Testosterone Treatment Mimics Seasonal Downregulation of Dopamine Innervation in the Auditory System of Female Midshipman Fish.

In seasonally breeding vertebrates, hormones coordinate changes in nervous system structure and function to facilitate reproductive readiness and success. Steroid hormones often exert their effects indirectly via regulation of neuromodulators, which in turn can coordinate the modulation of sensory input with appropriate motor output. Female plainfin midshipman fish (Porichthys notatus) undergo increased peripheral auditory sensitivity in time for the summer breeding season, improving their ability to detect mates, which is regulated by steroid hormones. Reproductive females also show differences in catecholaminergic innervation of auditory circuitry compared with winter, non-reproductive females as measured by tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholaminergic synthesis. Importantly, catecholaminergic input to the inner ear from a dopaminergic-specific forebrain nucleus is decreased in the summer and dopamine inhibits the sensitivity of the inner ear, suggesting that gonadal steroids may alter auditory sensitivity by regulating dopamine innervation. In this study, we gonadectomized non-reproductive females, implanted them with estradiol (E2) or testosterone (T), and measured TH immunoreactive (TH-ir) fibers in auditory nuclei where catecholaminergic innervation was previously shown to be seasonally plastic. We found that treatment with T, but not E2, reduced TH-ir innervation in the auditory hindbrain. T-treatment also reduced TH-ir fibers in the forebrain dopaminergic cell group that projects to the inner ear, and likely to the auditory hindbrain. Higher T plasma in the treatment group was correlated with reduced-ir TH terminals in the inner ear. These T-treatment induced changes in TH-ir fibers mimic the seasonal downregulation of dopamine in the midshipman inner ear and provide evidence that steroid hormone regulation of peripheral auditory sensitivity is mediated, in part, by dopamine.

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.

Effects of Hatchery Rearing on the Structure and Function of Salmonid Mechanosensory Systems.

This paper reviews recent studies on the effects of hatchery rearing on the auditory and lateral line systems of salmonid fishes. Major conclusions are that (1) hatchery-reared juveniles exhibit abnormal lateral line morphology (relative to wild-origin conspecifics), suggesting that the hatchery environment affects lateral line structure, perhaps due to differences in the hydrodynamic conditions of hatcheries versus natural rearing environments, and (2) hatchery-reared salmonids have a high proportion of abnormal otoliths, a condition associated with reduced auditory sensitivity and suggestive of inner ear dysfunction.

Saccular Transcriptome Profiles of the Seasonal Breeding Plainfin Midshipman Fish (Porichthys notatus), a Teleost with Divergent Sexual Phenotypes.

Acoustic communication is essential for the reproductive success of the plainfin midshipman fish (Porichthys notatus). During the breeding season, type I males use acoustic cues to advertise nest location to potential mates, creating an audible signal that attracts reproductive females. Type II (sneaker) males also likely use this social acoustic signal to find breeding pairs from which to steal fertilizations. Estrogen-induced changes in the auditory system of breeding females are thought to enhance neural encoding of the advertisement call, and recent anatomical data suggest the saccule (the main auditory end organ) as one possible target for this seasonal modulation. Here we describe saccular transcriptomes from all three sexual phenotypes (females, type I and II males) collected during the breeding season as a first step in understanding the mechanisms underlying sexual phenotype-specific and seasonal differences in auditory function. We used RNA-Seq on the Ion Torrent platform to create a combined transcriptome dataset containing over 79,000 assembled transcripts representing almost 9,000 unique annotated genes. These identified genes include several with known inner ear function and multiple steroid hormone receptors. Transcripts most closely matched to published genomes of nile tilapia and large yellow croaker, inconsistent with the phylogenetic relationship between these species but consistent with the importance of acoustic communication in their life-history strategies. We then compared the RNA-Seq results from the saccules of reproductive females with a separate transcriptome from the non-reproductive female phenotype and found over 700 differentially expressed transcripts, including members of the Wnt and Notch signaling pathways that mediate cell proliferation and hair cell addition in the inner ear. These data constitute a valuable resource for furthering our understanding of the molecular basis for peripheral auditory function as well as a range of future midshipman and cross-species comparative studies of the auditory periphery.

Neuroendocrine control of seasonal plasticity in the auditory and vocal systems of fish.

Seasonal changes in reproductive-related vocal behavior are widespread among fishes. This review highlights recent studies of the vocal plainfin midshipman fish, Porichthys notatus, a neuroethological model system used for the past two decades to explore neural and endocrine mechanisms of vocal-acoustic social behaviors shared with tetrapods. Integrative approaches combining behavior, neurophysiology, neuropharmacology, neuroanatomy, and gene expression methodologies have taken advantage of simple, stereotyped and easily quantifiable behaviors controlled by discrete neural networks in this model system to enable discoveries such as the first demonstration of adaptive seasonal plasticity in the auditory periphery of a vertebrate as well as rapid steroid and neuropeptide effects on vocal physiology and behavior. This simple model system has now revealed cellular and molecular mechanisms underlying seasonal and steroid-driven auditory and vocal plasticity in the vertebrate brain.

