Steroid-dependent plasticity of vocal motor systems: novel insights from teleost fish.

Vocal communication is a trait shared by most vertebrates. Non-mammalian model systems have provided exquisite examples of how motor and sensory systems, respectively, produce and encode the physical attributes of acoustic communication signals that play essential roles in mediating the dynamics of social behavior. These same models, mainly developed for a few species of fish, amphibians and birds, have proven to be equally important for demonstrating how steroids and other hormones shape the neural mechanisms of vocal communication. This review mainly considers recent studies in teleost fish demonstrating the role of steroids in the rapid modulation of the firing properties of a central pattern generator for vocalization. Thus, steroids, like other classes of neurochemicals, can play an instrumental role in reshaping the neurophysiological coding of motor patterning, in this case for social signaling behavior.

Directionality and frequency tuning of primary saccular afferents of a vocal fish, the plainfin midshipman (Porichthys notatus).

While particle motion is thought to directly stimulate the inner ear of most fish species, it is difficult to measure and might not be predictable from pressure measurements in a small tank. It is therefore important to replicate experiments conducted relative to pressure measurements using stimuli of known particle motion, to ensure that unmeasured components of the stimulus field do not produce misleading frequency response profiles. The frequency sensitivity of the inner ear of the plainfin midshipman fish, Porichthys notatus, in response to isopressure stimuli has been described. This study now examines the frequency and directional response properties of midshipman saccular afferents in response to whole-body displacements simulating acoustic particle motion. Best frequencies were distributed bimodally, with peaks at 50 Hz and 100 Hz. Most units had cosinusoidally shaped directional response profiles in the horizontal and vertical planes, though some units showed slight deviations from this pattern. A few units (probably saccular efferents) had omnidirectional directional response profiles and did not phase lock to the stimulus waveform. These results are consistent with responses of the midshipman saccular nerve to isopressure stimuli, and strengthen the hypothesis that the frequency sensitivity of the midshipman ear matches the frequency content of behaviorally relevant vocalizations.

Anatomical distribution and cellular basis for high levels of aromatase activity in the brain of teleost fish: aromatase enzyme and mRNA expression identify glia as source.

Although teleost fish have higher levels of brain aromatase activity than any other vertebrate group, its function remains speculative, and no study has identified its cellular basis. A previous study determined aromatase activity in a vocal fish, the plainfin midshipman (Porichthys notatus), and found highest levels in the telencephalon and lower levels in the sonic hindbrain, which was dimorphic between and within (males) sexes. We have now localized aromatase-containing cells in the midshipman brain both by immunocytochemistry using teleost-specific aromatase antibodies and by in situ hybridization using midshipman-specific aromatase probes. Aromatase-immuno-reactivity and mRNA hybridization signal are consistent with relative levels of aromatase activity in different brain regions: concentrated in the dimorphic sonic motor nucleus, in a band just beneath the periaqueductal gray in the midbrain, in ventricular regions in the hypothalamus, and highest levels in the telencephalon especially in preoptic and ventricular areas. Surprisingly, double-label immunofluorescence does not show aromatase-immunoreactive colocalization in neurons, but instead in radial glia throughout the brain. This is the first study to identify aromatase expression mostly, if not entirely, in glial cells under normal rather than brain injury-dependent conditions. The abundance of aromatase in teleosts may represent an adaptation linked to continual neurogenesis that is known to occur throughout an individual's lifetime among fishes. The localization of aromatase within the intersexually and intrasexually dimorphic vocal-motor circuit further implies a function in the expression of alternative male reproductive phenotypes and, more generally, the development of natural, individual variation of specific brain nuclei.

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.

Coding of concurrent vocal signals by the auditory midbrain: effects of duration.

