Characterization of anthropogenic noise and oyster toadfish (Opsanus tau) calling behavior in urban and small-town coastal soundscapesa).

The oyster toadfish (Opsanus tau) is an ideal model to examine the effects of anthropogenic noise on behavior because they rely on acoustic signals for mate attraction and social interactions. We predict that oyster toadfish have acclimated to living in noise-rich environments because they are common in waterways of urban areas, like New York City (NYC). We used passive acoustic monitoring at two locations to see if calling behavior patterns are altered in areas of typically high boat traffic versus low boat traffic (Pier 40, NYC, NY, and Eel Pond, Woods Hole, MA, respectively). We hypothesized that toadfish in NYC would adjust their circadian calling behavior in response to daily anthropogenic noise patterns. We quantified toadfish calls and ship noise over three 24-h periods in the summer reproductive period at both locations. We observed an inverse relationship between the duration of noise and the number of toadfish calls at Pier 40 in comparison to Eel Pond. Additionally, toadfish at Pier 40 showed significant differences in peak calling behavior compared to Eel Pond. Therefore, oyster toadfish may have acclimated to living in an urban environment by potentially altering their communication behavior in the presence of boat noise.

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.

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.

Neuroanatomical Evidence for Catecholamines as Modulators of Audition and Acoustic Behavior in a Vocal Teleost.

The plainfin midshipman fish (Porichthys notatus) is a well-studied model to understand the neural and endocrine mechanisms underlying vocal-acoustic communication across vertebrates. It is well established that steroid hormones such as estrogen drive seasonal peripheral auditory plasticity in female Porichthys in order to better encode the male's advertisement call. However, little is known of the neural substrates that underlie the motivation and coordinated behavioral response to auditory social signals. Catecholamines, which include dopamine and noradrenaline, are good candidates for this function, as they are thought to modulate the salience of and reinforce appropriate behavior to socially relevant stimuli. This chapter summarizes our recent studies which aimed to characterize catecholamine innervation in the central and peripheral auditory system of Porichthys as well as test the hypotheses that innervation of the auditory system is seasonally plastic and catecholaminergic neurons are activated in response to conspecific vocalizations. Of particular significance is the discovery of direct dopaminergic innervation of the saccule, the main hearing end organ, by neurons in the diencephalon, which also robustly innervate the cholinergic auditory efferent nucleus in the hindbrain. Seasonal changes in dopamine innervation in both these areas appear dependent on reproductive state in females and may ultimately function to modulate the sensitivity of the peripheral auditory system as an adaptation to the seasonally changing soundscape. Diencephalic dopaminergic neurons are indeed active in response to exposure to midshipman vocalizations and are in a perfect position to integrate the detection and appropriate motor response to conspecific acoustic signals for successful reproduction.

Catecholaminergic Fiber Innervation of the Vocal Motor System Is Intrasexually Dimorphic in a Teleost with Alternative Reproductive Tactics.

Catecholamines, which include the neurotransmitters dopamine and noradrenaline, are known modulators of sensorimotor function, reproduction, and sexually motivated behaviors across vertebrates, including vocal-acoustic communication. Recently, we demonstrated robust catecholaminergic (CA) innervation throughout the vocal motor system in the plainfin midshipman fish Porichthys notatus, a seasonal breeding marine teleost that produces vocal signals for social communication. There are 2 distinct male reproductive morphs in this species: type I males establish nests and court females with a long-duration advertisement call, while type II males sneak spawn to steal fertilizations from type I males. Like females, type II males can only produce brief, agonistic, grunt type vocalizations. Here, we tested the hypothesis that intrasexual differences in the number of CA neurons and their fiber innervation patterns throughout the vocal motor pathway may provide neural substrates underlying divergence in reproductive behavior between morphs. We employed immunofluorescence (-ir) histochemistry to measure tyrosine hydroxylase (TH; a rate-limiting enzyme in catecholamine synthesis) neuron numbers in several forebrain and hindbrain nuclei as well as TH-ir fiber innervation throughout the vocal pathway in type I and type II males collected from nests during the summer reproductive season. After controlling for differences in body size, only one group of CA neurons displayed an unequivocal difference between male morphs: the extraventricular vagal-associated TH-ir neurons, located just lateral to the dimorphic vocal motor nucleus (VMN), were significantly greater in number in type II males. In addition, type II males exhibited greater TH-ir fiber density within the VMN and greater numbers of TH-ir varicosities with putative contacts on vocal motor neurons. This strong inverse relationship between the predominant vocal morphotype and the CA innervation of vocal motor neurons suggests that catecholamines may function to inhibit vocal output in midshipman. These findings support catecholamines as direct modulators of vocal behavior, and differential CA input appears reflective of social and reproductive behavioral divergence between male midshipman morphs.

