Vocalizations by a sexually dimorphic isolated larynx: peripheral constraints on behavioral expression

The clawed frog Xenopus laevis uses sexually dimorphic vocalizations, mate calling and ticking, to advertise reproductive state. The basic unit of vocalization is a brief click, produced by the movement of cartilagenous disks located within the larynx. The rate of click production in the male-specific mate call (71 Hz) is an order of magnitude faster than the rate of click production in female typical ticking (6 Hz). To determine if vocalization rate is constrained by the periphery, male and female larynges were isolated and response of the muscles to nerve stimulation was studied. Laryngeal muscle response is markedly dimorphic in the 2 sexes, both in the amplitude potentiation of electromyograms and in the rate at which discrete tension transients can be produced. At 6 Hz (ticking), both sexes generate discrete tension transients in response to each stimulus pulse. In response to nerve stimulation at 71 Hz (mate calling), male laryngeal muscle generates discrete tension transients while female laryngeal muscle does not. Since expression of sex-specific vocalizations is regulated by androgenic hormones, responses of laryngeal muscle to nerve stimulation in androgen-treated adult females and castrated adult males were also examined. The responses of laryngeal muscle from castrated and intact males are similar. Androgen-treated female larynx is partially masculinized but does not produce tension transients at the mate call rate. These physiological results are in close agreement with behavioral observations. Sounds produced by the isolated larynx were nearly identical in spectral properties to those produced by an intact male. We determined that the production of a discrete tension transient is prerequisite to click production. Thus, one reason females do not mate call, even when treated with androgens, is that female laryngeal muscle cannot produce discrete tension transients at a rapid rate.     

Sexually differentiated central pattern generators in Xenopus laevis

Understanding the neural mechanisms that underlie the function of central pattern generators (CPGs) presents a formidable challenge requiring sophisticated tools and well-chosen model systems. In this article, we describe recent work on vocalizations of the African clawed frog Xenopus laevis. These behaviors are driven by sexually differentiated CPGs and are exceptionally well suited to this objective. In particular, a simplified mechanism of vocal production (independent of respiratory musculature) allows straightforward interpretations of nerve activity with respect to behavior. Furthermore, the development of a fictively vocalizing isolated brain, together with the finding of rapid androgen-induced masculinization of female vocalizations, provides an invaluable tool for determining how new behaviors arise from existing circuits.     

Breathing and calling: neuronal networks in the Xenopus laevis hindbrain

Xenopus laevis is an aquatic anuran with a complex vocal repertoire. Unlike terrestrial frogs, vocalizations are independent of respiration, and a single muscle group--the laryngeal dilators--produces underwater calls. We sought to identify the premotor neural network that underlies vocal behaviors. Vocal patterns generated by premotor networks control laryngeal motor neurons in cranial nucleus (n.) IX-X. Glottal motor neurons, active during respiration, are also present in n.IX-X. We used horseradish peroxidase (HRP), Lucifer yellow, and fluorescently conjugated dextrans to characterize the organization of n.IX-X and to trace premotor neuron projections. Premotor nuclei include the inferior reticular formation (Ri) adjacent to n.IX-X and the pretrigeminal nucleus of the dorsal tegmental area of the medulla (DTAM), the primary descending input to n.IX-X. Intramuscular HRP injections revealed a spatially segregated pattern, with glottal motor neurons in anterior n.IX-X and laryngeal motor neurons in the caudal portion of the nucleus. Dextran injections identified commissural n.IX-X neurons that project to the contralateral motor nucleus and DTAM-projecting n.IX-X neurons. Both neuronal types are clustered in anteromedial n.IX-X, closely associated with glottal motor neurons. Ri neurons project to ipsilateral and contralateral DTAM. Projections from DTAM target n.IX-X bilaterally, and all four identified subtypes receive DTAM input. In contrast, Ri neurons receive little input from DTAM. We hypothesize that connectivity between neurons in n.IX-X, Ri and DTAM may provide mechanisms to generate laryngeal and glottal activity patterns and that DTAM may coordinate vocal and respiratory motor pools, perhaps acting to switch between these two mutually exclusive behaviors.     

