Label-free, whole-brain in vivo mapping in an adult vertebrate with third harmonic generation microscopy.

Comprehensive understanding of interconnected networks within the brain requires access to high resolution information within large field of views and over time. Currently, methods that enable mapping structural changes of the entire brain in vivo are extremely limited. Third harmonic generation (THG) can resolve myelinated structures, blood vessels, and cell bodies throughout the brain without the need for any exogenous labeling. Together with deep penetration of long wavelengths, this enables in vivo brain-mapping of large fractions of the brain in small animals and over time. Here, we demonstrate that THG microscopy allows non-invasive label-free mapping of the entire brain of an adult vertebrate, Danionella dracula, which is a miniature species of cyprinid fish. We show this capability in multiple brain regions and in particular the identification of major commissural fiber bundles in the midbrain and the hindbrain. These features provide readily discernable landmarks for navigation and identification of regional-specific neuronal groups and even single neurons during in vivo experiments. We further show how this label-free technique can easily be coupled with fluorescence microscopy and used as a comparative tool for studies of other species with similar body features to Danionella, such as zebrafish (Danio rerio) and tetras (Trochilocharax ornatus). This new evidence, building on previous studies, demonstrates how small size and relative transparency, combined with the unique capabilities of THG microscopy, can enable label-free access to the entire adult vertebrate brain.

A tale of two males: Behavioral and neural mechanisms of alternative reproductive tactics in midshipman fish.

An amalgam of investigations at the interface of neuroethology and behavioral neuroendocrinology first established the most basic behavioral, neuroanatomical, and neurophysiological characters of vocal-acoustic communication morphs in the plainfin midshipman fish, Porichthys notatus Girard. This foundation has led, in turn, to the repeated demonstration that neuro-behavioral mechanisms driving reproductive-related, vocal-acoustic behaviors can be uncoupled from gonadal state for two adult male phenotypes that follow alternative reproductive tactics (ARTs).

Midbrain node for context-specific vocalisation in fish.

Vocalizations communicate information indicative of behavioural state across divergent social contexts. Yet, how brain regions actively pattern the acoustic features of context-specific vocal signals remains largely unexplored. The midbrain periaqueductal gray (PAG) is a major site for initiating vocalization among mammals, including primates. We show that PAG neurons in a highly vocal fish species (Porichthys notatus) are activated in distinct patterns during agonistic versus courtship calling by males, with few co-activated during a non-vocal behaviour, foraging. Pharmacological manipulations within vocally active PAG, but not hindbrain, sites evoke vocal network output to sonic muscles matching the temporal features of courtship and agonistic calls, showing that a balance of inhibitory and excitatory dynamics is likely necessary for patterning different call types. Collectively, these findings support the hypothesis that vocal species of fish and mammals share functionally comparable PAG nodes that in some species can influence the acoustic structure of social context-specific vocal signals.

Sound production biomechanics in three-spined toadfish and potential functional consequences of swim bladder morphology in the Batrachoididaea).

The relationship between sound complexity and the underlying morphology and physiology of the vocal organ anatomy is a fundamental component in the evolution of acoustic communication, particularly for fishes. Among vertebrates, the mammalian larynx and avian syrinx are the best-studied vocal organs, and their ability to produce complex vocalizations has been modeled. The range and complexity of the sounds in mammalian lineages have been attributed, in part, to the bilateral nature of the vocal anatomy. Similarly, we hypothesize that the bipartite swim bladder of some species of toadfish (family Batrachoididae) is responsible for complex nonlinear characters of the multiple call types that they can produce, supported by nerve transection experiments. Here, we develop a low-dimensional coupled-oscillator model of the mechanics underlying sound production by the two halves of the swim bladder of the three-spined toadfish, Batrachomoeus trispinosus. Our model was able to replicate the nonlinear structure of both courtship and agonistic sounds. The results provide essential support for the hypothesis that fishes and tetrapods have converged in an evolutionary innovation for complex acoustic signaling, namely, a relatively simple bipartite mechanism dependent on sonic muscles contracting around a gas filled structure.