Neural selectivity to signal duration within the auditory midbrain has been observed in several species and is thought to play a role in signal recognition. Here we examine the effects of signal duration on the coding of individual and concurrent vocal signals in a teleost fish with exceptionally long duration vocalizations, the plainfin midshipman, Porichthys notatus. Nesting males produce long-duration, multi-harmonic signals known as hums to attract females to their nests; overlapping hums produce acoustic beats at the difference frequency of their spectral components. Our data show that all midbrain neurons have sustained responses to long-duration hum-like tones and beats. Overall spike counts increase linearly with signal duration, although spike rates decrease dramatically. Neurons show varying degrees of spike rate decline and hence, differential changes in spike rate across the neuron population may code signal duration. Spike synchronization to beat difference frequency progressively increases throughout long-duration beats such that significant difference frequency coding is maintained in most neurons. The significance level of difference frequency synchronization coding increases by an order of magnitude when integrated over the entirety of long-duration signals. Thus, spike synchronization remains a reliable difference frequency code and improves with integration over longer time spans.

Acoustic nuclei in the medulla and midbrain of the vocalizing Gulf toadfish (Opsanus beta).

The organization of the descending and secondary octaval nuclei in the hindbrain of the Gulf toadfish, Opsanus beta, was revealed following the injection of biotin compounds into a physiologically identified auditory region of the torus semicircularis. The results show retrogradely-filled neurons mainly in a dorsomedial division of the descending octaval nucleus, and dorsal and ventral divisions of a secondary octaval nucleus; minor labeling also appeared in dorsolateral and rostromedial intermediate divisions of the descending nucleus. The pattern identified is consistent with that reported in other teleosts, including both vocal and non-vocal species, and clarifies earlier reports of the organization of hindbrain octaval nuclei in toadfish and the closely related midshipman fish.

Peripheral encoding of behaviorally relevant acoustic signals in a vocal fish: harmonic and beat stimuli.

Nesting male midshipman fish, Porichthys notatus, emit simple, long-duration sounds known as hums, which are attractive to gravid females. While hums share the multi-harmonic structure typical of many vertebrate communication sounds, their lack of amplitude modulation gives individual hums unusually simple temporal envelopes. However, hums often overlap, producing beats in the summed acoustic waveform. This study presents responses of individual saccular afferent fibers to two-tone harmonic and beat stimuli presented via an underwater loudspeaker. Spike activity was quantified as vector strength of synchronization and average spike rate. Responses to harmonic stimuli depended on harmonic phase; these effects apparently resulted primarily from variation in waveform fine temporal structure rather than auditory non-linearities. At most phases, addition of the harmonic enhanced afferent synchronization compared to the fundamental alone. Two-tone beat stimuli evoked stronger synchronization to the component frequencies than to the beat modulation rate. Vector strength tended to be higher to the lower frequency component, and this pattern appeared to derive from afferent tuning. Midshipman saccular afferents encoded both the temporal envelope and waveform fine structure of these naturalistic signals, information that may be important in conspecific communication.

Effects of temporal envelope modulation on acoustic signal recognition in a vocal fish, the plainfin midshipman.

Amplitude modulation is an important parameter defining vertebrate acoustic communication signals. Nesting male plainfin midshipman fish, Porichthys notatus, emit simple, long duration hums in which modulation is strikingly absent. Envelope modulation is, however, introduced when the hums of adjacent males overlap to produce acoustic beats. Hums attract gravid females and can be mimicked with continuous tones at the fundamental frequency. While individual hums have flat envelopes, other midshipman signals are amplitude modulated. This study used one-choice playback tests with gravid females to examine the role of envelope modulation in hum recognition. Various pulse train and two-tone beat stimuli resembling natural communication signals were presented individually, and the responses compared to those for continuous pure tones. The effectiveness of pulse trains was graded and depended upon both pulse duration and the ratio of pulse to gap length. Midshipman were sensitive to beat modulations from 0.5 to 10 Hz, with fewer fish approaching the beat than the pure tone. Reducing the degree of modulation increased the effectiveness of beat stimuli. Hence, the lack of modulation in the midshipman's advertisement call corresponds to the importance of envelope modulation for the categorization of communication signals even in this relatively simple system.

Social behavior functions and related anatomical characteristics of vasotocin/vasopressin systems in vertebrates.