Homogeneity of intrinsic properties of sexually dimorphic vocal motoneurons in male and female zebra finches

Sex differences in behavioral repertoires are often reflected in the underlying electrophysiological and morphological properties of motor neurons. Male zebra finches produce long, spectrally complex, learned songs and short calls, whereas female finches only produce short, innate, and spectrally simple calls. In both sexes, vocalizations are produced by using syringeal muscles controlled by motoneurons within the tracheosyringeal part of the hypoglossal motor nucleus (XIIts). We asked whether the sexually dimorphic vocal repertoire of adult zebra finches is paralleled by structural and functional differences in syringeal motoneurons. By using immunohistochemical and intracellular staining methods, we describe sex differences in the morphology of XIIts and its surrounding neuropil (suprahypoglossal region; SH). Although the overall number of XIIts neurons and the proportions of somata/neuropil were not sexually dimorphic, the volumes of both XIIts and SH were larger in males, in part because male XIIts neurons had larger somata. In contrast, female XIIts motoneurons had a more complex dendritic structure than did male neurons, suggesting that the larger volume of the male XIIts is due in part to increased numbers of afferents. Intracellular recordings in brain slices revealed that the intrinsic electrophysiological properties of female XIIts neurons were similar to published values for male XIIts motoneurons. We also show that female neurons received glycinergic inputs from the brainstem respiratory premotor column, similar to those described in males. These findings indicate that male and female zebra finches produce their disparate vocal repertoires using physiologically similar motoneurons. Thus, sites upstream of the motoneuron pool may be the major determinants of sexually dimorphic vocal behaviors in this species.     

Differential responsiveness in brain and behavior to sexually dimorphic long calls in male and female zebra finches

In zebra finches (Taeniopygia guttata), as in most other songbird species, there are robust sex differences in brain morphology and vocal behavior. First, male zebra finches have larger song system nuclei—involved in sensorimotor learning and production of song—than females. Second, male zebra finches learn their song from a tutor, whereas female zebra finches develop a learned preference for the song of their father but do not sing themselves. Third, female zebra finches produce an unlearned “long call,” while males learn their long call (which is different from that of females) from their song tutor. We investigated behavioral and molecular neuronal responsiveness to this sexually dimorphic communication signal. Behavioral responsiveness was quantified by measuring the number of calls and approaches in response to calls that were broadcast from a speaker. We quantified neuronal activation by measuring the number of neurons expressing Zenk, the protein product of the immediate early gene ZENK, in a number of different forebrain regions in response to male calls, to female calls, or to silence. In both sexes female calls evoked more calls and approaches than male calls. There was significantly greater Zenk expression in response to female calls compared to silence in the caudomedial nidopallium, caudomedial mesopallium, and the hippocampus in females, but not in males. Thus, male and female zebra finches both show a behavioral preference for female calls, but differential neuronal activation in response to sexually dimorphic calls. J. Comp. Neurol. 516:312–320, 2009. © 2009 Wiley‐Liss, Inc.     

Call patterns and basilar papilla tuning in cricket frogs. II. Intrapopulation variation and allometry.

We determined the influence of body size on the male advertisement call's dominant frequency and basilar papilla's (BP) tuning in male and female cricket frogs (Acris crepitans) in two Texas populations (Wimberley and Stengel Ranch). In both populations, call and tuning characters correlated negatively with body size; females were larger than males and their BPs were tuned to a lower frequency. Analysis of covariance showed that neither the sex difference in tuning nor the population differences in calls or tuning were due to the difference in body size alone, but instead represented differences in the allometric relationships of each character with body size. The analysis implied that differences between sexes or populations were due more to shifts in the Y-intercept rather than the slope of the relationship with body size. This suggests a developmental model in which sexes or populations possess resonant structures in the ear or larynx with similar growth rates but different starting points or initial growth phases, resulting in different frequency characteristics as adults. The examination of the relationship between female BP tuning and male call dominant frequency predicts potentially different patterns of sexual selection in the two populations, with the Wimberley population males subject to much greater directional selection for low frequency calls.     