California singing fish.

Bass describes the fascinating life history, behavior, and neurobiology of the California singing fish, including its remarkable vocal abilities.

Whole-brain optical access in a small adult vertebrate with two- and three-photon microscopy.

Although optical microscopy has allowed scientists to study the entire brain in early developmental stages, access to the brains of live, adult vertebrates has been limited. Danionella, a genus of miniature, transparent fish closely related to zebrafish has been introduced as a neuroscience model to study the adult vertebrate brain. However, the extent of optically accessible depth in these animals has not been quantitatively characterized. Here, we show that both two- and three-photon microscopy can access the entire depth and rostral-caudal extent of the adult wildtype Danionella dracula brain without any modifications to the animal other than mechanical stabilization. Three-photon microscopy provides higher signal-to-background ratio and optical sectioning of fluorescently labeled vasculature through the deepest part of the brain, the hypothalamus. Hence, we use multiphoton microscopy to penetrate the entire adult brain within the geometry of this genus' head structures and without the need for pigment removal.

Acoustic and postural displays in a miniature and transparent teleost fish, Danionella dracula.

Acoustic behavior is widespread across vertebrates, including fishes. We report robust acoustic displays during aggressive interactions for a laboratory colony of Danionella dracula, a miniature and transparent species of teleost fish closely related to zebrafish (Danio rerio), which are hypothesized to be sonic based on the presence of a hypertrophied muscle associated with the male swim bladder. Males produce bursts of pulsatile sounds and a distinct postural display - extension of a hypertrophied lower jaw, a morphological trait not present in other Danionella species - during aggressive but not courtship interactions. Females show no evidence of sound production or jaw extension in such contexts. Novel pairs of size-matched or -mismatched males were combined in resident-intruder assays where sound production and jaw extension could be linked to individuals. In both dyad contexts, resident males produced significantly more sound pulses than intruders. During heightened sonic activity, the majority of the highest sound producers also showed increased jaw extension. Residents extended their jaw more than intruders in size-matched but not -mismatched contexts. Larger males in size-mismatched dyads produced more sounds and jaw extensions compared with their smaller counterparts, and sounds and jaw extensions increased with increasing absolute body size. These studies establish D. dracula as a sonic species that modulates putatively acoustic and postural displays during aggressive interactions based on residency and body size, providing a foundation for further investigating the role of multimodal displays in a new model clade for neurogenomic and neuroimaging studies of aggression, courtship and other social interactions.

Noise-induced hearing loss correlates with inner ear hair cell decrease in larval zebrafish.

Anthropogenic noise can be hazardous for the auditory system and wellbeing of animals, including humans. However, very limited information is known on how this global environmental pollutant affects auditory function and inner ear sensory receptors in early ontogeny. The zebrafish (Danio rerio) is a valuable model in hearing research, including investigations of developmental processes of the vertebrate inner ear. We tested the effects of chronic exposure to white noise in larval zebrafish on inner ear saccular sensitivity and morphology at 3 and 5 days post-fertilization (dpf), as well as on auditory-evoked swimming responses using the prepulse inhibition (PPI) paradigm at 5 dpf. Noise-exposed larvae showed a significant increase in microphonic potential thresholds at low frequencies, 100 and 200 Hz, while the PPI revealed a hypersensitization effect and a similar threshold shift at 200 Hz. Auditory sensitivity changes were accompanied by a decrease in saccular hair cell number and epithelium area. In aggregate, the results reveal noise-induced effects on inner ear structure-function in a larval fish paralleled by a decrease in auditory-evoked sensorimotor responses. More broadly, this study highlights the importance of investigating the impact of environmental noise on early development of sensory and behavioural responsiveness to acoustic stimuli.

Oxytocin-like receptor expression in evolutionarily conserved nodes of a vocal network associated with male courtship in a teleost fish.

