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.

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.

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.

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.

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.