Distribution of aromatase in the brain of the African cichlid fish Astatotilapia burtoni: Aromatase expression,but not estrogen receptors,varies with female reproductive-state

Estrogen synthesis and signaling in the brains of vertebrates has pleotropic effects ranging from neurogenesis to modulation of behaviors. The majority of studies on brain-derived estrogens focus on males, but estrogenic signaling in females likely plays important roles in regulation of reproductive cycling and social behaviors. We used females of the mouth brooding African cichlid fish, Astatotilapia burtoni, to test for reproductive state-dependent changes in estrogenic signaling capacity within microdissected brain nuclei that are important for social behaviors. Expression levels of the rate-limiting enzyme aromatase, but not estrogen receptors, measured by qPCR changes across the reproductive cycle. Gravid females that are close to spawning had higher aromatase levels in all brain regions compared to females with lower reproductive potential. This brain aromatase expression was positively correlated with circulating estradiol levels and ovarian readiness. Using chromogenic in situ hybridization we localized aromatase-expressing cells to ependymal regions bordering the ventricles from the forebrain to the hindbrain, and observed more abundant staining in gravid compared to mouth brooding females in most regions. Staining was most prominent in subpallial telencephalic regions, and diencephalic regions of the preoptic area, thalamus, and hypothalamus, but was also observed in sensory and sensorimotor areas of the midbrain and hindbrain. Aromatase expression was observed in radial glial cells, revealed by co-localization with the glial marker GFAP and absence of co-localization with the neuronal marker HuC/D. Collectively these results support the idea that brain-derived estradiol in females may serve important functions in reproductive state-dependent physiological and behavioral processes across vertebrates.     

Saccular-specific hair cell addition correlates with reproductive state-dependent changes in the auditory saccular sensitivity of a vocal fish

The plainfin midshipman fish, Porichthys notatus, is a seasonal breeding teleost fish for which vocal-acoustic communication is essential for its reproductive success. Female midshipman use the saccule as the primary end organ for hearing to detect and locate "singing" males that produce multiharmonic advertisement calls during the summer breeding season. Previous work has shown that female auditory sensitivity changes seasonally with reproductive state; summer reproductive females become better suited than winter nonreproductive females to detect and encode the dominant higher harmonic components in the male's advertisement call, which are potentially critical for mate selection and localization. Here, we test the hypothesis that these seasonal changes in female auditory sensitivity are concurrent with seasonal increases in saccular hair cell receptors. We show that there is increased hair cell density in reproductive females and that this increase is not dependent on body size since similar changes in hair cell density were not found in the other inner ear end organs. We also observed an increase in the number of small, potentially immature saccular hair bundles in reproductive females. The seasonal increase in saccular hair cell density and smaller hair bundles in reproductive females was paralleled by a dramatic increase in the magnitude of the evoked saccular potentials and a corresponding decrease in the auditory thresholds recorded from the saccule. This demonstration of correlated seasonal plasticity of hair cell addition and auditory sensitivity may in part facilitate the adaptive auditory plasticity of this species to enhance mate detection and localization during breeding.     

Distribution of androgen receptor mRNA expression in vocal,auditory, and neuroendocrine circuits in a teleost fish

Across all major vertebrate groups, androgen receptors (ARs) have been identified in neural circuits that shape reproductive‐related behaviors, including vocalization. The vocal control network of teleost fishes presents an archetypal example of how a vertebrate nervous system produces social, context‐dependent sounds. We cloned a partial cDNA of AR that was used to generate specific probes to localize AR expression throughout the central nervous system of the vocal plainfin midshipman fish (Porichthys notatus). In the forebrain, AR mRNA is abundant in proposed homologs of the mammalian striatum and amygdala, and in anterior and posterior parvocellular and magnocellular nuclei of the preoptic area, nucleus preglomerulosus, and posterior, ventral and anterior tuberal nuclei of the hypothalamus. Many of these nuclei are part of the known vocal and auditory circuitry in midshipman. The midbrain periaqueductal gray, an essential link between forebrain and hindbrain vocal circuitry, and the lateral line recipient nucleus medialis in the rostral hindbrain also express abundant AR mRNA. In the caudal hindbrain‐spinal vocal circuit, high AR mRNA is found in the vocal prepacemaker nucleus and along the dorsal periphery of the vocal motor nucleus congruent with the known pattern of expression of aromatase‐containing glial cells. Additionally, abundant AR mRNA expression is shown for the first time in the inner ear of a vertebrate. The distribution of AR mRNA strongly supports the role of androgens as modulators of behaviorally defined vocal, auditory, and neuroendocrine circuits in teleost fish and vertebrates in general. J. Comp. Neurol. 518:493–512, 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Vocal-acoustic circuitry and descending vocal pathways in teleost fish: convergence with terrestrial vertebrates reveals conserved traits