Neuropeptides, including oxytocin-like peptides, are a conserved group of hormones that regulate a wide range of social behaviors, including vocal communication. In the current study, we evaluate whether putative brain sites for the actions of isotocin (IT), the oxytocin (OT) homolog of teleost fishes are associated with vocal courtship and circuitry in the plainfin midshipman fish (Porichthys notatus). During the breeding season, nesting males produce advertisement calls known as "hums" to acoustically court females at night and attract them to nests. We first identify IT receptor (ITR) mRNA in evolutionarily conserved regions of the forebrain preoptic area (POA), anterior hypothalamus (AH), and midbrain periaqueductal gray (PAG), and in two topographically separate populations within the hindbrain vocal pattern generator- duration-coding vocal prepacemaker (VPP) and amplitude-coding vocal motor nuclei (VMN) that also innervate vocal muscles. We also verify that ITR expression overlaps known distribution sites of OT-like immunoreactive fibers. Next, using phosphorylated ribosomal subunit 6 (pS6) as a marker for activated neurons, we demonstrate that ITR-containing neurons in the anterior parvocellular POA, AH, PAG, VPP, and VMN are activated in humming males. Posterior parvocellular and magno/gigantocellular divisions of the POA remain constitutively active in nonhumming males that are also in a reproductive state. Together with prior studies of midshipman fish and other vertebrates, our findings suggest that IT-signaling influences male courtship behavior, in part, by acting on brain regions that broadly influence behavioral state (POA) as well as the initiation (POA and PAG) and temporal structure (VPP and VMN) of advertisement hums.

Vocal and Electric Fish: Revisiting a Comparison of Two Teleost Models in the Neuroethology of Social Behavior.

The communication behaviors of vocal fish and electric fish are among the vertebrate social behaviors best understood at the level of neural circuits. Both forms of signaling rely on midbrain inputs to hindbrain pattern generators that activate peripheral effectors (sonic muscles and electrocytes) to produce pulsatile signals that are modulated by frequency/repetition rate, amplitude and call duration. To generate signals that vary by sex, male phenotype, and social context, these circuits are responsive to a wide range of hormones and neuromodulators acting on different timescales at multiple loci. Bass and Zakon (2005) reviewed the behavioral neuroendocrinology of these two teleost groups, comparing how the regulation of their communication systems have both converged and diverged during their parallel evolution. Here, we revisit this comparison and review the complementary developments over the past 16 years. We (a) summarize recent work that expands our knowledge of the neural circuits underlying these two communication systems, (b) review parallel studies on the action of neuromodulators (e.g., serotonin, AVT, melatonin), brain steroidogenesis (via aromatase), and social stimuli on the output of these circuits, (c) highlight recent transcriptomic studies that illustrate how contemporary molecular methods have elucidated the genetic regulation of social behavior in these fish, and (d) describe recent studies of mochokid catfish, which use both vocal and electric communication, and that use both vocal and electric communication and consider how these two systems are spliced together in the same species. Finally, we offer avenues for future research to further probe how similarities and differences between these two communication systems emerge over ontogeny and evolution.

To hum or not to hum: Neural transcriptome signature of male courtship vocalization in a teleost fish.

For many animal species, vocal communication is a critical social behavior and often a necessary component of reproductive success. Additionally, vocalizations are often demanding motor acts. Wanting to know whether a specific molecular toolkit might be required for vocalization, we used RNA-sequencing to investigate neural gene expression underlying the performance of an extreme vocal behavior, the courtship hum of the plainfin midshipman fish (Porichthys notatus). Single hums can last up to 2 h and may be repeated throughout an evening of courtship activity. We asked whether vocal behavioral states are associated with specific gene expression signatures in key brain regions that regulate vocalization by comparing transcript expression levels in humming versus non-humming males. We find that the circadian-related genes period3 and Clock are significantly upregulated in the vocal motor nucleus and preoptic area-anterior hypothalamus, respectively, in humming compared with non-humming males, indicating that internal circadian clocks may differ between these divergent behavioral states. In addition, we identify suites of differentially expressed genes related to synaptic transmission, ion channels and transport, neuropeptide and hormone signaling, and metabolism and antioxidant activity that together may support the neural and energetic demands of humming behavior. Comparisons of transcript expression across regions stress regional differences in brain gene expression, while also showing coordinated gene regulation in the vocal motor circuit in preparation for courtship behavior. These results underscore the role of differential gene expression in shifts between behavioral states, in this case neuroendocrine, motor and circadian control of courtship vocalization.