Coding Rate and Duration of Vocalizations of the Frog, Xenopus laevis

Vocalizations involve complex rhythmic motor patterns, but the underlying temporal coding mechanisms in the nervous system are poorly understood. Using a recently developed whole-brain preparation from which "fictive" vocalizations are readily elicited in vitro, we investigated the cellular basis of temporal complexity of African clawed frogs (Xenopus laevis). Male advertisement calls contain two alternating components-fast trills (～300 ms) and slow trills (～700 ms) that contain clicks repeated at ～60 and ～30 Hz, respectively. We found that males can alter the duration of fast trills without changing click rates. This finding led us to hypothesize that call rate and duration are regulated by independent mechanisms. We tested this by obtaining whole-cell patch-clamp recordings in the "fictively" calling isolated brain. We discovered a single type of premotor neuron with activity patterns correlated with both the rate and duration of fast trills. These "fast-trill neurons" (FTNs) exhibited long-lasting depolarizations (LLDs) correlated with each fast trill and action potentials that were phase-locked with motor output-neural correlates of call duration and rate, respectively. When depolarized without central pattern generator activation, FTNs produced subthreshold oscillations and action potentials at fast-trill rates, indicating FTN resonance properties are tuned to, and may dictate, the fast-trill rhythm. NMDA receptor (NMDAR) blockade eliminated LLDs in FTNs, and NMDAR activation in synaptically isolated FTNs induced repetitive LLDs. These results suggest FTNs contain an NMDAR-dependent mechanism that may regulate fast-trill duration. We conclude that a single premotor neuron population employs distinct mechanisms to regulate call rate and duration.     

The ontogeny of androgen receptors in the CNS of Xenopus laevis frogs

Androgenic steroids have been implicated in the development of sex differences in Xenopus laevis frogs. In order to determine when neurons first acquire the ability to concentrate androgen, we prepared autoradiograms of CNS in developing frogs following injection of tritiated dihydrotestosterone (DHT). X. laevis tadpoles and juveniles from stage 60 to 2 months post-metamorphosis (PM) were injected with [3H]DHT. Brain and spinal cord autoradiograms from these animals were examined for the presence of labelled cells. The pattern of [3H]DHT labelling in stage-64 tadpoles and in PM juveniles was similar but not identical to that seen in adults. Heavily labelled cells were seen in the motor nucleus of cranial nerves IX and X, medullary reticular formation, a presumed sensory nucleus of cranial nerve V, pretrigeminal nucleus of the dorsal tegmental area of the medulla, laminar nucleus of the torus semicircularis, anterior pituitary, ventral thalamus and anterior spinal cord. The vestibular sensory nucleus of cranial nerve VIII was the only area that concentrates DHT in adults but did not contain labelled cells in young animals. No [3H]DHT-labelled cells were found in stage-60 tadpoles. The onset of androgen concentrating capability in X. laevis CNS thus probably occurs between stages 60 and 64.     

Dimorphic male brains and alternative reproductive tactics in a vocalizing fish.

Brains and types of behavior among sexually mature vertebrates are portrayed as having two phenotypic states: male and female. However, mating systems and the behavioral tactics employed by the two sexes are far more diverse than conveyed by this simple intersexual dichotomy. For example, in many species, sexually mature males may practice one of two alternative mating tactics. Recent studies of a sound-producing teleost fish now show that intrasexual dimorphism of vocal motor phenotypes among males accompanies the intrasexual divergence of types of reproductive behavior. Thus, one group of males, like females and juveniles, lacks the behavioral, neurophysiological and morphological traits typical of the sexually differentiated vocal motor pathway of the second group. The results are an explicit demonstration that for any one species: (1) alternative mating tactics can be paralleled by alternative phenotypes for the neurons that determine tactic-specific types of behavior, and (2) reproductive maturation is not obligatorily linked to the expression of neuronal secondary sex characteristics.     