The neuropeptide arginine vasotocin (AVT; non-mammals) and its mammalian homologue, arginine vasopressin (AVP) influence a variety of sex-typical and species-specific behaviors, and provide an integrational neural substrate for the dynamic modulation of those behaviors by endocrine and sensory stimuli. Although AVT/AVP behavioral functions and related anatomical features are increasingly well-known for individual species, ubiquitous species-specificity presents ever increasing challenges for identifying consistent structure-function patterns that are broadly meaningful. Towards this end, we provide a comprehensive review of the available literature on social behavior functions of AVT/AVP and related anatomical characteristics, inclusive of seasonal plasticity, sexual dimorphism, and steroid sensitivity. Based on this foundation, we then advance three major questions which are fundamental to a broad conceptualization of AVT/AVP social behavior functions: (1) Are there sufficient data to suggest that certain peptide functions or anatomical characteristics (neuron, fiber, and receptor distributions) are conserved across the vertebrate classes? (2) Are independently-evolved but similar behavior patterns (e.g. similar social structures) supported by convergent modifications of neuropeptide mechanisms, and if so, what mechanisms? (3) How does AVT/AVP influence behavior - by modulation of sensorimotor processes, motivational processes, or both? Hypotheses based upon these questions, rather than those based on individual organisms, should generate comparative data that will foster cross-class comparisons which are at present underrepresented in the available literature.

Coding of concurrent vocal signals by the auditory midbrain: effects of stimulus level and depth of modulation.

The segregation of concurrent vocal signals is an auditory processing task faced by all vocal species. To segregate concurrent signals, the auditory system must encode the spectral and temporal features of the fused waveforms such that at least one signal can be individually detected. In the plainfin midshipman fish (Porichthys notatus), the overlapping mate calls of neighboring males produce acoustic beats with amplitude and phase modulations at the difference frequencies (dF) between spectral components. Prior studies in midshipman have shown that midbrain neurons provide a combinatorial code of the temporal and spectral characteristics of beats via synchronization of spike bursts to dF and changes in spike rate and interspike intervals with changes in spectral composition. In the present study we examine the effects of changes in signal parameters of beats (overall intensity level and depth of modulation) on the spike train outputs of midbrain neurons. The observed changes in spike train parameters further support the hypothesis that midbrain neurons provide a combinatorial code of the spectral and temporal features of concurrent vocal signals.

Temporal population code of concurrent vocal signals in the auditory midbrain.

Unique patterns of spike activity across neuron populations have been implicated in the coding of complex sensory stimuli. Delineating the patterns of neural activity in response to varying stimulus parameters and their relationships to the tuning characteristics of individual neurons is essential to ascertaining the nature of population coding within the brain. Here, we address these points in the midbrain coding of concurrent vocal signals of a sound-producing fish, the plainfin midshipman. Midshipman produce multiharmonic vocalizations which frequently overlap to produce beats. We used multivariate statistical analysis from single-unit recordings across multiple animals to assess the presence of a temporal population code. Our results show that distinct patterns of temporal activity emerge among midbrain neurons in response to concurrent signals that vary in their difference frequency. These patterns can serve to code beat difference frequencies. The patterns directly result from the differential temporal coding of difference frequency by individual neurons. Difference frequency encoding, based on temporal patterns of activity, could permit the segregation of concurrent vocal signals on time scales shorter than codes requiring averaging. Given the ubiquity across vertebrates of auditory midbrain tuning to the temporal structure of acoustic signals, a similar temporal population code is likely present in other species.

Sonic/vocal motor pathways in squirrelfish (Teleostei, Holocentridae).

Similar to many teleost fish, squirrelfish (family Holocentridae) produce vocalizations by the contraction of muscles that lead to vibration of the swimbladder. We used biotinylated compounds to identify the position and extent of vocal motor neurons in comparison to additional motor neuron groups, namely those of red and white dorsal epaxial muscle and opercular muscle that are located adjacent to or near the sonic muscle. The sonic motor nucleus (SMN) was located in the caudal medulla and rostral spinal cord in a ventrolateral position with dendrites extending dorsally in a dense bundle along the lateral edge of the medulla and axons exiting via ventral occipital nerve roots. Transneuronal transport of biocytin identified premotor neurons within the SMN and in the medially adjacent reticular formation that projected to the contralateral SMN and more rostrally to the octavolateralis efferent nucleus and nucleus praeeminentialis, suggesting interactions between vocal and octavolateralis systems as seen in other teleosts. Motor neurons innervating the red and white dorsal muscle formed a loose aggregate in the dorsal motor column, adjacent to the medial longitudinal fasciculus, sending fibers bilaterally throughout the spinal cord with axons exiting via ventral spinal nerve roots. Opercular motor neurons were located within the facial motor nucleus. The anatomical characteristics of the SMN of squirrelfish, a representative member of the order Beryciformes, are similar to those of representative members of the closely related order Scorpaeniformes, but diverge from the SMN of more distantly related orders of paracanthopterygiian and ostariophysan teleosts. These results therefore suggest a possible homology among the SMNs of acanthopterygiian fishes.