Activation of noradrenergic locus coeruleus and social behavior network nuclei varies with duration of male midshipman advertisement calls.

Vocal courtship is vital to the reproductive success of many vertebrates and is therefore a highly-motivated behavioral state. Catecholamines have been shown to play an essential role in the expression and maintenance of motivated vocal behavior, such as the coordination of vocal-motor output in songbirds. However, it is not well-understood if this relationship applies to anamniote vocal species. Using the plainfin midshipman fish model, we tested whether specific catecholaminergic (i.e., dopaminergic and noradrenergic) nuclei and nodes of the social behavior network (SBN) are differentially activated in vocally courting (humming) versus non-humming males. Herein, we demonstrate that tyrosine hydroxylase immunoreactive (TH-ir) neuron number in the noradrenergic locus coeruleus (LC) and induction of cFos (an immediate early gene product and proxy for neural activation) in the preoptic area differentiated humming from non-humming males. Furthermore, we found relationships between activation of the LC and SBN nuclei with the total amount of time that males spent humming, further reinforcing a role for these specific brain regions in the production of motivated reproductive-related vocalizations. Finally, we found that patterns of functional connectivity between catecholaminergic nuclei and nodes of the SBN differed between humming and non-humming males, supporting the notion that adaptive behaviors (such as the expression of advertisement hums) emerge from the interactions between various catecholaminergic nuclei and the SBN.

Neural and hormonal mechanisms of reproductive-related arousal in fishes.

The major classes of chemicals and brain pathways involved in sexual arousal in mammals are well studied and are thought to be of an ancient, evolutionarily conserved origin. Here we discuss what is known of these neurochemicals and brain circuits in fishes, the oldest and most species-rich group of vertebrates from which tetrapods arose over 350 million years ago. Highlighted are case studies in vocal species where well-delineated sensory and motor pathways underlying reproductive-related behaviors illustrate the diversity and evolution of brain mechanisms driving sexual motivation between (and within) sexes. Also discussed are evolutionary insights from the neurobiology and reproductive behavior of elasmobranch fishes, the most ancient lineage of jawed vertebrates, which are remarkably similar in their reproductive biology to terrestrial mammals.

Gap junction-mediated glycinergic inhibition ensures precise temporal patterning in vocal behavior.

Precise neuronal firing is especially important for behaviors highly dependent on the correct sequencing and timing of muscle activity patterns, such as acoustic signaling. Acoustic signaling is an important communication modality for vertebrates, including many teleost fishes. Toadfishes are well known to exhibit high temporal fidelity in synchronous motoneuron firing within a hindbrain network directly determining the temporal structure of natural calls. Here, we investigated how these motoneurons maintain synchronous activation. We show that pronounced temporal precision in population-level motoneuronal firing depends on gap junction-mediated, glycinergic inhibition that generates a period of reduced probability of motoneuron activation. Super-resolution microscopy confirms glycinergic release sites formed by a subset of adjacent premotoneurons contacting motoneuron somata and dendrites. In aggregate, the evidence supports the hypothesis that gap junction-mediated, glycinergic inhibition provides a timing mechanism for achieving synchrony and temporal precision in the millisecond range for rapid modulation of acoustic waveforms.