Vocal circuitry in Xenopus laevis: telencephalon to laryngeal motor neurons

Sexually differentiated calling patterns of Xenopus laevis are conveyed to the vocal organ by a dedicated neuromuscular system. Here, we define afferents to vocal motor neurons and determine whether the connectivity of the vocal pathway is sexually differentiated. The use of fluorescent dextran amines and the isolated brain preparation readily permitted identification of anterograde and retrograde connectivity patterns. The whole-mount preparation allowed us to observe projections in their entirety, including cells of origin of a projection (for retrograde projections), terminal fields (for anterograde connections), and fiber tracts. Major findings are the confirmation of a robust and reciprocal connection between cranial nucleus (n.) IX-X and the pretrigeminal nucleus of the dorsal tegmental area of the medulla (DTAM) as well as between DTAM and the ventral striatum (VS). Newly revealed is the extensive connectivity between the rostral subdivision of the dorsal nucleus raphe (rRpd) and candidate vocal nuclei. In contrast to previous results using peroxidase, we did not observe dramatic sex differences in connectivity, although some connections were less robust in female than in male brains. Some retrograde connections previously observed (e.g., anterior preoptic area to DTAM) were not confirmed. Plausible hypotheses are that a set of rhombencephalic neurons located in DTAM, the inferior reticular formation and n.IX-X are responsible for generating patterned vocal activity, that activity is modulated by neurons in rRpd, and that activity in VS (particularly that evoked by conspecific calls), together with effects of steroid hormones at many sites in the vocal circuit, contribute to the initiation of calling.     

A comparative neurological approach to emotional expressions in primate vocalizations

Different approaches from different research domains have crystallized debate over primate emotional processing and vocalizations in recent decades. On one side, researchers disagree about whether emotional states or processes in animals truly compare to those in humans. On the other, a long-held assumption is that primate vocalizations are innate communicative signals over which nonhuman primates have limited control and a mirror of the emotional state of the individuals producing them, despite growing evidence of intentional production for some vocalizations. Our goal is to connect both sides of the discussion in deciphering how the emotional content of primate calls compares with emotional vocal signals in humans. We focus particularly on neural bases of primate emotions and vocalizations to identify cerebral structures underlying emotion, vocal production, and comprehension in primates, and discuss whether particular structures or neuronal networks solely evolved for specific functions in the human brain. Finally, we propose a model to classify emotional vocalizations in primates according to four dimensions (learning, control, emotional, meaning) to allow comparing calls across species.     

Vocal parameters that indicate threat level correlate with FOS immunolabeling in social and vocal control brain regions

Transmitting information via communicative signals is integral to interacting with conspecifics, and some species achieve this task by varying vocalizations to reflect context. Although signal variation is critical to social interactions, the underlying neural control has not been studied. In response to a predator, black-capped chickadees (Poecile atricapilla) produce mobbing calls (chick-a-dee calls) with various parameters, some of which convey information about the threat stimulus. We predicted that vocal parameters indicative of threat would be associated with distinct patterns of neuronal activity within brain areas involved in social behavior and those involved in the sensorimotor control of vocal production. To test this prediction, we measured the syntax and structural aspects of chick-a-dee call production in response to a hawk model and assessed the protein product of the immediate early gene FOS in brain regions implicated in context-specific vocal and social behavior. These regions include the medial preoptic area (POM) and lateral septum (LS), as well as regions involved in vocal motor control, including the dorsomedial nucleus of the intercollicular complex and the HVC. We found correlations linking call rate (previously demonstrated to reflect threat) to labeling in the POM and LS. Labeling in the HVC correlated with the number of D notes per call, which may also signal threat level. Labeling in the call control region dorsomedial nucleus was associated with the structure of D notes and the overall number of notes, but not call rate or type of notes produced. These results suggest that the POM and LS may influence attributes of vocalizations produced in response to predators and that the brain region implicated in song control, the HVC, also influences call production. Because variation in chick-a-dee call rate indicates predator threat, we speculate that these areas could integrate with motor control regions to imbue mobbing signals with additional information about threat level.     