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.

Rhythmic midbrain-evoked vocalization is inhibited by vasoactive intestinal polypeptide in the teleost Porichthys notatus.

Vasoactive intestinal polypeptide (VIP) is distributed in vocal midbrain areas of multiple vertebrate taxa, suggesting that VIP may modulate midbrain-evoked vocalization. To test this hypothesis, neurophysiological experiments were conducted in the teleost Porichthys notatus which generates vocalizations in mating and agonistic contexts. Electrical stimulation of the paralemniscal midbrain and local delivery of VIP were conducted in conjunction with occipital nerve recordings that reflect the patterned output of hindbrain vocal circuitry. Consistent with our hypothesis, VIP significantly reduced the duration and number of rhythmic vocal-motor bursts obtained in a dose-dependent manner; vocalization latency was concomitantly increased. These results provide the first evidence for VIP modulation of midbrain vocal function.

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.

Central lateral line pathways in a vocalizing fish.

The organization of the central lateral line pathways in the midshipman fish, Porichthys notatus, was identified following biotin injections into physiologically identified sites in the lateral line-recipient nucleus ventrolateralis in the midbrain. Retrogradely filled neurons are located primarily in nucleus medialis, the principal termination site of lateral line nerve afferents in the medulla, whereas terminal fields are mainly identified in isthmal (nucleus praeeminentialis) and diencephalic (posterior thalamic) nuclei. Compared to other teleosts, nucleus medialis has a distinctive cytoarchitecture in that most of its somata are confined to a dense cell plate adjacent to the fourth ventricle. Injections into nucleus ventrolateralis reveal a caudal (MEDc) and a rostral (MEDr) division of nucleus medialis which are separated by a dorsomedial division of the descending octaval nucleus. MEDc is further divisible into a caudal spherical and a more extensive rostral Purkinje-like cell division. MEDr includes a caudal division of Purkinje-like cells and a rostral division of round and fusiform-shaped cells that form a lateral band under the cerebellar crest. In addition to labeling terminals in nucleus ventrolateralis, biotin injections into MEDc and MEDr further distinguish intrinsic connectivity within nucleus medialis, and also label somata and terminals within other octavolateralis nuclei in the medulla. Injections into both nucleus ventrolateralis and nucleus medialis identify sites which may be processing information from both the auditory and lateral line systems, including the eighth nerve-recipient descending octaval nucleus, the acoustic division of the midbrain, and nucleus praeeminentialis which receives auditory input from the midbrain in midshipman.

Forebrain peptides modulate sexually polymorphic vocal circuitry.

The peptide arginine-vasopressin (mammals) and its evolutionary precursor arginine-vasotocin (non-mammals) modulate reproductive physiology and numerous related social behaviours, as do oxytocin (mammals) and its homologues mesotocin and isotocin (fish). The distributions in the brain and/or the behavioural functions of these peptides often differ between the sexes, and between species with divergent social structures. Here we present neurophysiological evidence that males with vocal characteristics typical of females share a pattern of neuropeptide function with females rather than conspecific males. The plainfin midshipman fish (Porichthys notatus) has two male morphs with different reproductive tactics and vocalizations (a key species-typical behaviour which varies in its physical attributes and contextual usage, depending on the morph's social strategy). Forebrain-evoked, rhythmic vocal-motor activity that precisely mimics natural vocalizations was modulated by arginine-vasotocin, isotocin and their antagonists delivered to the preoptic area-anterior hypothalamus, a primary site for behavioural integration in all vertebrates. Peptides had different effects in males that acoustically court females (arginine-vasotocin-sensitive) than in females and sneak-spawning males (isotocin-sensitive), showing that the neuromodulatory mechanisms that establish reproduction-related behaviour can be dissociated from gonadal sex.

