Neural control of fluid homeostasis is engaged below 10°C in hibernation.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) hibernate for several months each winter without access to water,1 but the mechanisms that maintain fluid homeostasis during hibernation are poorly understood. In torpor, when body temperature (TB) reaches 4°C, squirrels decrease metabolism, slow heart rate, and reduce plasma levels of the antidiuretic hormones arginine vasopressin (AVP) and oxytocin (OXT).1 Squirrels spontaneously undergo interbout arousal (IBA) every 2 weeks, temporarily recovering an active-like metabolism and a TB of 37°C for up to 48 h.1,2 Despite the low levels of AVP and OXT during torpor, profound increases in blood pressure and heart rate during the torpor-IBA transition are not associated with massive fluid loss, suggesting the existence of a mechanism that protects against diuresis at a low TB. Here, we demonstrate that the antidiuretic hormone release pathway is activated by hypothalamic supraoptic nucleus (SON) neurons early in the torpor-arousal transition. SON neuron activity, dense-core vesicle release from the posterior pituitary, and plasma hormone levels all begin to increase before TB reaches 10°C. In vivo fiber photometry of SON neurons from hibernating squirrels, together with RNA sequencing and c-FOS immunohistochemistry, confirms that SON is electrically, transcriptionally, and translationally active to monitor blood osmolality throughout the dynamic torpor-arousal transition. Our work emphasizes the importance of the antidiuretic pathway during the torpor-arousal transition and reveals that the neurophysiological mechanism that coordinates the hormonal response to retain fluid is active at an extremely low TB, which is prohibitive for these processes in non-hibernators.

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.

Osmolyte Depletion and Thirst Suppression Allow Hibernators to Survive for Months without Water.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) are obligatory hibernators who can survive over 6 months of the year in underground burrows or laboratory hibernaculum without access to food or water [1]. Hibernation consists of prolonged periods of torpor, lasting up to 18 days, which are characterized by low body temperature and suppressed metabolism. This torpidity is interspersed with short periods of interbout arousal, lasting up to 48 h, during which squirrels temporarily return to an active-like state and lose small amounts of water to urination and evaporation [2]. Water is also lost during torpor due to a positive vapor pressure difference created by the slightly higher temperature of the body compared to its surroundings [2, 3]. Here, we investigate the physiological mechanism of survival during prolonged water loss and deprivation throughout hibernation. By measuring hydration status during hibernation, we show that squirrels remain hydrated during torpor by depleting osmolytes from the extracellular fluid. During brief periods of arousal, serum osmolality and antidiuretic hormone levels are restored, but thirst remains suppressed. This decoupling of thirst and diuresis enables water retention by the kidney while suppressing the drive to leave the safety of the underground burrow in search of water. An acute increase in serum osmolality reinstates water-seeking behavior, demonstrating preservation of the physiological thirst circuit during hibernation. Better mechanistic understanding of internal osmolyte regulation and thirst suppression could translate to advancements in human medicine and long-term manned spaceflight. VIDEO ABSTRACT.

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.

Behavioural tactic predicts preoptic-hypothalamic gene expression more strongly than developmental morph in fish with alternative reproductive tactics.

Reproductive success relies on the coordination of social behaviours, such as territory defence, courtship and mating. Species with extreme variation in reproductive tactics are useful models for identifying the neural mechanisms underlying social behaviour plasticity. The plainfin midshipman (Porichthys notatus) is a teleost fish with two male reproductive morphs that follow widely divergent developmental trajectories and display alternative reproductive tactics (ARTs). Type I males defend territories, court females and provide paternal care, but will resort to cuckoldry if they cannot maintain a territory. Type II males reproduce only through cuckoldry. We sought to disentangle gene expression patterns underlying behavioural tactic, in this case ARTs, from those solely reflective of developmental morph. Using RNA-sequencing, we investigated differential transcript expression in the preoptic area-anterior hypothalamus (POA-AH) of courting type I males, cuckolding type I males and cuckolding type II males. Unexpectedly, POA-AH differential expression was more strongly coupled to behavioural tactic than morph. This included a suite of transcripts implicated in hormonal regulation of vertebrate social behaviour. Our results reveal that divergent expression patterns in a conserved neuroendocrine centre known to regulate social-reproductive behaviours across vertebrate lineages may be uncoupled from developmental history to enable plasticity in the performance of reproductive tactics.

"Singing" Fish Rely on Circadian Rhythm and Melatonin for the Timing of Nocturnal Courtship Vocalization.