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.

Serotonin distribution in the brain of the plainfin midshipman: Substrates for vocal-acoustic modulation and a reevaluation of the serotonergic system in teleost fishes.

Serotonin (5-HT) is a modulator of neural circuitry underlying motor patterning, homeostatic control, and social behavior. While previous studies have described 5-HT distribution in various teleosts, serotonergic raphe subgroups in fish are not well defined and therefore remain problematic for cross-species comparisons. Here we used the plainfin midshipman fish, Porichthys notatus, a well-studied model for investigating the neural and hormonal mechanisms of vertebrate vocal-acoustic communication, to redefine raphe subgroups based on both stringent neuroanatomical landmarks as well as quantitative cell measurements. In addition, we comprehensively characterized 5-HT-immunoreactive (-ir) innervation throughout the brain, including well-delineated vocal and auditory nuclei. We report neuroanatomical heterogeneity in populations of the serotonergic raphe nuclei of the brainstem reticular formation, with three discrete subregions in the superior raphe, an intermediate 5-HT-ir cell cluster, and an extensive inferior raphe population. 5-HT-ir neurons were also observed within the vocal motor nucleus (VMN), forming putative contacts on those cells. In addition, three major 5-HT-ir cell groups were identified in the hypothalamus and one group in the pretectum. Significant 5-HT-ir innervation was found in components of the vocal pattern generator and cranial motor nuclei. All vocal midbrain nuclei showed considerable 5-HT-ir innervation, as did thalamic and hindbrain auditory and lateral line areas and vocal-acoustic integration sites in the preoptic area and ventral telencephalon. This comprehensive atlas offers new insights into the organization of 5-HT nuclei in teleosts and provides neuroanatomical evidence for serotonin as a modulator of vocal-acoustic circuitry and behavior in midshipman fish, consistent with findings in vocal tetrapods.

A versatile macro-based neurohistological image analysis suite for ImageJ focused on automated and standardized user interaction and reproducible data output.

BACKGROUND: The development and increasing adoption of advanced microscopy imaging technologies, including high resolution, multi-dimensional digital photography and multiple fluorescence channel acquisition, as well as the availability of inexpensive terabyte-capacity storage, have enabled research laboratories to pursue neurohistological imaging experiments involving multiple neurochemical probes and experimental conditions covering a variety of brain regions. Analyzing and processing the resulting datasets, composed of hundreds of micrographs, presents challenges in ensuring accuracy and reproducibility under demanding time and training constraints. NEW METHOD: The 'Custom Macros' plugin suite for ImageJ automates and systematizes user interaction in neurohistological image analysis tasks, including region selection and thresholding, point/object counts, area measurement, batch filter processing, and data review. Written in the accessible ImageJ macro language, the plugin implements a user login-based data storage framework and facilitates inter-laboratory collaboration over cloud file server clients. RESULTS: A macro-based interface approach integrates dozens of novel operations, software interactions, algorithm calls, and background tasks into individual shortcut commands. Every completed procedure generates image, region, and calibrated measurement records that are saved in a standardized folder structure. COMPARISONS WITH EXISTING METHODS: Plugin installation adds startup access to a persistent interface layer of extensive and streamlined functionality that is generalizable to a variety of neurohistological contexts, thus providing an efficient and reliable alternative to the use of analysis software in an unstructured, provisional manner that necessitates repeated menu and plugin interaction. CONCLUSIONS: Our free/open-source software provides researchers a straightforward solution to addressing daunting usability and data oversight issues, ultimately making efficient, accessible, and reproducible image analysis methodology attainable for many laboratories.

Forebrain Dopamine System Regulates Inner Ear Auditory Sensitivity to Socially Relevant Acoustic Signals.