Preoptic GnRH and AVT: axes for sexual plasticity in teleost fish.

Alternative reproductive tactics within one sex, adult sex or role change, and reproductive suppression are all forms of reproductive plasticity commonly exhibited among teleost fishes. The two neuropeptides that have been most extensively studied with regard to such behavioral plasticity are gonadotropin releasing hormone (GnRH) and arginine vasotocin (AVT). Here, we review intra- and intersexual variation in the number and size of GnRH and AVT neurons along with gonadal phenotype in those species of teleosts showing intraspecific plasticity in reproductive behavior. In several species, male dimorphisms in the number and/or size of GnRH neurons in the forebrain's preoptic area parallel a divergence in relative gonad size and reproductive tactics. The available studies of AVT-containing neurons in the preoptic area also indicate intrasexual dimorphisms among males, although a proximate link to other reproductive traits and behavioral outcomes is more difficult to recognize. For both GnRH and AVT, there are also species-typical patterns in the coupling between structural (e.g., neuronal and gonadal) traits and reproductive tactic expressed, which likely reflect distinct patterns of adaptation to particular ecological environments. As discussed, neurophysiological, biochemical, and receptor density studies are now essential to establish the functional significance of the diverse organizational patterns of GnRH and AVT neurons in teleosts. Similar studies also need to be carried out in species of other vertebrate groups that show comparable behavioral plasticity.

Peripheral encoding of behaviorally relevant acoustic signals in a vocal fish: single tones.

The midshipman fish, Porichthys notatus, generates acoustic signals for intraspecific communication. Nesting males produce long-duration "hums" which attract gravid females and can be effectively mimicked by pure tones. In this study we examine the encoding of tonal signals by the midshipman peripheral auditory system. Single-unit recordings were made from afferents innervating the sacculus while presenting sounds via an underwater loudspeaker. Units were characterized by iso-intensity spike rate and vector strength of synchronization curves, as well as by peristimulus time histograms. Additionally, response-intensity curves and responses to long-duration (up to 10 s) stimuli were obtained. As has been seen in other teleosts, afferents had highly variable activity profiles. Excitatory frequencies ranged from 60 to over 300 Hz with most units responding best around 70 or 140 Hz. Thresholds at 90 Hz ranged from 95 to 145 dB re 1 microPa. Strong synchronization provided a robust temporal code of frequency, comparable to that described for goldfish. Spike rate showed varying degrees of adaptation but high rates were generally maintained even for 10-s stimuli. The midshipman peripheral auditory system is well suited to encoding conspecific communication signals, but nonetheless shares many response patterns with the auditory system of other teleosts.

Early development of the motor and premotor circuitry of a sexually dimorphic vocal pathway in a teleost fish.

The plainfin midshipman fish (Porichthys notatus) has a caudal hindbrain vocal motor circuit that has been proposed to share a common embryonic origin with the hindbrain vocal networks of other vertebrates. In midshipman, this vocal circuit includes three groups of neurons: sonic motor, pacemaker, and ventral medullary. Here, transneuronal transport of biocytin or neurobiotin was used to delineate the early ontogeny of the three hindbrain vocal nuclei and their pattern of connectivity. The organization of the vocal nuclei was studied in animals beginning soon after hatching until the nuclei have the adult phenotype at the time fish become free-swimming. There is a clear sequence of events whereby motoneurons establish their connections with the sonic muscle prior to establishing connections with premotor neurons; developmental milestones of the vocal pathway parallel those of the sonic muscle. The results also indicate that sexual differentiation of the vocal motor system in midshipman begins early in development, well before any evidence of sexual maturation. Embryonic males and females differ in the relationship between soma size and body length for the three hindbrain nuclei. Males are also more variable than females in body mass, volume of the sonic motor nucleus, and motoneuron cell size.