Functional subdivisions of the olfactory system correlate with lectin-binding properties in Xenopus.

Soybean agglutinin (SBA) is known to selectively label a portion of neurons in amphibian and mammalian primary olfactory systems. Hitherto, no other distinctive features have been found to correlate with the two neuronal populations. Investigating SBA-HRP binding in olfactory mucosa and CNS of Xenopus, we noted that labelled and unlabelled structures can readily be assigned to different olfactory subsystems. The SBA negative one is utilized to detect air-borne odors, whereas major SBA-positive structures serve a role in the perception of water dissolved molecules. Some labelled fibers by-pass the olfactory bulb, traverse the telencephalon and innervate prosencephalic structures. They are considered to be aberrant olfactory nerve fibers, rather than being part of the terminal nerve.     

Brain pathways for learned and unlearned vocalizations differ in zebra finches

Male zebra finches sing, females do not. However, both sexes produce the "long call" when placed in visual isolation. This call is sexually dimorphic; it includes learned components in males but not in females. The 3 learned features of the male long call are a high fundamental frequency, a fast frequency modulation, and a short, stable duration. These features are learned by the male during development, as is song. Since similar features are also found in song syllables, we wanted to know whether long-call production depends on the same CNS pathway that controls song production. Three critical components of the song pathway are telencephalic nuclei HVC, RA, and the tracheosyringeal (ts) nerves innervating the syrinx. In male zebra finches, bilateral section of the ts nerves affected the fundamental frequency and fast frequency modulations of both the long call and song but left the temporal features intact. Ts nerve section had no effect on the female long call. Bilateral lesions of either HVC or RA in males affected the fundamental frequency, fast frequency modulations, and temporal structure of both the long call and song. Similar lesions had no effect on the female long call. These results demonstrate that HVC, RA, and the ts nerves make critical contributions to the acoustic features of the male long call and song, while the temporal pattern depends on HVC and RA but not the ts nerves. HVC and RA lesions remove all the learned features that distinguish the male call and reveal a simple unlearned vocalization shared by both sexes. We suggest that the learned features of oscine songbird vocalizations are controlled by a telencephalic pathway that acts in concert with other pathways responsible for simpler, unlearned vocalizations.     

Distribution of GABA-like immunoreactive cell bodies in the brains of two amphibians, Rana catesbeiana and Xenopus laevis

The distribution of the neurotransmitter gamma-aminobutyric acid (GABA) is not well understood for non-mammalian vertebrates. We thus used immunocytochemistry to locate putative GABAergic cells in the brains of male bullfrogs (Rana catesbeiana) and South African clawed frogs (Xenopus laevis). GABA-immunoreactive cell bodies were broadly distributed throughout the brains of both species with similar general patterns. In both, the greatest numbers of GABA-positive cells were found in the olfactory bulb, thalamus, and optic tectum, but virtually no major brain region lacked GABAergic cells. Species differences were also apparent. The density of GABA-immunoreactive cells was substantially higher in many areas of the bullfrog brain, compared to Xenopus. Bullfrogs possessed extensive cell populations in the medial pallium, preoptic area, optic tectum, torus semicircularis and tegmentum but cells were fewer in these locations in Xenopus. In the bullfrog hindbrain, GABA-immunoreactive cell bodies were restricted to very narrow and distinct populations. In Xenopus, however, cells in a similar position were fewer and spread more extensively. The distribution of GABA cells in the brain of these two species supports the hypotheses that GABA is involved in control of olfaction, audition, vision and vocalization. However, differences in the distribution of GABA between the bullfrog and Xenopus suggest that the extent of the GABAergic influence might vary between species.     