Dopamine is integral to attentional and motivational processes, but studies are largely restricted to the central nervous system. In mammals [1, 2] and fishes [3, 4], central dopaminergic neurons project to the inner ear and could modulate acoustic signals at the earliest stages of processing. Studies in rodents show dopamine inhibits cochlear afferent neurons and protects against noise-induced acoustic injury [5-10]. However, other functions for inner ear dopamine have not been investigated, and the effect of dopamine on peripheral auditory processing in non-mammalians remains unknown [11, 12]. Insights could be gained by studies conducted in the context of intraspecific acoustic communication. We present evidence from a vocal fish linking reproductive-state-dependent changes in auditory sensitivity with seasonal changes in the dopaminergic efferent system in the saccule, their primary organ of hearing. Plainfin midshipman (Porichthys notatus) migrate from deep-water winter habitats to the intertidal zone in the summer to breed. Nesting males produce nocturnal vocalizations to attract females [13]. Both sexes undergo seasonal enhancement of hearing sensitivity at the level of the hair cell [14-16], increasing the likelihood of detecting conspecific signals [17, 18]. Importantly, reproductive females concurrently have reduced dopaminergic input to the saccule [19]. Here, we show that dopamine decreases saccule auditory sensitivity via a D2-like receptor. Saccule D2a receptor expression is reduced in the summer and correlates with sensitivity within and across seasons. We propose that reproductive-state-dependent changes to the dopaminergic efferent system provide a release of inhibition in the saccule, enhancing peripheral encoding of social-acoustic signals.

Dopaminergic neurons are preferentially responsive to advertisement calls and co-active with social behavior network nuclei in sneaker male midshipman fish.

Vocal species use acoustic signals to facilitate diverse behaviors such as mate attraction and territorial defense. However, little is known regarding the neural substrates that interpret such divergent conspecific signals. Using the plainfin midshipman fish model, we tested whether specific catecholaminergic (i.e., dopaminergic and noradrenergic) nuclei and nodes of the social behavior network (SBN) are differentially responsive following exposure to playbacks of divergent social signals in sneaker males. We chose sneaker (type II) males since they attempt to steal fertilizations from territorial type I males who use an advertisement call (hum) to attract females yet are also subjected to vocal agonistic behavior (grunts) by type I males. We demonstrate that induction of cFos (an immediate early gene product and proxy for neural activation) in two forebrain dopaminergic nuclei is greater in sneaker males exposed to hums but not grunts compared to ambient noise, suggesting hums preferentially activate these nuclei, further asserting dopamine as an important regulator of social-acoustic behaviors. Moreover, acoustic exposure to social signals with divergent salience engendered contrasting shifts in functional connectivity between dopaminergic nuclei and nodes of the SBN, supporting the idea that interactions between these two circuits may underlie adaptive decision-making related to intraspecific male competition.

Conserved neurochemical pathways involved in hypothalamic control of energy homeostasis.

The melanocortin system, which includes alpha-melanocyte-stimulating hormone (alpha-MSH) and its endogenous antagonist, agouti-related protein (AgRP), is fundamental for the central control of energy homeostasis in mammals. Recent studies have demonstrated that many neuropeptides involved in the control of ingestive behavior and energy expenditure, including melanocortins, are also expressed and functional in teleost fishes. To test the hypothesis that the underlying neural pathways involved in energy homeostasis are conserved throughout vertebrate evolution, the neuroanatomical distribution of alpha-MSH in relation to AgRP was mapped in a teleost (zebrafish, Danio rerio) by double-label immunocytochemistry. Zebrafish alpha-MSH- and AgRP-immunoreactive (ir) cells are found in discrete populations in the ventral periventricular hypothalamus, the proposed arcuate homologue in teleosts. Major ascending projections are similar for both peptides, and dense ir-fibers innervate preoptic and ventral telencephalic nuclei homologous to paraventricular, lateral septal, and amygdala nuclei in mammals. Furthermore, alpha-MSH and AgRP-ir somata and fibers are pronounced at 5 days post fertilization when yolk reserves are depleted and larvae begin to feed actively, which supports the functional significance of these peptides for feeding behavior. The conservation of melanocortin peptide function and projection pathways further support zebrafish as an excellent genetic model system to investigate basic mechanisms involved in the central regulation of energy homeostasis.