Vocal behavior is multifaceted and requires that vocal-motor patterning be integrated at multiple brain levels with auditory, neuroendocrine, and other social behavior processes (e.g., courtship and aggression). We now provide anatomical evidence for an extensive vocal network in teleost fishes (Batrachoididae: Porichthys notatus; Opsanus beta) that is strongly integrated with neuroendocrine and auditory pathways and that exhibits striking similarities to the vocal-acoustic circuitry known for mammals. Biotin compound injections into neurophysiologically identified vocal regions of the forebrain (preoptic area and anterior hypothalamus) and of the midbrain (periaqueductal gray and paralemniscal tegmentum) reveal extensive connectivity within and between these regions, as well as reciprocal relationships with the auditory thalamus and/or auditory midbrain (torus semicircularis). Thus, specific components of the basal forebrain and midbrain are here designated as the forebrain vocal-acoustic complex (fVAC) and midbrain vocal-acoustic complex (mVAC), respectively. Biotin injections into the mVAC and a previously identified hindbrain vocal pattern generator likewise provide anatomical evidence for a distributed network of descending projections to the vocal pacemaker-motoneuron circuitry. Together, the present experiments establish a vocal-auditory-neuroendocrine network in teleost fish that links the forebrain and midbrain to the hindbrain vocal pattern generator (i.e., fVAC --> mVAC --> pattern generator) and provides an anatomical framework for the previously identified neuropeptide modulation of vocal activity elicited from the forebrain and midbrain, which contributes to the expression of sex- and male morph-specific behavior. We conclude with a broad comparison of these findings with those for other vertebrate taxa and suggest that the present findings provide novel insights into the structure of conserved behavioral regulatory circuits that have led to evolutionary convergence in vocal-acoustic systems.     

Inhibitory and modulatory inputs to the vocal central pattern generator of a teleost fish

Vocalization is a behavioral feature that is shared among multiple vertebrate lineages, including fish. The temporal patterning of vocal communication signals is set, in part, by central pattern generators (CPGs). Toadfishes are well‐established models for CPG coding of vocalization at the hindbrain level. The vocal CPG comprises three topographically separate nuclei: pre‐pacemaker, pacemaker, motor. While the connectivity between these nuclei is well understood, their neurochemical profile remains largely unexplored. The highly vocal Gulf toadfish, Opsanus beta, has been the subject of previous behavioral, neuroanatomical and neurophysiological studies. Combining transneuronal neurobiotin‐labeling with immunohistochemistry, we map the distribution of inhibitory neurotransmitters and neuromodulators along with gap junctions in the vocal CPG of this species. Dense GABAergic and glycinergic label is found throughout the CPG, with labeled somata immediately adjacent to or within CPG nuclei, including a distinct subset of pacemaker neurons co‐labeled with neurobiotin and glycine. Neurobiotin‐labeled motor and pacemaker neurons are densely co‐labeled with the gap junction protein connexin 35/36, supporting the hypothesis that transneuronal neurobiotin‐labeling occurs, at least in part, via gap junction coupling. Serotonergic and catecholaminergic label is also robust within the entire vocal CPG, with additional cholinergic label in pacemaker and prepacemaker nuclei. Likely sources of these putative modulatory inputs are neurons within or immediately adjacent to vocal CPG neurons. Together with prior neurophysiological investigations, the results reveal potential mechanisms for generating multiple classes of social context‐dependent vocalizations with widely divergent temporal and spectral properties.     

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.     

Neuron types in the zebrafish optic tectum labeled by an id2b transgene

The larval zebrafish optic tectum has emerged as a prominent model for understanding how neural circuits control visually guided behaviors. Further advances in this area will require tools to monitor and manipulate tectal neurons with cell type specificity. Here, we characterize the morphology and neurotransmitter phenotype of tectal neurons labeled by an id2b:gal4 transgene. Whole-brain imaging of stable transgenic id2b:gal4 larvae revealed labeling in a subset of neurons in optic tectum, cerebellum, and hindbrain. Genetic mosaic labeling of single neurons within the id2b:gal4 expression pattern enabled us to characterize three tectal neuron types with distinct morphologies and connectivities. The first is a neuron type previously identified in the optic tectum of other teleost fish: the tectal pyramidal neuron (PyrN). PyrNs are local interneurons that form two stratified dendritic arbors and one stratified axonal arbor in the tectal neuropil. The second tectal neuron type labeled by the id2b:gal4 transgene is a projection neuron that forms a stratified dendritic arbor in the tectal neuropil and an axon that exits tectum to form a topographic projection to torus longitudinalis (TL). A third neuron type labeled is a projection neuron with a nonstratified dendritic arbor and a descending axonal projection to tegmentum. These findings establish the id2b:gal4 transgenic as a useful tool for future studies aimed at elucidating the functional role of tectum, TL, and tegmentum in visually guided behaviors.     