Gap junction-mediated glycinergic inhibition ensures precise temporal patterning in vocal behavior.

Precise neuronal firing is especially important for behaviors highly dependent on the correct sequencing and timing of muscle activity patterns, such as acoustic signaling. Acoustic signaling is an important communication modality for vertebrates, including many teleost fishes. Toadfishes are well known to exhibit high temporal fidelity in synchronous motoneuron firing within a hindbrain network directly determining the temporal structure of natural calls. Here, we investigated how these motoneurons maintain synchronous activation. We show that pronounced temporal precision in population-level motoneuronal firing depends on gap junction-mediated, glycinergic inhibition that generates a period of reduced probability of motoneuron activation. Super-resolution microscopy confirms glycinergic release sites formed by a subset of adjacent premotoneurons contacting motoneuron somata and dendrites. In aggregate, the evidence supports the hypothesis that gap junction-mediated, glycinergic inhibition provides a timing mechanism for achieving synchrony and temporal precision in the millisecond range for rapid modulation of acoustic waveforms.

Morphological diversity of acoustic and electric communication systems of mochokid catfish.

Mochokid catfish offer a distinct opportunity to study a communication system transitioning to a new signaling channel because some produce sounds and others electric discharges. Both signals are generated using an elastic spring system (ESS), which includes a protractor muscle innervated by motoneurons within the protractor nucleus that also has a motoneuron afferent population. Synodontis grandiops and S. nigriventris produce sounds and electric discharges, respectively, and their ESSs show several morphological and physiological differences. The extent to which these differences explain different signal types remains unclear. Here, we compare ESS morphologies and behavioral phenotypes among five mochokids. S. grandiops and S. nigriventris were compared with Synodontis eupterus that is known to produce both signal types, and representative members of two sister genera, Microsynodontis cf. batesii and Mochokiella paynei, for which no data were available. We provide support for the hypothesis that peripheral and central components of the ESS are conserved among mochokids. We also show that the two nonsynodontids are only sonic, consistent with sound production being an ancestral character for mochokids. Even though the three sound producing-only species differ in some ESS characters, several are similar and likely associated with only sound production. We propose that the ability of S. eupterus to generate both electric discharges and sounds may depend on a protractor muscle intermediate in morphology between sound producing-only and electric discharge-only species, and two separate populations of protractor motoneurons. Our results further suggest that an electrogenic ESS in synodontids is an exaptation of a sound producing ESS.

Neuroanatomical and neurophysiological mechanisms of acoustic and weakly electric signaling in synodontid catfish.

To what extent do modifications in the nervous system and peripheral effectors contribute to novel behaviors? Using a combination of morphometric analysis, neuroanatomical tract-tracing, and intracellular neuronal recording, we address this question in a sound-producing and a weakly electric species of synodontid catfish, Synodontis grandiops, and Synodontis nigriventris, respectively. The same peripheral mechanism, a bilateral pair of protractor muscles associated with vertebral processes (elastic spring mechanism), is involved in both signaling systems. Although there were dramatic species differences in several morphometric measures, electromyograms provided strong evidence that simultaneous activation of paired protractor muscles accounts for an individual sound and electric discharge pulse. While the general architecture of the neural network and the intrinsic properties of the motoneuron population driving each target was largely similar, differences could contribute to species-specific patterns in electromyograms and the associated pulse repetition rate of sounds and electric discharges. Together, the results suggest that adaptive changes in both peripheral and central characters underlie the transition from an ancestral sound to a derived electric discharge producing system, and thus the evolution of a novel communication channel among synodontid catfish. Similarities with characters in other sonic and weakly electric teleost fish provide a striking example of convergent evolution in functional adaptations underlying the evolution of the two signaling systems among distantly related taxa.