Connectivity and ultrastructure of dopaminergic innervation of the inner ear and auditory efferent system of a vocal fish.

Dopamine (DA) is a conserved modulator of vertebrate neural circuitry, yet our knowledge of its role in peripheral auditory processing is limited to mammals. The present study combines immunohistochemistry, neural tract tracing, and electron microscopy to investigate the origin and synaptic characteristics of DA fibers innervating the inner ear and the hindbrain auditory efferent nucleus in the plainfin midshipman, a vocal fish that relies upon the detection of mate calls for reproductive success. We identify a DA cell group in the diencephalon as a common source for innervation of both the hindbrain auditory efferent nucleus and saccule, the main hearing endorgan of the inner ear. We show that DA terminals in the saccule contain vesicles but transmitter release appears paracrine in nature, due to the apparent lack of synaptic contacts. In contrast, in the hindbrain, DA terminals form traditional synaptic contacts with auditory efferent neuronal cell bodies and dendrites, as well as unlabeled axon terminals, which, in turn, form inhibitory-like synapses on auditory efferent somata. Our results suggest a distinct functional role for brain-derived DA in the direct and indirect modulation of the peripheral auditory system of a vocal nonmammalian vertebrate.

Attention and Motivated Response to Simulated Male Advertisement Call Activates Forebrain Dopaminergic and Social Decision-Making Network Nuclei in Female Midshipman Fish.

Little is known regarding the coordination of audition with decision-making and subsequent motor responses that initiate social behavior including mate localization during courtship. Using the midshipman fish model, we tested the hypothesis that the time spent by females attending and responding to the advertisement call is correlated with the activation of a specific subset of catecholaminergic (CA) and social decision-making network (SDM) nuclei underlying auditory- driven sexual motivation. In addition, we quantified the relationship of neural activation between CA and SDM nuclei in all responders with the goal of providing a map of functional connectivity of the circuitry underlying a motivated state responsive to acoustic cues during mate localization. In order to make a baseline qualitative comparison of this functional brain map to unmotivated females, we made a similar correlative comparison of brain activation in females who were unresponsive to the advertisement call playback. Our results support an important role for dopaminergic neurons in the periventricular posterior tuberculum and ventral thalamus, putative A11 and A13 tetrapod homologues, respectively, as well as the posterior parvocellular preoptic area and dorsomedial telencephalon, (laterobasal amygdala homologue) in auditory attention and appetitive sexual behavior in fishes. These findings may also offer insights into the function of these highly conserved nuclei in the context of auditory-driven reproductive social behavior across vertebrates.

Intra- and Intersexual swim bladder dimorphisms in the plainfin midshipman fish (Porichthys notatus): Implications of swim bladder proximity to the inner ear for sound pressure detection.

The plainfin midshipman fish, Porichthys notatus, is a nocturnal marine teleost that uses social acoustic signals for communication during the breeding season. Nesting type I males produce multiharmonic advertisement calls by contracting their swim bladder sonic muscles to attract females for courtship and spawning while subsequently attracting cuckholding type II males. Here, we report intra- and intersexual dimorphisms of the swim bladder in a vocal teleost fish and detail the swim bladder dimorphisms in the three sexual phenotypes (females, type I and II males) of plainfin midshipman fish. Micro-computerized tomography revealed that females and type II males have prominent, horn-like rostral swim bladder extensions that project toward the inner ear end organs (saccule, lagena, and utricle). The rostral swim bladder extensions were longer, and the distance between these swim bladder extensions and each inner-ear end organ type was significantly shorter in both females and type II males compared to that in type I males. Our results revealed that the normalized swim bladder length of females and type II males was longer than that in type I males while there was no difference in normalized swim bladder width among the three sexual phenotypes. We predict that these intrasexual and intersexual differences in swim bladder morphology among midshipman sexual phenotypes will afford greater sound pressure sensitivity and higher frequency detection in females and type II males and facilitate the detection and localization of conspecifics in shallow water environments, like those in which midshipman breed and nest.