Partial deletion of p75NTR in large-diameter DRG neurons exerts no influence upon the survival of peripheral sensory neurons in vivo

The p75 neurotrophin receptor (p75^NTR) is required for maintaining peripheral sensory neuron survival and function; however, the underlying cellular mechanism remains unclear. The general view is that expression of p75^NTR by the neuron itself is required for maintaining sensory neuron survival and myelination in the peripheral nervous system (PNS). Adopting a neuronal-specific conditional knockout strategy, we demonstrate the partial depletion of p75^NTR in neurons exerts little influence upon maintaining sensory neuron survival and peripheral nerve myelination in health and after demyelinating neuropathy. Our data show that the density and total number of dorsal root ganglion (DRG) neurons in 2-month-old mice is not affected following the deletion of p75^NTR in large-diameter myelinating neurons, as assessed by stereology. Adopting experimental autoimmune neuritis induced in adult male mice, an animal model of demyelinating peripheral neuropathy, we identify that deleting p75^NTR in myelinating neurons exerts no influence upon the disease progression, the total number of DRG neurons, and the extent of myelin damage in the sciatic nerve, indicating that the expression of neuronal p75^NTR is not essential for maintaining peripheral neuron survival and myelination after a demyelinating insult in vivo. Together, results of this study suggest that the survival and myelination of peripheral sensory neurons is independent of p75^NTR expressed by a subtype of neurons in vivo. Thus, our findings provide new insights into the mechanism underpinning p75^NTR-mediated neuronal survival in the PNS.     

Lumbar spinal cord neurons putatively involved in ejaculation are sexually dimorphic in early postnatal mice

A crucial role in ejaculation is thought to be played by a population of lumbar spino-thalamic neurons (LSt), which express galanin and other neuropeptides. In rats, these neurons are activated with ejaculation and their lesion selectively abolishes ejaculation but not other mating behaviors. Consistently with their role, in adult rats and humans, LSt neurons are sexually dimorphic, being more numerous in males. Here we examined whether sexual dimorphism arises early in development, using a transgenic mouse line in which the expression of fluorescent protein is driven by the galanin promoter. We focused on postnatal day 4, shortly after a transient perinatal androgen surge in males that could play an organizational role in LSt development. We found a population of brightly fluorescent neurons organized in bilateral columns dorsolateral to the central canal in segments L1–L5, the expected location of the LSt group. Their number was close to that of adult preparations and significantly greater in male than in female siblings (+19%; CI_95%: +13% to +27%; p < .01). This was not due to a generalized higher galanin expression in the male since fluorescent L4 DRG neurons, innervating the hindlimbs and lower back, were not significantly dimorphic (−4%; CI_95%: −10% to +8%; p = .92). Unexpectedly, we found in cervical segments a population of fluorescent neurons having a location relative to the central canal similar to the LSt. Thus, the LSt group is sexually dimorphic soon after birth. However, it is possible that only a subset of its neurons participate in the control of ejaculation.     

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.     

Dorsal pallidal neurons directly link the nidopallium and midbrain in the zebra finch (Taeniopygia guttata)

The dorsal pallidum in birds is considered similar, if not homologous, to the globus pallidus (GP) of mammals. The dorsal pallidum projects to both thalamic and midbrain targets similar to the direct and indirect pathways arising from the internal and external segments of the GP. In the present study, retrograde and anterograde tracing studies revealed a previously undescribed projection of the avian dorsal pallidum. This arises from a specific dorsomedial component, which terminates in the intercollicular nucleus and partly surrounds the avian equivalent of the central nucleus of the inferior colliculus. The respiratory‐vocal dorsomedial nucleus of the intercollicular complex, however, does not receive these projections. The somata of the pallidal neurons retrogradely labeled from injections in the intercollicular nucleus were large and generally multipolar and had extensive, sparsely branching central processes (presumptive dendrites) that together extended up to 2 mm dorsally into the intermediate and caudomedial nidopallium. The size and morphology of these neurons were similar to those of large pallidal neurons labeled by calretinin immunoreactivity, which could be co‐localized to the same cells. Thus, rather than being directly involved in the control of movement, the large dorsomedial neurons of the caudal dorsal pallidum may be involved in sensory processing, in that they provide an unusual direct link between sensory (auditory/somatosensory) regions of the nidopallium and sensory regions of the intercollicular nucleus of the midbrain. J. Comp. Neurol. 525:1731–1742, 2017. © 2016 Wiley Periodicals, Inc.     

