Vasotocin innervation and modulation of vocal-acoustic circuitry in the teleost Porichthys notatus

Arginine vasotocin (AVT) and its mammalian homologue arginine vasopressin (AVP) modulate reproduction-related and other social behaviors in a broad range of vertebrate species. These functions of AVT/AVP may be in part achieved through the modulation of sensorimotor integration, although experimental evidence supporting this hypothesis remains limited. In the present experiments, we demonstrate (1) AVT innervation of candidate vocal-acoustic brain regions, and (2) AVT modulation of vocal-motor physiology in the plainfin midshipman fish (Porichthys notatus), which uses vocalizations in both mate attraction and agonistic contexts. AVT distribution was compared with known vocally active brain regions and to central auditory and vocal pathways. AVT-immunoreactive fibers and putative terminals descend almost exclusively from the preoptic area and are found in two primary candidate sites for vocal-acoustic integration - the anterior tuberal hypothalamus and paralemniscal midbrain tegmentum. AVT immunoreactivity is also located in several other vocally active regions, including the ventral tuberal nucleus, periaqueductal gray, and paraventricular regions of the isthmus and rostral hindbrain. The parvocellular preoptic area itself is also vocally active, although thresholds are substantially higher than for other regions. The functional significance of AVT input to vocal-acoustic regions was demonstrated in the paralemniscal midbrain where local delivery of AVT modulated electrically evoked, rhythmic vocal-motor output, which precisely mimicked natural vocalizations. AVT produced dose-dependent inhibitions of parameters associated with call initiation (burst latency and number of vocal-motor bursts elicited) but not of vocal-motor patterning (fundamental frequency and burst duration). Together, these findings provide support for the proposal that AVT modulates sensorimotor processes underlying social/acoustic communication.     

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.     

Coexistence of oxytocin and tyrosine hydroxylase in the rat hypothalamus,an immunocytochemical study

Summary Immunocytochemical double labelling was used to determine the structural relationship of oxytocin (OT) and tyrosine hydroxylase (TH) containing perikarya and processes in the rat hypothalamus. Extrahypothalamic TH fibers, as well as parvocellular TH neurons were found to form contacts with OT cells. A fraction of the OT neurons contained TH immunoreactivity. It is likely that in addition to the classical mesencephalic afferences also hypothalamic interneurons and magnocellular dopaminergic neurons control the hypothalamo neurohypophysial system.     

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.     

Social and neural modulation of sexual plasticity in teleost fish

Teleost fishes are the 'champions' of sexual plasticity among vertebrates. Several species have two male reproductive morphs with distinct suites of behavioral, somatic, neuronal, endocrinological, and life history traits. Here, we consider recent studies of the social and neural modulation of sexual plasticity for such species with a focus on two neuropeptides, gonadotropin releasing hormone (GnRH) and arginine vasotocin (AVT, teleostean analogue of mammalian arginine vasopressin). The major premise of this review is that phenotypic changes in GnRH and AVT expression in the brain can orchestrate events leading to changes in either sexual status or the expression of morph specific display behaviors important in reproduction.     

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.     

Brain Vasotocin Pathways and the Control of Sexual Behaviors in the Bullfrog

The neurohypophysial peptide arginine vasotocin (AVT) alters the display of several sexually dimorphic behaviors in the bullfrog (Rana catesbeiana). These behaviors include mate calling, release calling, call phonotaxis, and locomotor activity. Populations of AVT-immunoreactive cells are present in six areas of bullfrog brain and fibers are widespread. Neural areas involved in vocalization, in particular, contain AVT cells and fibers. As well, AVT concentrations in a subset of brain areas are sexually dimorphic and steroid sensitive. Effects of gonadectomy and gonadal steroid treatment vary, depending on the brain area and sex of the frog. For example, some anterior areas are sensitive to changes in both dihydrotestosterone (DHT) and estradiol. In some posterior brain areas, on the other hand, AVT levels are affected only by DHT. A similar situation exists for putative AVT receptors in bullfrogs. Receptors are widespread, occurring in many areas that have been linked to behavior. Receptor concentrations are sexually dimorphic in the amygdala pars lateralis, hypothalamus, pretrigeminal nucleus, and dorsolateral nucleus. Estradiol alters AVT receptor level in the amygdala of both sexes of bullfrog and both estradiol and DHT alter the receptor number in the pretrigeminal nucleus, but only in males. The mechanisms responsible for steroid effects on vasotocin neurons and their targets are unknown. Specific AVT cells, fiber terminal fields, and receptor populations are likely influenced by gonadal steroids for effective timing of individual behaviors displayed by bullfrogs.     

