphology of sensory neurons in zebrafish were compared between wild type and touché mutants. In zebrafish, tactile stimuli are conferred by two groups of touch sensitive neurons: trigeminal neurons relay touch to the craniofacial region and Rohon-Beard cells (RBs) relay touch to the trunk and tail regions. To facilitate the identification and characterization of trigeminal neurons and RBs the touché mutation was crossed into a stable transgenic line (Uemura et al., 2005) expressing eGFP under the control of a sensory neuron enhancer-promoter (ssx-mini-ICP:eGFP). When compared to wild type siblings, touché mutants were found to possess a similar number of RBs per somite, trigeminal neurons per ganglia, and an indistinguishable amount of neurite coverage by sensory neurons (Fig. 5). Thus there does not appear to be a gross morphological difference between the sensory neurons of wild type and touché mutants.

Figure 5
Gross morphology of sensory neurons in touché mutants appears normal. (A) Whole-mount labeling of eGFP positive sensory neurons from a touché mutant larvae at 52hpf. Scale bar 200μm. (B) Craniofacial labeling with trigeminal ganglia ...

Light-evoked activation of sensory neurons triggers motor behaviors in touché mutants

Given that sensory neurons appear normal in touché mutants we examined whether the exogenous activation of sensory neurons could trigger motor behaviors. Recently, exposure to blue light in embryos expressing Channelrhodopsin-2 fused to enhanced Yellow Fluorescent Protein (ChR2-eYFP) under the control of a sensory neuron promoter was shown to be sufficient to trigger action potentials within trigeminal neurons, and activation of motor behaviors (Douglass et al., 2008). The injection of the ChR2-eYFP construct resulted in embryos with several fluorescent cells in locations consistent with sensory neurons (Fig. 6A). We found that exposure to blue light (~1s) at ~27hpf triggered motor behaviors in both wild type and touché mutant embryos positive for eYFP expression (Fig. 6B). In contrast, exposure to red light failed to evoke motor behaviors in either wild type or touché mutant embryos positive for eYFP expression (Fig. 6C). As a control, embryos expressing eGFP within the same neurons failed to respond to blue light exposure. Thus the reaction to blue light required the injection of the ChR2-eYFP construct. These findings indicate that electrical transduction downstream of action potential generation in sensory neurons appears present, and capable of activating motor behaviors within touché mutants.

Figure 6
Light-evoked activation of motor behaviors in touché mutants. (A) Lateral view of a 28hpf touché mutant embryo with several RBs positive for expression of ChannelRhodopsin-2 conjugated to eYFP (ChR2-eYFP). Scale bar 200μm. (B) ...

Excitable properties of touché mutant sensory neurons are normal

Previously the development of ionic currents in zebrafish RBs were examined (Ribera and Nusslein-Volhard, 1998), wherein the maturation of an overshooting action potential in sensory neurons was suggested to underlie the development of touch responsiveness. This hypothesis was supported by the examination of sensory neuron membrane properties from the touch-unresponsive mutant macho, which revealed that macho mutants fail to develop overshooting action potentials despite the normal development of other ionic currents. Based on these findings the excitable properties of touché RBs were examined in vivo employing a preparation wherein the skin and muscle contralateral to target RBs was removed leaving the peripheral neurites intact (Fig. 7A). Using this preparation we found that RBs from both wild type and touché mutants initiate action potentials in response to depolarizing current injections to the cell body (Fig. 7B). When compared wild type and touché mutant RBs were found to exhibit similar resting membrane potentials, action potential thresholds, and amplitudes of overshoot and undershoot (Fig. 7C).

Figure 7
Electrical stimulation of peripheral neurites triggers action potentials within touché mutant RBs. (A) Schematic of the in vivo recording preparation which allows for electrical and mechanical stimulation of the same peripheral neurite. (RB) Rohon-Beard ...

Next a bipolar stimulating probe, attached to a piezoelectric motor, was placed upon eGFP positive neurites ~100μm from the cell body in the stable transgenic line described above (Fig. 5). This approach allowed for the electrical and mechanical stimulation (see below) of the same neurite belonging to one RB. In both wild type and touché mutant RBs depolarization of the peripheral neurite evoked action potentials detectable within the cell body (Fig. 7B). A similar stimulus applied ~15μm rostral or caudal to the peripheral neurite failed to activate sensory neurons indicating that the bipolar stimulating probe was focally exciting neurites. When again compared wild type and touché mutant RBs were found to exhibit similar action potential thresholds, and generated action potentials with similar amplitudes of overshoot and undershoot (Fig. 7C). Thus the lack of responsiveness to light-touch in touché mutants cannot be explained by a difference in the excitability of touché mutant sensory neurons, or by a breakdown in electrical transduction from the peripheral neurites to the cell body.