Distribution of estrogen receptor alpha mRNA in the brain and inner ear of a vocal fish with comparisons to sites of aromatase expression.

Among vertebrates, teleost fish have the greatest capacity for estrogen production in the brain. Previously, we characterized the distribution of the estrogen-synthesizing enzyme aromatase in the brain of the midshipman fish. Here, we investigated the distribution of estrogen receptor alpha (ERalpha). A partial cDNA of ERalpha was cloned and used to generate midshipman-specific primers for RT and real-time PCR which identified transcripts in liver and ovary, the CNS, and the sensory epithelium of the main auditory endorgan (sacculus). In situ hybridization revealed abundant expression throughout the preoptic area, a vocal-acoustic site in the hypothalamus, amygdala homologs of the dorsal pallium, the pineal organ, the inner ear, the pituitary, and the ovary. Weaker expression was found in the midbrain's nucleus of the medial longitudinal fasciculus and in the dimorphic vocal motor nucleus. ERalpha expression in the pineal, gonad, and pituitary axis may function to time seasonal abiotic cues to reproductive state, while expression in the vocal motor and auditory systems support neurophysiological evidence for estrogen as a modulator of vocal motor and auditory encoding mechanisms in midshipman fish. While ERalpha is restricted to specific nuclei, aromatase expression is abundant in glial cells throughout the entire forebrain, and high in midbrain and hindbrain - spinal vocal regions. The only site of aromatase-containing neurons is in the peripheral auditory system, where it is localized to ganglion cells in the auditory nerve. Estrogen production proximal to ERalpha-positive neurons may provide for focal sites of estrogen effects on reproductive-, vocal-, and auditory-related neurons.

Catecholaminergic innervation of central and peripheral auditory circuitry varies with reproductive state in female midshipman fish, Porichthys notatus.

In seasonal breeding vertebrates, hormone regulation of catecholamines, which include dopamine and noradrenaline, may function, in part, to modulate behavioral responses to conspecific vocalizations. However, natural seasonal changes in catecholamine innervation of auditory nuclei is largely unexplored, especially in the peripheral auditory system, where encoding of social acoustic stimuli is initiated. The plainfin midshipman fish, Porichthys notatus, has proven to be an excellent model to explore mechanisms underlying seasonal peripheral auditory plasticity related to reproductive social behavior. Recently, we demonstrated robust catecholaminergic (CA) innervation throughout the auditory system in midshipman. Most notably, dopaminergic neurons in the diencephalon have widespread projections to auditory circuitry including direct innervation of the saccule, the main endorgan of hearing, and the cholinergic octavolateralis efferent nucleus (OE) which also projects to the inner ear. Here, we tested the hypothesis that gravid, reproductive summer females show differential CA innervation of the auditory system compared to non-reproductive winter females. We utilized quantitative immunofluorescence to measure tyrosine hydroxylase immunoreactive (TH-ir) fiber density throughout central auditory nuclei and the sensory epithelium of the saccule. Reproductive females exhibited greater density of TH-ir innervation in two forebrain areas including the auditory thalamus and greater density of TH-ir on somata and dendrites of the OE. In contrast, non-reproductive females had greater numbers of TH-ir terminals in the saccule and greater TH-ir fiber density in a region of the auditory hindbrain as well as greater numbers of TH-ir neurons in the preoptic area. These data provide evidence that catecholamines may function, in part, to seasonally modulate the sensitivity of the inner ear and, in turn, the appropriate behavioral response to reproductive acoustic signals.