Dimorphic male brains and alternative reproductive tactics in a vocalizing fish.

Brains and types of behavior among sexually mature vertebrates are portrayed as having two phenotypic states: male and female. However, mating systems and the behavioral tactics employed by the two sexes are far more diverse than conveyed by this simple intersexual dichotomy. For example, in many species, sexually mature males may practice one of two alternative mating tactics. Recent studies of a sound-producing teleost fish now show that intrasexual dimorphism of vocal motor phenotypes among males accompanies the intrasexual divergence of types of reproductive behavior. Thus, one group of males, like females and juveniles, lacks the behavioral, neurophysiological and morphological traits typical of the sexually differentiated vocal motor pathway of the second group. The results are an explicit demonstration that for any one species: (1) alternative mating tactics can be paralleled by alternative phenotypes for the neurons that determine tactic-specific types of behavior, and (2) reproductive maturation is not obligatorily linked to the expression of neuronal secondary sex characteristics.     

Caudal mesopallial neurons in female songbirds bridge sensory and motor brain regions

Female songbirds use male song as an indicator of fitness and use that information to select their mate. Investigations of the female auditory system have provided evidence that the neurons within the caudal mesopallium (CM) are involved in the processing of songs that a female finds attractive, however, it is not clear how CM may exert its influence on behavioral indicators of mate choice. In the present study, anterograde tracing revealed the efferent connections of the female songbird CM. The results demonstrate connections to other auditory regions previously described in males, as well as novel connections to brain regions implicated in motor control. As in males, CM neurons in females project robustly to the lateral and medial extents of the caudal nidopallium, and to the ventral intermediate arcopallium. In a novel finding that is not present in males, CM neurons also project to the robust nucleus of the arcopallium and to the caudal striatum. Calling behavior and the expression of copulation solicitation displays are key indicators of female mate choice, and the projections found here bridge critical gaps necessary to understand how auditory perception can influence circuits related to the expression of those affiliative behaviors in female songbirds.     

Distribution of LPXRFa,a gonadotropin‐inhibitory hormone ortholog peptide,and LPXRFa receptor in the brain and pituitary of the tilapia

In vertebrates, gonadotropin‐releasing hormone (GnRH) and gonadotropin‐inhibitory hormone (GnIH), respectively, regulate reproduction in positive and negative manners. GnIH belongs to the LPXRFa family of peptides previously identified in mammalian and nonmammalian vertebrates. Studying the detailed distribution of LPXRFa as well as its receptor (LPXRFa‐R) in the brain and pituitary is important for understanding their multiple action sites and potential functions. However, the distribution of LPXRFa and LPXRFa‐R has not been studied in teleost species, partially because of the lack of fish‐specific antibodies. Therefore, in the present study, we generated specific antibodies against LPXRFa and its receptor from Nile tilapia (Oreochromis niloticus), and examined their distributions in the brain and pituitary by immunohistochemistry. Tilapia LPXRFa‐immunoreactive neurons lie in the posterior ventricular nucleus of the caudal preoptic area, whereas LPXRFa‐R‐immunoreactive cells are distributed widely. Double immunofluorescence showed that neither LPXRFa‐immunoreactive fibers nor LPXRFa‐R is closely associated or coexpressed with GnRH1, GnRH3, or kisspeptin (Kiss2) neurons. In the pituitary, LPXRFa fibers are closely associated with gonadotropic endocrine cells [expressing luteinizing hormone (LH) and follicle‐stimulating hormone (FSH)], with adrenocorticomelanotropic cells [corticotropin (ACTH) and α‐melanotropin (α‐MSH)], and with somatolactin endocrine cells. In contrast, LPXRFa‐R are expressed only in LH, ACTH, and α‐MSH cells. These results suggest that LPXRFa and LPXRFa‐R signaling acts directly on the pituitary cells independent from GnRH or kisspeptin and could play multiple roles in reproductive and nonreproductive functions in teleosts. J. Comp. Neurol. 524:2753–2775, 2016. © 2016 Wiley Periodicals, Inc.