Zenk expression in auditory regions changes with breeding condition in male Black-capped chickadees (Poecile atricapillus)

Black-capped chickadees (Poecile atricapillus) produce different vocalizations at different times of year: the fee-bee song is produced by males primarily in spring, whereas the chick-a-dee call is produced year-round but most frequently in the fall and winter. We wanted to determine if neural response to playback in auditory regions of the songbird brain varied with season in parallel to production. We captured adult male black-capped chickadees from the wild in either breeding condition or non-breeding condition and within 24-48 h of bringing them into the laboratory setting, played them recordings of either conspecific vocalizations (fee-bee songs or chick-a-dee calls), heterospecific vocalizations (Song Sparrow, Melospiza melodia, song), or silence. We then measured ZENK protein immunoreactivity (Zenk-ir) in caudomedial nidopallium (NCM) and caudomedial mesopallium (CMM), two regions important for perception of conspecific vocalizations. We found that, overall, non-breeding birds had greater Zenk-ir than breeding birds. In addition, we found that birds in non-breeding condition had significantly greater Zenk-ir to heterospecific song than birds in breeding condition, but this difference was not seen in birds that heard conspecific songs or calls. Finally, in NCMd chickadees had greater response to playback of conspecific vocalizations (when combining song and call groups) than playback of heterospecific vocalizations but only while in breeding condition. Our results qualify the claim that Zenk-ir is biased toward conspecific vocalizations, and indicate that specificity of neural response varies with season. Variation could be a result of increased production and perceptual demand in spring, or hormonal changes in breeding birds, possibly because chickadees display vocal plasticity in chick-a-dee calls in the fall, requiring a degree of neural plasticity across seasons.     

Audio-vocal interaction in the pontine brainstem during self-initiated vocalization in the squirrel monkey

The adjustment of the voice by auditory input happens at several brain levels. The caudal pontine brainstem, though rarely investigated, is one candidate area for such audio-vocal integration. We recorded neuronal activity in this area in awake, behaving squirrel monkeys (Saimiri sciureus) during vocal communication, using telemetric single-unit recording techniques. We found audio-vocal neurons at locations not described before, namely in the periolivary region of the superior olivary complex and the adjacent pontine reticular formation. They showed various responses to external sounds (noise bursts) and activity increases (excitation) or decreases (inhibition) to self-produced vocalizations, starting prior to vocal onset and continuing through vocalizations. In most of them, the responses to noise bursts and self-produced vocalizations were similar, with the only difference that neuronal activity started prior to vocal onset. About one-third responded phasically to noise bursts, independent of whether they increased or decreased their activity to vocalization. The activity of most audio-vocal neurons correlated with basic acoustic features of the vocalization, such as call duration and/or syllable structure. Auditory neurons near audio-vocal neurons showed significantly more frequent phasic response patterns than those in areas without audio-vocal activity. Based on these findings, we propose that audio-vocal neurons showing similar activity to external acoustical stimuli and vocalization play a role in olivocochlear regulation. Specifically, audio-vocal neurons with a phasic response to external auditory stimuli are candidates for the mediation of basal audio-vocal reflexes such as the Lombard reflex. Thus, our findings suggest that complex audio-vocal integration mechanisms exist in the ventrolateral pontine brainstem.     

Glial-defined boundaries in Xenopus CNS.

Regional specificity, which arises early during central nervous system (CNS) development, reflects the generation of boundary regions that define the domains of distinct neural cell types and the guidance of axonal growth. The boundaries between discrete CNS domains often appear to be established by specialized glial cells. Boundary glia have been implicated in supporting neurite extension by providing mechanical and chemical barriers during development and regeneration. The present study demonstrates biochemical and morphological differences in boundary glial cells in the hindbrain and spinal cord of developing Xenopus laevis. DM gamma, a membrane protein of the proteolipid protein family, is localized to radial glial processes in hindbrain boundary regions. By contrast, DM beta, a neuronal protein that bears significant homology to DM gamma in primary sequence and that promotes neurite outgrowth, is expressed in hindbrain axonal pathways. In addition, the expression of two intermediate filament proteins, glial fibrillary acidic protein and vimentin, are progressively restricted to glial cells in the rhombomere center and boundary regions, respectively. Those two intermediate filament proteins show distinct expression domains in the spinal cord as well. The present study suggests that a glial surface protein, DM gamma, may act as a boundary molecule in developing Xenopus hindbrain and that a distinct subpopulation of glial cells may define functional domains within the CNS.