Ashwin A. Bhandiwad

Auditory sensitivity of larval zebrafish (Danio rerio) measured using a behavioral prepulse inhibition assay.

SUMMARY Zebrafish (Danio rerio) have become a valuable model for investigating the molecular genetics and development of the inner ear in vertebrates. In this study, we employed a prepulse inhibition (PPI) paradigm to assess hearing in larval wild-type (AB) zebrafish during early development at 5–6 days post-fertilization (d.p.f.). We measured the PPI of the acoustic startle response in zebrafish using a 1-dimensional shaker that simulated the particle motion component of sound along the fishs dorsoventral axis. The thresholds to startle-inducing stimuli were determined in 5–6 d.p.f. zebrafish, and their hearing sensitivity was then characterized using the thresholds of prepulse tone stimuli (90–1200 Hz) that inhibited the acoustic startle response to a reliable startle stimulus (820 Hz at 20 dB re. 1 m s−2). Hearing thresholds were defined as the minimum prepulse tone level required to significantly reduce the startle response probability compared with the baseline (no-prepulse) condition. Larval zebrafish showed greatest auditory sensitivity from 90 to 310 Hz with corresponding mean thresholds of −19 to −10 dB re. 1 m s−2, respectively. Hearing thresholds of prepulse tones were considerably lower than previously predicted by startle response assays. The PPI assay was also used to investigate the relative contribution of the lateral line to the detection of acoustic stimuli. After aminoglycoside-induced neuromast hair-cell ablation, we found no difference in PPI thresholds between treated and control fish. We propose that this PPI assay can be used to screen for novel zebrafish hearing mutants and to investigate the ontogeny of hearing in zebrafish and other fishes.

Exposure to Advertisement Calls of Reproductive Competitors Activates Vocal-Acoustic and Catecholaminergic Neurons in the Plainfin Midshipman Fish, Porichthys notatus

While the neural circuitry and physiology of the auditory system is well studied among vertebrates, far less is known about how the auditory system interacts with other neural substrates to mediate behavioral responses to social acoustic signals. One species that has been the subject of intensive neuroethological investigation with regard to the production and perception of social acoustic signals is the plainfin midshipman fish, Porichthys notatus, in part because acoustic communication is essential to their reproductive behavior. Nesting male midshipman vocally court females by producing a long duration advertisement call. Females localize males by their advertisement call, spawn and deposit all their eggs in their mate’s nest. As multiple courting males establish nests in close proximity to one another, the perception of another male’s call may modulate individual calling behavior in competition for females. We tested the hypothesis that nesting males exposed to advertisement calls of other males would show elevated neural activity in auditory and vocal-acoustic brain centers as well as differential activation of catecholaminergic neurons compared to males exposed only to ambient noise. Experimental brains were then double labeled by immunofluorescence (-ir) for tyrosine hydroxylase (TH), an enzyme necessary for catecholamine synthesis, and cFos, an immediate-early gene product used as a marker for neural activation. Males exposed to other advertisement calls showed a significantly greater percentage of TH-ir cells colocalized with cFos-ir in the noradrenergic locus coeruleus and the dopaminergic periventricular posterior tuberculum, as well as increased numbers of cFos-ir neurons in several levels of the auditory and vocal-acoustic pathway. Increased activation of catecholaminergic neurons may serve to coordinate appropriate behavioral responses to male competitors. Additionally, these results implicate a role for specific catecholaminergic neuronal groups in auditory-driven social behavior in fishes, consistent with a conserved function in social acoustic behavior across vertebrates.

Use of the swim bladder and lateral line in near-field sound source localization by fish

We investigated the roles of the swim bladder and the lateral line system in sound localization behavior by the plainfin midshipman fish (Porichthys notatus). Reproductive female midshipman underwent either surgical deflation of the swim bladder or cryoablation of the lateral line and were then tested in a monopolar sound source localization task. Fish with nominally ‘deflated’ swim bladders performed similar to sham-deflated controls; however, post-experiment evaluation of swim bladder deflation revealed that a majority of ‘deflated’ fish (88%, seven of the eight fish) that exhibited positive phonotaxis had partially inflated swim bladders. In total, 95% (21/22) of fish that localized the source had at least partially inflated swim bladders, indicating that pressure reception is likely required for sound source localization. In lateral line experiments, no difference was observed in the proportion of females exhibiting positive phonotaxis with ablated (37%) versus sham-ablated (47%) lateral line systems. These data suggest that the lateral line system is likely not required for sound source localization, although this system may be important for fine-tuning the approach to the sound source. We found that midshipman can solve the 180 deg ambiguity of source direction in the shallow water of our test tank, which is similar to their nesting environment. We also found that the potential directional cues (phase relationship between pressure and particle motion) in shallow water differs from a theoretical free-field. Therefore, the general question of how fish use acoustic pressure cues to solve the 180 deg ambiguity of source direction from the particle motion vector remains unresolved.