Mating Behavioral Function of Preoptic Galanin Neurons Is Shared between Fish with Alternative Male Reproductive Tactics and Tetrapods.

Understanding the contribution of neuropeptide-containing neurons to variation in social behavior remains critically important. Galanin has gained increased attention because of the demonstration that galanin neurons in the preoptic area (POA) promote mating and parental care in mammals. How widespread these mechanisms are among vertebrates essentially remains unexplored, especially among teleost fishes, which comprise nearly one-half of living vertebrate species. Teleosts with alternative reproductive tactics exhibit stereotyped patterns of social behavior that diverge widely between individuals within a sex. This includes midshipman that have two male morphs. Type I males mate using either acoustic courtship to attract females to enter a nest they guard or cuckoldry during which they steal fertilizations from a nest-holding male using a sneak or satellite spawning tactic, whereas type II males only cuckold. Using the neural activity marker phospho-S6, we show increased galanin neuron activation in courting type I males during mating that is not explained by their courtship vocalizations, parental care of eggs, or nest defense against cuckolders. This increase is not observed during mating in cuckolders of either morph or females (none of which show parental care). Together with their role in mating in male mammals, the results demonstrate an unexpectedly specific and deep-rooted, phylogenetically shared behavioral function for POA galanin neurons. The results also point to galanin-dependent circuitry as a potential substrate for the evolution of divergent phenotypes within one sex and provide new functional insights into how POA populations in teleosts compare to the POA and anterior hypothalamus of tetrapods.SIGNIFICANCE STATEMENT Studies of neuropeptide regulation of vertebrate social behavior have mainly focused on the vasopressin-oxytocin family. Recently, galanin has received attention as a regulator of social behavior largely because of studies demonstrating that galanin neurons in the preoptic area (POA) promote mating and parental care in mammals. Species with alternative reproductive tactics (ARTs) exhibit robust, consistent differences in behavioral phenotypes between individuals within a sex. Taking advantage of this trait, we show POA galanin neurons are specifically active during mating in one of two male reproductive tactics, but not other mating-related behaviors in a fish with ARTs. The results demonstrate a deep, phylogenetically shared role for POA galanin neurons in reproductive-related social behaviors with implications for the evolution of ARTs.

Galanin immunoreactivity is sexually polymorphic in neuroendocrine and vocal-acoustic systems in a teleost fish.

Galanin is a peptide that regulates pituitary hormone release, feeding, and reproductive and parental care behaviors. In teleost fish, increased galanin expression is associated with territorial, reproductively active males. Prior transcriptome studies of the plainfin midshipman (Porichthys notatus), a highly vocal teleost fish with two male morphs that follow alternative reproductive tactics, show that galanin is upregulated in the preoptic area-anterior hypothalamus (POA-AH) of nest-holding, courting type I males during spawning compared to cuckolding type II males. Here, we investigate possible differences in galanin immunoreactivity in the brain of both male morphs and females with a focus on vocal-acoustic and neuroendocrine networks. We find that females differ dramatically from both male morphs in the number of galanin-expressing somata and in the distribution of fibers, especially in brainstem vocal-acoustic nuclei and other sensory integration sites that also differ, though less extensively, between the male morphs. Double labeling shows that primarily separate populations of POA-AH neurons express galanin and the nonapeptides arginine-vasotocin or isotocin, homologues of mammalian arginine vasopressin and oxytocin that are broadly implicated in neural mechanisms of vertebrate social behavior including morph-specific actions on vocal neurophysiology in midshipman. Finally, we report a small population of POA-AH neurons that coexpress galanin and the neurotransmitter γ-aminobutyric acid. Together, the results indicate that galanin neurons in midshipman fish likely modulate brain activity at a broad scale, including targeted effects on vocal motor, sensory and neuroendocrine systems; are unique from nonapeptide-expressing populations; and play a role in male-specific behaviors.