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

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 fish's 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.

Differences in lateral line morphology between hatchery- and wild-origin steelhead.

Despite identification of multiple factors mediating salmon survival, significant disparities in survival-to-adulthood among hatchery- versus wild-origin juveniles persist. In the present report, we explore the hypothesis that hatchery-reared juveniles might exhibit morphological defects in vulnerable mechanosensory systems prior to release from the hatchery, potentiating reduced survival after release. Juvenile steelhead (Oncorhynchus mykiss) from two different hatcheries were compared to wild-origin juveniles on several morphological traits including lateral line structure, otolith composition (a proxy for auditory function), and brain weight. Wild juveniles were found to possess significantly more superficial lateral line neuromasts than hatchery-reared juveniles, although the number of hair cells within individual neuromasts was not significantly different across groups. Wild juveniles were also found to possess primarily normal, aragonite-containing otoliths, while hatchery-reared juveniles possessed a high proportion of crystallized (vaterite) otoliths. Finally, wild juveniles were found to have significantly larger brains than hatchery-reared juveniles. These differences together predict reduced sensitivity to biologically important hydrodynamic and acoustic signals from natural biotic (predator, prey, conspecific) and abiotic (turbulent flow, current) sources among hatchery-reared steelhead, in turn predicting reduced survival fitness after release. Physiological and behavioral studies are required to establish the functional significance of these morphological differences.

Adaptive hearing in the vocal plainfin midshipman fish: getting in tune for the breeding season and implications for acoustic communication.

The plainfin midshipman fish (Porichthys notatus Girard, 1854) is a vocal species of batrachoidid fish that generates acoustic signals for intraspecific communication during social and reproductive activity and has become a good model for investigating the neural and endocrine mechanisms of vocal-acoustic communication. Reproductively active female plainfin midshipman fish use their auditory sense to detect and locate "singing" males, which produce a multiharmonic advertisement call to attract females for spawning. The seasonal onset of male advertisement calling in the midshipman fish coincides with an increase in the range of frequency sensitivity of the female's inner ear saccule, the main organ of hearing, thus leading to enhanced encoding of the dominant frequency components of male advertisement calls. Non-reproductive females treated with either testosterone or 17ß-estradiol exhibit a dramatic increase in the inner ear's frequency sensitivity that mimics the reproductive female's auditory phenotype and leads to an increased detection of the male's advertisement call. This novel form of auditory plasticity provides an adaptable mechanism that enhances coupling between sender and receiver in vocal communication. This review focuses on recent evidence for seasonal reproductive-state and steroid-dependent plasticity of auditory frequency sensitivity in the peripheral auditory system of the midshipman fish. The potential steroid-dependent mechanism(s) that lead to this novel form of auditory and behavioral plasticity are also discussed.

Steroid-dependent auditory plasticity leads to adaptive coupling of sender and receiver.

For seasonally breeding vertebrates, reproductive cycling is often coupled with changes in vocalizations that function in courtship and territoriality. Less is known about changes in auditory sensitivity to those vocalizations. Here, we show that nonreproductive female midshipman fish treated with either testosterone or 17beta-estradiol exhibit an increase in the degree of temporal encoding of the frequency content of male vocalizations by the inner ear that mimics the reproductive female's auditory phenotype. This sensory plasticity provides an adaptable mechanism that enhances coupling between sender and receiver in vocal communication.

Seasonal variation of steroid hormone levels in an intertidal-nesting fish, the vocal plainfin midshipman.

This study characterized the seasonal variation of the steroid hormones testosterone (T), 11-ketotestosterone (11-KT), 17beta-estradiol (E2), and cortisol (F) as they relate to the gonadal development and reproductive behavior of the plainfin midshipman fish, Porichthys notatus. The plainfin midshipman is a deep-water teleost that seasonally migrates into the shallow intertidal zone where type I, or "singing," males build nests, acoustically court and spawn with females. The gonadosomatic index and plasma steroid levels were measured from adult type I males and females collected over four time periods (non-reproductive, pre-nesting, nesting, and post-nesting) that corresponded to seasonal fluctuations in midshipman reproductive biology and behavior. Among type I males, plasma levels of T and 11-KT were low during the winter non-reproductive period, gradually increased during seasonal recrudescence of the testes in the spring pre-nesting period, and then peaked at the beginning of the summer nesting period. In the latter half of the nesting period and during the fall post-nesting period, plasma levels of T and 11-KT were low or non-detectable. Low, detectable levels of E2 were also found in the plasma of 50% or more type I males during every seasonal period except the winter non-reproductive period. Among females, plasma levels of T and E2 were low throughout the year but briefly peaked in April during the spring pre-nesting period when ovaries underwent seasonal recrudescence. Plasma F levels were correlated with collection depth and were lower in males than females when fish were collected deeper than 120 m. The sex-specific peaks of steroid hormone levels in male and female midshipman may serve differential functions related to the physiology, reproductive behavior, and vocal communication of this species.