The patterning of social acoustic signaling at multiple timescales, from day-night rhythms to acoustic temporal properties, enhances sender-receiver coupling and reproductive success [1-8]. In diurnal birds, the nocturnal production of melatonin, considered the major vertebrate timekeeping hormone [9, 10], suppresses vocal activity but increases song syllable duration over circadian and millisecond timescales, respectively [11, 12]. Comparable studies are lacking for nocturnal vertebrates, including many teleost fish species that are also highly vocal during periods of reproduction [4, 13-20]. Utilizing continuous sound recordings, light cycle manipulations, hormone implants, and in situ hybridization, we demonstrate in a nocturnally breeding teleost fish that (1) courtship vocalization exhibits an endogenous circadian rhythm under constant dark conditions that is suppressed under constant light, (2) exogenous delivery of a melatonin analog under inhibitory constant light conditions rescues courtship vocal activity as well as the duration of single calls, and (3) melatonin receptor 1b is highly expressed in evolutionarily conserved neuroendocrine and vocal-acoustic networks crucial for patterning reproductive and vocal behaviors in fishes and tetrapods. Our findings, together with those in birds, show melatonin's remarkable versatility as a timing signal in distantly related lineages. It exerts opposing effects on vocalization in nocturnal versus diurnal species at the circadian timescale but comparable effects at the finer timescale of acoustic features. We propose that melatonin's separable effects at different timescales depends on its actions within distinct neural networks that control circadian rhythms, reproduction, and vocalization, which may be selected upon over evolutionary time as dissociable modules to pattern and coordinate social behaviors. VIDEO ABSTRACT.

Gene expression underlying enhanced, steroid-dependent auditory sensitivity of hair cell epithelium in a vocal fish.

BACKGROUND: Successful animal communication depends on a receiver's ability to detect a sender's signal. Exemplars of adaptive sender-receiver coupling include acoustic communication, often important in the context of seasonal reproduction. During the reproductive summer season, both male and female midshipman fish (Porichthys notatus) exhibit similar increases in the steroid-dependent frequency sensitivity of the saccule, the main auditory division of the inner ear. This form of auditory plasticity enhances detection of the higher frequency components of the multi-harmonic, long-duration advertisement calls produced repetitively by males during summer nights of peak vocal and spawning activity. The molecular basis of this seasonal auditory plasticity has not been fully resolved. Here, we utilize an unbiased transcriptomic RNA sequencing approach to identify differentially expressed transcripts within the saccule's hair cell epithelium of reproductive summer and non-reproductive winter fish. RESULTS: We assembled 74,027 unique transcripts from our saccular epithelial sequence reads. Of these, 6.4 % and 3.0 % were upregulated in the reproductive and non-reproductive saccular epithelium, respectively. Gene ontology (GO) term enrichment analyses of the differentially expressed transcripts showed that the reproductive saccular epithelium was transcriptionally, translationally, and metabolically more active than the non-reproductive epithelium. Furthermore, the expression of a specific suite of candidate genes, including ion channels and components of steroid-signaling pathways, was upregulated in the reproductive compared to the non-reproductive saccular epithelium. We found reported auditory functions for 14 candidate genes upregulated in the reproductive midshipman saccular epithelium, 8 of which are enriched in mouse hair cells, validating their hair cell-specific functions across vertebrates. CONCLUSIONS: We identified a suite of differentially expressed genes belonging to neurotransmission and steroid-signaling pathways, consistent with previous work showing the importance of these characters in regulating hair cell auditory sensitivity in midshipman fish and, more broadly, vertebrates. The results were also consistent with auditory hair cells being generally more physiologically active when animals are in a reproductive state, a time of enhanced sensory-motor coupling between the auditory periphery and the upper harmonics of vocalizations. Together with several new candidate genes, our results identify discrete patterns of gene expression linked to frequency- and steroid-dependent plasticity of hair cell auditory sensitivity.

Neural transcriptome reveals molecular mechanisms for temporal control of vocalization across multiple timescales.

BACKGROUND: Vocalization is a prominent social behavior among vertebrates, including in the midshipman fish, an established model for elucidating the neural basis of acoustic communication. Courtship vocalizations produced by territorial males are essential for reproductive success, vary over daily and seasonal cycles, and last up to hours per call. Vocalizations rely upon extreme synchrony and millisecond precision in the firing of a homogeneous population of motoneurons, the vocal motor nucleus (VMN). Although studies have identified neural mechanisms driving rapid, precise, and stable neuronal firing over long periods of calling, little is known about underlying genetic/molecular mechanisms. RESULTS: We used RNA sequencing-based transcriptome analyses to compare patterns of gene expression in VMN to the surrounding hindbrain across three daily and seasonal time points of high and low sound production to identify candidate genes that underlie VMN's intrinsic and network neuronal properties. Results from gene ontology enrichment, enzyme pathway mapping, and gene category-wide expression levels highlighted the importance of cellular respiration in VMN function, consistent with the high energetic demands of sustained vocal behavior. Functionally important candidate genes upregulated in the VMN, including at time points corresponding to high natural vocal activity, encode ion channels and neurotransmitter receptors, hormone receptors and biosynthetic enzymes, neuromodulators, aerobic respiration enzymes, and antioxidants. Quantitative PCR and RNA-seq expression levels for 28 genes were significantly correlated. Many candidate gene products regulate mechanisms of neuronal excitability, including those previously identified in VMN motoneurons, as well as novel ones that remain to be investigated. Supporting evidence from previous studies in midshipman strongly validate the value of transcriptomic analyses for linking genes to neural characters that drive behavior. CONCLUSIONS: Transcriptome analyses highlighted a suite of molecular mechanisms that regulate vocalization over behaviorally relevant timescales, spanning milliseconds to hours and seasons. To our knowledge, this is the first comprehensive characterization of gene expression in a dedicated vocal motor nucleus. Candidate genes identified here may belong to a conserved genetic toolkit for vocal motoneurons facing similar energetic and neurophysiological demands.