Touché mutants lack sensory neurons with generator potentials

To examine if tactile stimuli delivered to the peripheral neurite could evoke action potentials detectable within the cell body in touché mutants the bipolar stimulating probe was driven into the peripheral neurite with a piezoelectric motor. Previously the movement of probes with the piezoelectric motor was shown to be controllable on the μm level, with movements being completed within hundreds of microseconds (Moffatt and Hume, 2007). We found that in wild type RBs, wherein electrical stimulation of the peripheral neurite evoked action potentials detectable within the cell body (Fig. 7B), mechanical stimulation also triggered action potentials detectable within the cell body (n = 19/19; Fig. 8A). A closer examination revealed that RBs in wild type larvae could be subdivided into two groups: those possessing generator potentials (Type I, n = 9/19), and those wherein a generator potential was not observed (Type II, n = 10/19; Fig. 8A). Employing the same stimulating technique in touché mutants, which had also responded to electrical stimulation of the peripheral neurite (n = 16/16), failed to uncover RBs with generator potentials (Type I, n = 0/16).

Figure 8
Touché mutants lack sensory neurons with generator potentials. (A) (Top) in a subset of RBs (Type I, n = 9/19) a subthreshold mechanical stimulus (black) applied to the peripheral neurite results in a generator potential (arrows) at the onset ...

Generator potentials are graded membrane depolarizations induced in the termini of touch sensitive neurons, which upon reaching sufficient amplitudes, trigger action potentials in sensory afferents. To examine whether the membrane depolarizations observed in zebrafish RBs in response to mechanical stimuli were generator potentials a series of subthreshold mechanical stimuli were delivered to the peripheral neurite of several RBs (n = 6). In response to increasing mechanical stimuli membrane depolarizations were found to be graded (Fig. 8B), and after normalizing for RB input resistances (Fig. 8C) strongly correlated with displacement (r2 = 0.97). Finally to ensure that the graded membrane depolarizations were not failed action potentials we applied tetrodotoxin to the recording chamber which prevented spiking (n = 2), but failed to block the graded membrane depolarizations indicating that the observed graded membrane depolarizations were in fact generator potentials. Collectively these findings suggest that the touché mutant phenotype results from a lack of sensory neurons with generator potentials, and implies that light-touch in zebrafish is conveyed by mechanosensitive neurons with generator potentials.

Discussion

In a screen for novel touch-unresponsive zebrafish mutants we uncovered one mutant, subsequently name touché, which was unresponsive to light-touch. Although rough mapping indicated that touché could be a new allele of macho, a previously identified touch-unresponsive mutant (Granato et al., 1996), pair wise crosses with macho carriers complemented the touché mutant phenotype. Thus touché represents a new touch-unresponsive mutant.

Upon closer examination of the touché mutant phenotype it was noted that touché mutants also lacked a stopping response, a behavior known to require input from trigeminal touch-sensitive neurons (Boothby and Roberts, 1992a, b). This finding suggested that the touché mutation might affect all mechanosensitive processes, however touché mutants were found to exhibit acoustic-vestibular behaviors which rely on input from mechanosensitive hair cells. Coupled with the normal responsiveness of touché mutants to noxious stimuli (mustard oil and low pH) indicates that the touché mutation only affects behaviors dependent upon input from touch-sensitive neurons.

Potentially in support of a requirement for touché in the regulation of behaviors dependent upon input from touch-sensitive neurons were the findings that touché mutants swam for longer durations following electrical stimulation, and were more spontaneously active at four days, a time point wherein larvae are rather immotile and often found to be stuck to the sides of objects. These findings may reflect an absence of sensory feedback from mechanosensitive neurons such as those that innervate mucous cells which likely alert zebrafish larvae that they have encountered an object suitable for adherence similar to Xenopus laevis embryos (Boothby and Roberts, 1992a, b). Alternatively the increase in spontaneous activity in mutants could reflect the absence of an inhibitory influence of touché on the locomotor network which underlies swimming in zebrafish, or a requirement of sensory input in modulating locomotor network activity.

In an attempt to understand the contribution of touché to the activation of touch-evoked behaviors we first considered that the inability of touché mutants to respond to light-touch could be the result of absent, or aberrant touch-sensitive neurons. However comparisons between wild type and touché mutants revealed that mutants possessed a similar number of RBs per somite, trigeminal neurons, and extent of skin innervation by peripheral neurites. While these findings suggest that the mutant phenotype is the result of a functional loss of touché within touch-sensitive neurons a limitation of this comparison exists which stems from a general lack of knowledge concerning sensory neurons in zebrafish. Presently it is unknown whether zebrafish sensory neurons are a heterogenous or homogenous population of mono or polymodal sensitive neurons. Therefore while touché mutants have a similar overall number of sensory neurons, touché mutants could lack a subtype of sensory neuron responsive to light-touch while having more of another type. A detailed analysis of sensory neuron responsiveness to various stimuli in zebrafish is needed to examine this possibility.