The effects of salinity and temperature on the transparency of the grass shrimp Palaemonetes pugio

SUMMARY Transparency is an effective form of camouflage, but it must be present throughout the entire volume of an animal to succeed. Certain environmental stressors may cause physiological responses that increase internal light scattering, making tissue less transparent and more conspicuous to predators. We tested this in the transparent grass shrimp, Palaemonetes pugio, which is found in shallow estuaries where both salinity and temperature change rapidly because of tidal cycles, evaporation and runoff. Animals originally kept at a salinity of 15 p.p.t. and a temperature of 20°C were placed into solutions with salinities of 0, 15, 25 or 30 p.p.t. and temperatures of 13, 20 or 27°C for 12 h (N=26 for each of 12 treatments). Under the control conditions of 15 p.p.t. at 20°C, the transparency of grass shrimp tails was 54±3% (mean ± s.e.). At higher salinities and at both higher and lower temperatures, transparency dropped significantly (P<0.001, two-way ANOVA), reaching 0.04±0.01% at 30 p.p.t. at 27°C. Confocal microscopy of P. pugios tail suggested that the observed loss of transparency was due to the pooling of low refractive index hemolymph between the high index muscle fibers, creating many index boundaries that increased light scattering. Analysis of a year-long salinity and temperature record from a North Carolina estuary showed that changes of the order of those found in this study are relatively common, suggesting that P. pugio may undergo periods of reduced crypsis, potentially leading to increased predation.

Revisiting psychoacoustic methods for the assessment of fish hearing

Behavioral methods have been critical in the study of auditory perception and discrimination in fishes. In this chapter, we review some of the common methods used in fish psychoacoustics. We discuss associative methods, such as operant, avoidance, and classical conditioning, and their use in constructing audiograms, measuring frequency selectivity, and auditory stream segregation. We also discuss the measurement of innate behavioral responses, such as the acoustic startle response (ASR), prepulse inhibition (PPI), and phonotaxis, and their use in the assessment of fish hearing to determine auditory thresholds and in the testing of mechanisms for sound source localization. For each psychoacoustic method, we provide examples of their use and discuss the parameters and situations where such methods can be best utilized. In the case of the ASR, we show how this method can be used to construct and compare audiograms between two species of larval fishes, the three-spined stickleback (Gasterosteus aculeatus) and the zebrafish (Danio rerio). We also discuss considerations for experimental design with respect to stimulus presentation and threshold criteria and how these techniques can be used in future studies to investigate auditory perception in fishes.

Larval Zebrafish Lateral Line as a Model for Acoustic Trauma

Visual Abstract Excessive noise exposure damages sensory hair cells, leading to permanent hearing loss. Zebrafish are a highly tractable model that have advanced our understanding of drug-induced hair cell death, yet no comparable model exists for noise exposure research. We demonstrate the utility of zebrafish as model to increase understanding of hair cell damage from acoustic trauma and develop protective therapies. We created an acoustic trauma system using underwater cavitation to stimulate lateral line hair cells. We found that acoustic stimulation resulted in exposure time- and intensity-dependent lateral line and saccular hair cell damage that is maximal at 48–72 h post-trauma. The number of TUNEL+ lateral line hair cells increased 72 h post-exposure, whereas no increase was observed in TUNEL+ supporting cells, demonstrating that acoustic stimulation causes hair cell-specific damage. Lateral line hair cells damaged by acoustic stimulation regenerate within 3 d, consistent with prior regeneration studies utilizing ototoxic drugs. Acoustic stimulation-induced hair cell damage is attenuated by pharmacological inhibition of protein synthesis or caspase activation, suggesting a requirement for translation and activation of apoptotic signaling cascades. Surviving hair cells exposed to acoustic stimulation showed signs of synaptopathy, consistent with mammalian studies. Finally, we demonstrate the feasibility of this platform to identify compounds that prevent acoustic trauma by screening a small redox library for protective compounds. Our data suggest that acoustic stimulation results in lateral line hair cell damage consistent with acoustic trauma research in mammals, providing a highly tractable model for high-throughput genetic and drug discovery studies.