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.     

A novel approach to AVT and IT studies in fish brain and pituitary: in vitro perfusion technique

The study was designed to develop a new procedure for perfusion of brain and pituitary explants collected from three-spined stickleback (Gasterosteus aculeatus) and round goby (Neogobius melanostomus). The procedure was elaborated for studies of arginine vasotocin (AVT) and isotocin (IT) release from explants of both species. AVT and IT, analogs of mammalian vasopressin and oxytocin, are neurohormones produced in hypothalamus and released in neurohypophysis of Teleostei. Both nonapeptides are used as biomarkers of fish well being. Three perfusion sets were applied to test the method of medium transport into gradient container, without or with aeration. Medium supply to the gradient container from the top, without aeration is recommended only for short-term studies. Aeration of the medium with a mixture of 95% O(2) and 5% CO(2) at a pressure of 127.51 mm Hg is necessary for a long-term research. Transport of one or two media in the gradient container from the top and the bottom, simultaneously, requires aeration with a mixture of 95% O(2) and 5% CO(2) at a pressure of 315.03 mm Hg. Although the presented procedure has been elaborated for studies of AVT and IT in fish explants, after only minor modification, if any, it can serve many other purposes.     

The Effects of Melatonin on Brain Arginine Vasotocin: Relationship with Sex and Seasonal Differences in Melatonin Receptor Type 1 in Green Treefrogs (Hyla cinerea)

The neuroendocrine mechanisms by which animals synchronise their physiological state with environmental cues are vital to timing life‐history events appropriately. One important endocrine transducer of environmental cues in vertebrates is the pineal hormone melatonin, the secretion of which is directly sensitive to photoperiod and temperature. Melatonin modulates arginine vasotocin (AVT)‐immunoreactive (‐IR) cell number in the brain of green treefrogs (Hyla cinerea) during the summer breeding season, and this modulation is sexually dimorphic. In the present study, we investigated whether the influence of melatonin on vasotocin varies seasonally. We show that treatment of nonreproductive male green treefrogs with melatonin‐filled silastic implants for 4 weeks during the winter does not alter vasotocin‐IR cell number in any brain region (i.e. nucleus accumbens, amygdala, preoptic area, suprachiasmatic nucleus or ventral hypothalamus). Taken together, these results suggest that the influence of melatonin on AVT is associated with sex and seasonal variation in melatonin receptor expression. We tested this hypothesis by using immunohistochemistry to characterise the distribution of melatonin receptor type 1 (MT1, also known as Mel1a) in the brain of reproductive and nonreproductive male and female frogs. We quantified MT1‐IR cell number in regions known to contain AVT cell populations. Reproductive males had significantly more MT1‐IR cells than nonreproductive males in all brain regions, including the combined nucleus accumbens, diagonal band of Broca and septum, striatum, amygdala, combined preoptic area and suprachiasmatic nucleus, as well as the ventral hypothalamus. In the accumbens region, where the effect of melatonin on AVT is known to be sexually dimorphic, males had significantly more MT1‐IR cells than females during the summer breeding season. Based on these findings, we suggest that MT1 plays a role in mediating the interactions between melatonin and vasotocin that regulate seasonal and sexually dimorphic changes in sociosexual behaviour.     

Opposite effects of vasotocin and of a specific vasotocin antiserum on active sleep of kittens

Summary The effects of intracerebroventricularly administered synthetic arginine vasotocin (AVT) or undiluted AVT, vasopressin (AVP) and oxytocin (OT) antisera on active sleep (AS) of newborn kittens have been investigated in comparison with rabbit serum control. In contrast to AVP and OT antisera, AVT antiserum has produced opposite effects on AS as AVT itself. Since after 10 l of undiluted AVT antiserum the percentage of AS did not decrease under 20% and even after 100 l AS did not decrease under 5%, it is concluded that, at least during perinatal life, AVT could be considered as a neuromodulator with AS-promoting effect.     