We next considered that touché might be required functionally within touch-sensitive neurons for the transduction of tactile stimuli to second order neurons. This requirement could be (but not limited to) within the mechanotransduction complex responsible for the transformation of tactile stimuli into an electrical signal within touch-sensitive neurons, in neurites for the propagation of the action potentials from the peripheral neurite to the central synapse, or at the level of the central synapse in transmitter release. In an attempt to discern between these possibilities we attempted to drive motor behaviors in touché mutants optogentically with ChannelRhodopsin-2. We found that wild type and touché mutants expressing ChannelRhodopsin-2 in sensory neurons responded to blue light (480nm) with motor behaviors. These findings indicate that touché mutant sensory neurons can activate second order neurons leading to motor behaviors, suggestive of a defect within sensory neuron activation.

To explore the activation of sensory neurons in vivo we developed and employed a novel recording preparation which allowed for electrical and mechanical stimulation of the same peripheral neurite. This preparation revealed that the transduction of an electrical signal from the peripheral neurite to the cell body was intact in both wild type and touché mutants. Furthermore tactile stimuli applied to the same neurite triggered action potentials detectable within the cell bodies of both wild type and touché mutants. However in contrast to wild type RBs we did not observe any touché mutant RBs wherein mechanically evoked action potentials exhibited generator potentials (Type I).

The generator potentials observed in Type I zebrafish RBs are rapidly adapting with activation at both the onset and offset of a stimulus. When combined with the apparent morphology of zebrafish RBs, this suggests that the Type I cutaneous mechanoreceptors described here are most similar to mammalian Aδ “Free Nerve Endings”, which in addition to mediating tactile stimuli are thought to communicate thermo and nociceptive stimuli. In support of this comparison, are reports suggesting that RBs are polymodal in nature, as they express multiple receptors (Kucenas et al., 2003; Prober et al., 2008), which are known to participate in the integration of various sensory stimuli in other vertebrates (Cockayne et al., 2000; Cockayne et al., 2005; Bautista et al., 2006; Kwan et al., 2006). In contrast, Type II neurons which lack generator potentials may represent nociceptive neurons which are responding indirectly to tissue damage rather than directly to tactile stimuli. Further experiments involving calibrated low force probes and conduction velocity assays in zebrafish will provide insight into the comparison with mammalian sensory neurons.

Collectively the findings presented here indicate that touché is required either developmentally for the differentiation/retention of light-touch sensitive neurons, or functionally within a subset of touch-sensitive neurons for the conversion tactile stimuli into action potentials via a generator potential dependent process. Discerning between these two possibilities will require a better understanding of the zebrafish sensory neuron population, insights into the genetic pathways governing the development of sensory neuron subtypes, and ultimately the molecular identification of the touché locus.

Acknowledgments

We would like to thank Pierre Drapeau, Mathieu Lachance, and members of the Kuwada and Drapeau laboratories for helpful comments regarding the preparation of this manuscript. We thank Guy Laliberté and Marina Drits for fish care. We would also like to thank Dr. Hitohsi Okamoto (RIKEN, Wako-city Saitama, Japan) for the ssx-mini-ICP:eGFP stable transgenic line of zebrafish, Dr. Florian Engert (Harvard University, Boston, MA USA) for the ChR2-eYFP construct, and Dr. Angeles B. Ribera (University of Colorado, Denver, CO USA) for the mutant macho (maott261). Present address of HH: Division of Biological Science, Nagoya University, Nagoya, Japan. Present address of WWC: University of California, San Francisco School of Medicine, San Francisco, CA. Present address of WZ: Life Science Institute, University of Michigan, Ann Arbor, MI.

The work reported here was supported by a grant from the National Science and Engineering Research Council of Canada (to L.S.-A), a Canadian Institutes of Health Research operating grant (L.S.-A), a chercheur boursier award and the GRSNC from the Fond de Recherche en Santé du Québec (to L.S.-A), a grant from the National Institute of NeurologicalDisorders and Stroke, Grant NS054731 (to J.Y.K.), the National Science Foundation, Grant #0725976 (to J.Y.K.), a Long-Term Fellowshipfrom the Human Frontier Science Program (to H.H.), and in part by a Center for OrganogenesisTraining Grant 5-T32-HD007505 (to W.W.C.).

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