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.     

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.     

Midbrain acoustic circuitry in a vocalizing fish

The mapping of auditory circuitry and its interface with vocal motor systems is essential to the investigation of the neural processing of acoustic signals and its relationship to sound production. Here we delineate the circuitry of a midbrain auditory center in a vocal fish, the plainfin midshipman. Biotin injections into physiologically identified auditory sites in nucleus centralis (NC) in the torus semicircularis show a medial column of retrogradely filled neurons in the medulla mainly in a dorsomedial division of a descending octaval nucleus (DO), dorsal and ventral divisions of a secondary octaval nucleus (SO), and the reticular formation (RF) near the lateral lemniscus. Biotin-filled neurons are also located at midbrain-pretectal levels in a medial pretoral nucleus. Terminal fields are identified in the medulla (ventral SO, RF), isthmus (nucleus praeeminentialis), midbrain (nucleus of the lateral lemniscus, medial pretoral nucleus, contralateral NC, tectum), diencephalon (lateral preglomerular, central posterior, and anterior tuber nuclei), and telencephalon (area ventralis). The medial column of toral afferent neurons is adjacent to and overlapping the positions of DO and SO neurons shown previously to be linked to the vocal pacemaker circuitry of the medulla. Midshipman are considered "hearing generalists" because they lack the peripheral adaptations of "specialists" that enhance the detection of the pressure component of acoustic signals. Whereas the results indicate a general pattern of acoustic circuitry similar to that of specialists, they also show central adaptations, namely, a vocal-acoustic interface in DO and SO related to this species' vocal abilities.     

Vocal‐motor and auditory connectivity of the midbrain periaqueductal gray in a teleost fish

The midbrain periaqueductal gray (PAG) plays a central role in the descending control of vocalization across vertebrates. The PAG has also been implicated in auditory‐vocal integration, although its precise role in such integration remains largely unexplored. Courtship and territorial interactions in plainfin midshipman fish depend on vocal communication, and the PAG is a central component of the midshipman vocal‐motor system. We made focal neurobiotin injections into the midshipman PAG to both map its auditory‐vocal circuitry and allow evolutionary comparisons with tetrapod vertebrates. These injections revealed an extensive bidirectional pattern of connectivity between the PAG and known sites in both the descending vocal‐motor and the ascending auditory systems, including portions of the telencephalon, dorsal thalamus, hypothalamus, posterior tuberculum, midbrain, and hindbrain. Injections in the medial PAG produced dense label within hindbrain auditory nuclei, whereas those confined to the lateral PAG preferentially labeled hypothalamic and midbrain auditory areas. Thus, the teleost PAG may have functional subdivisions playing different roles in vocal‐auditory integration. Together the results confirm several pathways previously identified by injections into known auditory or vocal areas and provide strong support for the hypothesis that the teleost PAG is centrally involved in auditory‐vocal integration. J. Comp. Neurol. 521:791–812, 2013. © 2012 Wiley Periodicals, Inc.     

Synaptic ultrastructure of Drosophila Johnston's organ axon terminals as revealed by an enhancer trap

The role of auditory circuitry is to decipher relevant information from acoustic signals. Acoustic parameters used by different insect species vary widely. All these auditory systems, however, share a common transducer: tympanal organs as well as the Drosophila flagellar ears use chordotonal organs as the auditory mechanoreceptors. We here describe the central neural projections of the Drosophila Johnston's organ (JO). These neurons, which represent the antennal auditory organ, terminate in the antennomechanosensory center. To ensure correct identification of these terminals we made use of a beta-galactosidase-expressing transgene that labels JO neurons specifically. Analysis of these projection pathways shows that parallel JO fibers display extensive contacts, including putative gap junctions. We find that the synaptic boutons show both chemical synaptic structures as well as putative gap junctions, indicating mixed synapses, and belong largely to the divergent type, with multiple small postsynaptic processes. The ultrastructure of JO fibers and synapses may indicate an ability to process temporally discretized acoustic information.     

Organization of eighth nerve afferent projections from individual endorgans of the inner ear in the teleost, Astronotus ocellatus

Eighth nerve fibers from the saccule, utricle, lagena, and the anterior, horizontal, and posterior semicircular canals of a cichlid fish were traced to the octavolateralis region of the brainstem using HRP and degeneration methods. The anterior, magnocellular, descending, and posterior nuclei of the octavus column receive inputs from all endorgans, whereas the tangential nucleus receives projections only from the utricle and semicircular canals. The most rostral projections only from the utricle and semicircular canals. The most rostral projection from each endorgan is found in the eminentia granularis of the vestibulolateral lobe of the cerebellum. Sparse terminals are found in the medial reticular formation from the utricle adn semicircular canals, and utricular and saccular remain terminate in the vicinity of the lateral dendrite of the Mauthner cell. Utricular and semicircular canal projections consistently overlap centrally as do saccular and lagenar inputs. Afferent fibers from all endorgans end within relatively distinct regions throughout the octavus column of nuclei. Saccular and lagenar inputs lie dorsal to the semicircular canal terminations. Utricular endings are complex, however, in that they overlap dorsally with saccular and lagenar terminals and ventrally with the semicircular canal inputs. Cerebellar inputs are found only in the eminentia granularis of the vestibulolateral lobe, and the densest terminals are from the utricle and the semicircular canals; the sparsest are from the saccule. Previous studies in fish have indicated that generally the utricle and semicircular canals are concerned with he maintenance of static and dynamic equilibrium whereas the saccule and lagena are concerned with auditory reception. There is recent evidence, however, for multiple functions within individual endorgans. Our anatomical findings suggest that in Astronotus each otolithic endorgan carries more than one modality; that the semicircular canals are concerned solely with an equilibrium function; and that acoustic information is processed dorsally and vestibular information ventrally along the octavus column of nuclei. No single nucleus appears to be solely auditory in function and only the tangential nucleus, situated ventrally in the octavus column, appears to be solely vestibular.     