Anatomical and functional implications of corticotrophin‐releasing hormone neurones in a septal nucleus of the avian brain: an emphasis on glial‐neuronal interaction via V1a receptors in vitro

Previously, we showed that corticotrophin‐releasing hormone immunoreactive (CRH‐IR) neurones in a septal structure are associated with stress and the hypothalamic‐pituitary‐adrenal axis in birds. In the present study, we focused upon CRH‐IR neurones located within the septal structure called the nucleus of the hippocampal commissure (NHpC). Immunocytochemical and gene expression analyses were used to identify the anatomical and functional characteristics of cells within the NHpC. A comparative morphometry analysis showed that CRH‐IR neurones in the NHpC were significantly larger than CRH‐IR parvocellular neurones in the paraventricular nucleus of the hypothalamus (PVN) and lateral bed nucleus of the stria terminalis. Furthermore, these large neurones in the NHpC usually have more than two processes, showing characteristics of multipolar neurones. Utilisation of an organotypic slice culture method enabled testing of how CRH‐IR neurones could be regulated within the NHpC. Similar to the PVN, CRH mRNA levels in the NHpC were increased following forskolin treatment. However, dexamethasone decreased forskolin‐induced CRH gene expression only in the PVN and not in the NHpC, indicating differential inhibitory mechanisms in the PVN and the NHpC of the avian brain. Moreover, immunocytochemical evidence also showed that CRH‐IR neurones reside in the NHpC along with the vasotocinergic system, comprising arginine vasotocin (AVT) nerve terminals and immunoreactive vasotocin V1a receptors (V1aR) in glia. Hence, we hypothesised that AVT acts as a neuromodulator within the NHpC to modulate activity of CRH neurones via glial V1aR. Gene expression analysis of cultured slices revealed that AVT treatment increased CRH mRNA levels, whereas a combination of AVT and a V1aR antagonist treatment decreased CRH mRNA expression. Furthermore, an attempt to identify an intercellular mechanism in glial‐neuronal communication in the NHpC revealed that brain‐derived neurotrophic factor (BDNF) and its receptor (TrkB) could be involved in the signalling mechanism. Immunocytochemical results further showed that both BDNF and TrkB receptors were found in glia of the NHpC. Interestingly, in cultured brain slices containing the NHpC, the use of a selective TrkB antagonist decreased the AVT‐induced increase in CRH gene expression levels. The results from the present study collectively suggest that CRH neuronal activity is modulated by AVT via V1aR involving BDNF and TrkB glia in the NHpC.     

Fluorescent Visualisation of Oxytocin in the Hypothalamo‐neurohypophysial/‐spinal Pathways After Chronic Inflammation in Oxytocin‐Monomeric Red Fluorescent Protein 1 Transgenic Rats

Oxytocin (OXT) is a well‐known neurohypophysial hormone that is synthesised in the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus. The projection of magnocellular neurosecretory cells, which synthesise OXT and arginine vasopressin in the PVN and SON, to the posterior pituitary plays an essential role in mammalian labour and lactation through its peripheral action. However, previous studies have shown that parvocellular OXTergic cells in the PVN, which project to the medulla and spinal cord, are involved in various physiological functions (e.g. sensory modulation and autonomic). In the present study, we examined OXT expression in the PVN, SON and spinal cord after chronic inflammation from adjuvant arthritis (AA). We used transgenic rats that express OXT and the monomeric red fluorescent protein 1 (mRFP1) fusion gene to visualise both the magnocellular and parvocellular OXTergic pathways. OXT‐mRFP1 fluorescence intensity was significantly increased in the PVN, SON, dorsal horn of the spinal cord and posterior pituitary in AA rats. The levels of OXT‐mRFP1 mRNA were significantly increased in the PVN and SON of AA rats. These results suggested that OXT was up‐regulated in both hypothalamic magnocellular neurosecretory cells and parvocellular cells by chronic inflammation, and also that OXT in the PVN‐spinal pathway may be involved in sensory modulation. OXT‐mRFP1 transgenic rats are a very useful model for visualising the OXTergic pathways from vesicles in a single cell to terminals in in vitro preparations.