Brain Activation Patterns in Response to Conspecific and Heterospecific Social Acoustic Signals in Female Plainfin Midshipman Fish, Porichthys notatus

While the peripheral auditory system of fish has been well studied, less is known about how the fish’s brain and central auditory system process complex social acoustic signals. The plainfin midshipman fish, Porichthys notatus, has become a good species for investigating the neural basis of acoustic communication because the production and reception of acoustic signals is paramount for this species’ reproductive success. Nesting males produce long-duration advertisement calls that females detect and localize among the noise in the intertidal zone to successfully find mates and spawn. How female midshipman are able to discriminate male advertisement calls from environmental noise and other acoustic stimuli is unknown. Using the immediate early gene product cFos as a marker for neural activity, we quantified neural activation of the ascending auditory pathway in female midshipman exposed to conspecific advertisement calls, heterospecific white seabass calls, or ambient environment noise. We hypothesized that auditory hindbrain nuclei would be activated by general acoustic stimuli (ambient noise and other biotic acoustic stimuli) whereas auditory neurons in the midbrain and forebrain would be selectively activated by conspecific advertisement calls. We show that neural activation in two regions of the auditory hindbrain, i.e., the rostral intermediate division of the descending octaval nucleus and the ventral division of the secondary octaval nucleus, did not differ via cFos immunoreactive (cFos-ir) activity when exposed to different acoustic stimuli. In contrast, female midshipman exposed to conspecific advertisement calls showed greater cFos-ir in the nucleus centralis of the midbrain torus semicircularis compared to fish exposed only to ambient noise. No difference in cFos-ir was observed in the torus semicircularis of animals exposed to conspecific versus heterospecific calls. However, cFos-ir was greater in two forebrain structures that receive auditory input, i.e., the central posterior nucleus of the thalamus and the anterior tuberal hypothalamus, when exposed to conspecific calls versus either ambient noise or heterospecific calls. Our results suggest that higher-order neurons in the female midshipman midbrain torus semicircularis, thalamic central posterior nucleus, and hypothalamic anterior tuberal nucleus may be necessary for the discrimination of complex social acoustic signals. Furthermore, neurons in the central posterior and anterior tuberal nuclei are differentially activated by exposure to conspecific versus other acoustic stimuli.

Hearing sensitivity differs between zebrafish lines used in auditory research.

Zebrafish are increasingly used in auditory studies, in part due to the development of several transgenic lines that express hair cell-specific fluorescent proteins. However, it is largely unknown how transgene expression influences auditory phenotype. We previously observed reduced auditory sensitivity in adult Brn3c:mGFP transgenic zebrafish, which express membrane-bound green fluorescent protein (GFP) in sensory hair cells. Here, we examine the auditory sensitivity of zebrafish from multiple transgenic and background strains. We recorded auditory evoked potentials in adult animals and observed significantly higher auditory thresholds in three lines that express hair cell-specific GFP. There was no obvious correlation between hair cell density and auditory thresholds, suggesting that reduced sensitivity was not due to a reduction in hair cell density. FM1-43 uptake was reduced in Brn3c:mGFP fish but not in other lines, suggesting that a mechanotransduction defect may be responsible for the auditory phenotype in Brn3c animals, but that alternate mechanisms underlie the increased AEP thresholds in other lines. We found reduced prepulse inhibition (a measure of auditory-evoked behavior) in larval Brn3c animals, suggesting that auditory defects develop early in this line. We also found significant differences in auditory sensitivity between adults of different background strains, akin to strain differences observed in mouse models of auditory function. Our results suggest that researchers should exercise caution when selecting an appropriate zebrafish transgenic or background strain for auditory studies.

Lights, camera, science: The utility and growing popularity of film festivals at scientific meetings

Scientific publications have traditionally been viewed as the fruit of a scientist’s labor. Publishing in the peer-reviewed literature is the gold-standard method for communicating research products to other researchers. Yet today, the greater population of academics and researchers are increasingly recognizing the value of non-traditional scientific research products (Bickford et al. 2012, Ecklund et al. 2012), and certain funding agencies are now asking scientists to list ‘products’ rather than just ‘publications’ on their proposals (Piwowar 2013). But how can scientists gather and learn about these different research products and use this as a

A new model for underwater noise research in larval fishes: Biomedical and ecological implications

In humans, excessive noise exposure from occupational or recreational sources causes permanent hearing loss. Similarly, exposure to underwater anthropogenic noise can cause hearing loss in aquatic organisms, including fish. While fish can recover from noise-induced hearing loss, underwater noise exposure can cause behavioral changes that reduce organismal fitness. In all vertebrates, acoustic trauma can cause damage to sensory hair cells. To better study the effects of noise on hair cells, we have developed a noise exposure system that uses broadband sound to damage hair cells of the inner ear and lateral line of larval zebrafish. Acoustic over-exposure kills hair cells in an intensity- and time-dependent manner, with maximum hair cell damage observed 72 hours after noise exposure. This time course is consistent with mammalian studies, where hair cell death occurs days to weeks after noise exposure. Other features of acoustic trauma are also conserved between zebrafish and mammals, including activation of apoptotic signaling cascades and changes in hair cell-afferent synapses. These studies demonstrate that larval zebrafish are a tractable new model for studies of noise-induced hair cell death. However, our acoustic trauma system could also be used in other species, allowing for new studies of underwater noise in larval fishes.