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.     

Projections from the sacculus to the cochlear nuclei in the Mongolian gerbil

The projections of the saccule, an otolith end organ, to the cochlear nuclei were studied using both transganglionic transport and intracellular injection techniques. Labeled fibers and terminals were observed in the anterior and posterior portions of the ventral cochlear nucleus and the dorsal cochlear nucleus. Most terminals were present in the granule cell domain, especially in the subpeduncular corner between the anteroventral cochlear nucleus and the floccular peduncle of the cerebellum. It has been hypothesized that the cochlea in mammals may have developed phylogenetically from the saccule. The projections from the saccule to the cochlear nuclei were investigated in a mammalian species, the Mongolian gerbil, in an attempt to obtain initial information supporting or refuting this hypothesis. The presence of an otolith end organ projection to the cochlear nuclei in rodents should encourage comparative studies in additional aspects of the evolution of the auditory system.     

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.     

Development of tyrosine hydroxylase-immunoreactive cell populations and fiber pathways in the brain of the dogfish Scyliorhinus canicula: New perspectives on the evolution of the vertebrate catecholaminergic system

Developmental studies of the central catecholaminergic (CA) system are essential for understanding its evolution. To obtain knowledge about the CA system in chondrichthyans, an ancient gnathostome group, we used immunohistochemical techniques for detecting tyrosine hydroxylase (TH), the initial rate‐limiting enzyme of the CA synthesis, to study: 1) the neuromery of developing TH‐immunoreactive (ir) neuronal populations, 2) the development of TH‐ir innervation, and 3) the organization of TH‐ir cells and fibers in the brain of postembryonic stages of the shark Scyliorhinus canicula. The first TH‐ir cells appeared in the hypothalamus and rostral diencephalon (suprachiasmatic, posterior recess and posterior tubercle nuclei at embryonic stage 26, and dorsomedial hypothalamus at stage 28); then in more caudal basal regions of the diencephalon and rostral mesencephalon (substantia nigra/ventral tegmental area); and later on in the anterior (locus coeruleus/nucleus subcoeruleus) and posterior (vagal lobe and reticular formation) rhombencephalon. The appearance of TH‐ir cells in the telencephalon (pallium) was rather late (stage [S]31) with respect to the other TH‐ir prosencephalic populations. The first TH‐ir fibers arose from cells of the posterior tubercle (S30) and formed recognizable ascending (toward dorsal and rostral territories) and descending pathways at S31. When the second half of embryonic development started (S32), TH‐ir fibers innervated most brain areas, and nearly all TH‐ir cell groups of the postembryonic brain were already established. This study provides key information about the evolution of the developmental patterns of central CA systems in fishes and thus may help in understanding how the vertebrate CA systems have evolved. J. Comp. Neurol. 520:3574–3603, 2012. © 2012 Wiley Periodicals, Inc.     

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.     

Central projections of octavolateralis nerves in the brain of a soniferous damselfish (Abudefduf abdominalis)

Sounds and hydrodynamic stimuli are important cues detected by the octavolateralis system in fishes. The central organization of auditory, mechanosensory, and vestibular projections is known for only a few phylogenetically diverse fishes, and less is known about projections in derived perciforms that use sounds for acoustic communication. We used neuronal labeling to provide a detailed analysis of octavolateralis endorgan projections in a soniferous perciform that does not have accessory morphological structures to enhance hearing. Octavolateralis nerves terminate ipsilaterally within seven medullary octaval nuclei: caudal (CON) and medial (MON) octavolateralis, anterior (AON), descending (DON), magnocellular (MgON), tangential (TON), and posterior (PON) octaval nuclei, and the eminentia granularis (EG). Anterior and posterior lateral line nerves project to the CON and MON, with dense projections to the EG. Semicircular canal nerves project primarily to ventral regions including the TON, ventral DON, intermediate DON (DONi), and MgON. Otolithic, semicircular canal, and anterior lateral line nerves all project to the MgON, which may serve a sensorimotor integration function. The DONi receives primarily segregated projections from all otolithic and semicircular canal nerves, whereas the ventral DON and TON receive principally utricular and semicircular canal afferents. The AON receives dense lateral and ventral projections from the saccule and utricle, and medial and dorsal projections from the lagena. These projection patterns are similar to those reported for non‐sonic perciforms, and indicate the absence of neuroanatomical modifications in first‐order octavolateralis nuclei in species that use acoustic communication. Thus patterns of central projections may be conserved among vocal and non‐vocal perciforms. J. Comp. Neurol. 512:628–650, 2009. © 2008 Wiley‐Liss, Inc.     

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.