Melatonin action in a midbrain vocal-acoustic network.

Melatonin is a well-documented time-keeping hormone that can entrain an individual's physiology and behavior to the day-night cycle, though surprisingly little is known about its influence on the neural basis of social behavior, including vocalization. Male midshipman fish (Porichthys notatus) produce several call types distinguishable by duration and by daily and seasonal cycles in their production. We investigated melatonin's influence on the known nocturnal- and breeding season-dependent increase in excitability of the midshipman's vocal network (VN) that directly patterns natural calls. VN output is readily recorded from the vocal nerve as a 'fictive call'. Five days of constant light significantly increased stimulus threshold levels for calls electrically evoked from vocally active sites in the medial midbrain, supporting previous findings that light suppresses VN excitability, while 2-iodomelatonin (2-IMel; a melatonin analog) implantation decreased threshold. 2-IMel also increased fictive call duration evoked from medial sites as well as lateral midbrain sites that produced several-fold longer calls irrespective of photoregime or drug treatment. When stimulus intensity was incrementally increased, 2-IMel increased duration only at lateral sites, suggesting that melatonin action is stronger in the lateral midbrain. For animals receiving 5 days of constant darkness, known to increase VN excitability, systemic injections of either of two mammalian melatonin receptor antagonists increased threshold and decreased duration for calls evoked from medial sites. Our results demonstrate melatonin modulation of VN excitability and suggest that social context-dependent call types differing in duration may be determined by neuro-hormonal action within specific regions of a midbrain vocal-acoustic network.

Spinal motor and sensory neurons are androgen targets in an acrobatic bird.

Sex steroids affect the motivation to court mates, but less is known about how they influence motor movements associated with courtship behavior. Steroidal control of motor function may be especially important for species in which courtship requires superior strength, stamina, and neuromuscular coordination. Here we use the golden-collared manakin (Manacus vitellinus) to examine whether the neuromuscular circuitry that controls motoric aspects of courtship activity is sensitive to androgens. Males of this tropical species attract mates by rapidly jumping among branches in a courtship arena and using their wings to produce loud wing snaps. Testosterone activates this display via the androgen receptor (AR), and past work reveals that manakins injected with radio-labeled T ((3)H-T) accumulate radioactivity in the spinal cord. Thus, we used quantitative PCR to measure AR, estrogen receptor-α (ER-α) subtype, and aromatase (AROM) mRNA in spinal cords of male and female manakins and zebra finches. Expression of AR, but not ER-α or aromatase, was higher throughout the manakin spinal cord compared with the zebra finch. Next, we tested whether AR-expressing skeletal muscles are innervated by motor and sensory neurons that also express AR. To do this, we backfilled spinal neurons by injecting fluorescent tracers into select AR-sensitive wing and leg muscles of wild caught male and female manakins. We then removed these spinal cords and measured AR expression with in situ hybridization. Both sexes showed abundant AR mRNA in the cervical and lumbosacral spinal enlargements as well as in dorsal root ganglia attached to these enlargements. Together our findings suggest that androgens act widely on peripheral motor and sensory circuits in golden-collared manakins to influence wing snapping displays.

Limb muscles are androgen targets in an acrobatic tropical bird.

Spectacular athleticism is a conspicuous feature of many animal courtship displays yet surprisingly little is known about androgen dependence of skeletal muscles underlying these displays. Testosterone (T) acts through androgen receptors (ARs) to stimulate muscular male Golden-collared manakins of Panama to perform a remarkably athletic courtship display that includes loud wingsnaps generated by the rapid and forceful lifting of the wings. We tested the hypothesis that androgen sensitivity, reflected in the expression levels of AR mRNA, is a muscular adaptation supporting these courtship displays. Quantitative PCR showed substantially greater AR mRNA expression in all limb muscles of wild male and female manakins compared with two other avian species that do not perform athletic displays, zebra finches and ochre-bellied flycatchers. AR expression levels in the massive skeletal muscles were comparable with the minute oscine syringeal muscle but greater than levels in nonmuscular androgen targets that did not differ across species. Compared with zebra finches, male manakins also had greater activity of the T-activating enzyme 5 alpha-reductase in a wing-lifting muscle. In addition, low levels of estrogen receptor alpha (ER) mRNA were detected in all muscles of control, T-treated, and estradiol-treated manakins. Treatment of manakins with T, but not estradiol, significantly increased skeletal muscle ER expression, suggesting that ER expression is AR-dependent. These results confirm manakin limb muscles as important androgen targets where T may act to promote the speed, force, and/or endurance required for the manakin display. Androgen-sensitive muscular phenotypes may adapt males of many species to perform impressive athletic displays.