Co-opting evo-devo concepts for new insights into mechanisms of behavioural diversity.

We propose that insights from the field of evolutionary developmental biology (or 'evo-devo') provide a framework for an integrated understanding of the origins of behavioural diversity and its underlying mechanisms. Towards that goal, in this Commentary, we frame key questions in behavioural evolution in terms of molecular, cellular and network-level properties with a focus on the nervous system. In this way, we highlight how mechanistic properties central to evo-devo analyses - such as weak linkage, versatility, exploratory mechanisms, criticality, degeneracy, redundancy and modularity - affect neural circuit function and hence the range of behavioural variation that can be filtered by selection. We outline why comparative studies of molecular and neural systems throughout ontogeny will provide novel insights into diversity in neural circuits and behaviour.

Melatonin receptor expression in vocal, auditory, and neuroendocrine centers of a highly vocal fish, the plainfin midshipman (Porichthys notatus).

Melatonin plays a central role in entraining activity to the day-night cycle in vertebrates. Here, we investigate neuroanatomical substrates of melatonin-dependent vocal-acoustic behavior in the nocturnal and highly vocal teleost fish, the plainfin midshipman (Porichthys notatus). Using in situ hybridization (ISH) and quantitative real-time PCR (qPCR), we assess the mRNA distribution and transcript abundance of melatonin receptor subtype 1B (mel1b), shown to be important for vocalization in midshipman fish and songbirds. ISH shows robust mel1b expression in major nodes of the central vocal and auditory networks in the subpallium, preoptic area (POA), anterior hypothalamus, dorsal thalamus, posterior tuberculum, midbrain torus semicircularis and periaqueductal gray, and hindbrain. Mel1b label is also abundant in secondary targets of the olfactory, visual, and lateral line systems, as well as telencephalic regions that have been compared to the amygdala, extended amygdala, striatum, septum, and hippocampus of tetrapods. Q-PCR corroborates mel1b abundance throughout the brain and shows significant increases in the morning compared with nighttime in tissue samples inclusive of the telencephalon and POA, but remains stable in other brain regions. Plasma melatonin levels show expected increase at night. Our findings support the hypothesis that melatonin's stimulatory effects on vocal-acoustic mechanisms in midshipman is mediated, in part, by melatonin binding in vocal, auditory, and neuroendocrine centers. Together with robust mel1b expression in multiple telencephalic nuclei and sensory systems, the results further indicate an expression pattern comparable to that in birds and mammals that is indicative of melatonin's broad involvement in the modulation of physiology and behavior.

FoxP2 Expression in a Highly Vocal Teleost Fish with Comparisons to Tetrapods.

Motivated by studies of speech deficits in humans, several studies over the past two decades have investigated the potential role of a forkhead domain transcription factor, FoxP2, in the central control of acoustic signaling/vocalization among vertebrates. Comparative neuroanatomical studies that mainly include mammalian and avian species have mapped the distribution of FoxP2 expression in multiple brain regions that imply a greater functional significance beyond vocalization that might be shared broadly across vertebrate lineages. To date, reports for teleost fish have been limited in number and scope to nonvocal species. Here, we map the neuroanatomical distribution of FoxP2 mRNA expression in a highly vocal teleost, the plainfin midshipman (Porichthys notatus). We report an extensive overlap between FoxP2 expression and vocal, auditory, and steroid-signaling systems with robust expression at multiple sites in the telencephalon, the preoptic area, the diencephalon, and the midbrain. Label was far more restricted in the hindbrain though robust in one region of the reticular formation. A comparison with other teleosts and tetrapods suggests an evolutionarily conserved FoxP2 phenotype important to vocal-acoustic and, more broadly, sensorimotor function among vertebrates.

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.