Hormones and the Regulation of Vocal Patterns in Amphibians: Xenopus laevis Vocalizations as a Model System

20 Hormones and the Regulation of Vocal Patterns in Amphibians: Xenopus laevis Vocalizations as a Model System E-J Yang, Harvard University, Cambridge, MA, USA D B Kelley, Columbia University, New York, NY, USA ß 2009 Elsevier Inc. All rights reserved.

Chapter Outline 20.1 20.2 20.3 20.4 20.5 20.5.1 20.5.2 20.5.3 20.5.4 20.5.5 20.5.5.1 20.5.5.2 20.6 20.7 20.8 20.8.1 20.8.2 20.8.3 20.9 References

Introduction Vocal Communication Mechanisms of Sound Production and the Hindbrain Circuit Underlying Vocal Production Hormonal Control of Vocal Behaviors in Adult Males Sex Differences and the Roles of Sex Steroid Hormones Laryngeal Muscle The Male Neuromuscular Synapse Vocal Nerve Activity Laryngeal Motoneurons Sexually Differentiated Vocal Patterns and Vocal Circuitry in X. laevis Masculinization Generating female vocalizations Neuromodulators and the Vocal Circuit Singing and Breathing Generating Patterned Vocal Output Xenopus laevis Vocal Pattern Generation in Terrestrial Amphibians The Evolution of Hindbrain Vocal Pattern Generators Evolution and the Sexual Differentiation of Neuroeffectors for Vocalization

Glossary arytenoid disks Cartilaginous disks within the vocal organ (larynx) of Xenopus frogs. satellite cells Subclass of myoblasts that can be recruited for muscle fiber growth and repair in muscle. x1ERa2 Novel estrogen receptor expressed in laryngeal muscle in Xenopus frogs.

20.1 Introduction Most terrestrial vertebrates produce vocal behaviors using complex motor programs that combine respiratory control with phonation (Bass and Baker, 1997). The South African clawed frog, Xenopus laevis, uses a simpler program for vocal production. These frogs are fully aquatic and sing underwater without breathing.

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The isolated vocal organ can produce calls in vitro if the laryngeal nerve is stimulated with the appropriate pattern (Tobias and Kelley, 1987). The isolated brainstem can generate the patterned activity that produces different calls (Rhodes et al., 2007; Zornik and Kelley, 2008). Sex differences in vocal signaling are prominent and matched to the cell biology and biophysical properties of vocal muscle and motoneurons (Kelley, 1996). Sex differences arise as the result of hormonally controlled developmental programs (Tobias et al., 1998a; Kay et al., 1999). Xenopus thus provides unique access to the biomechanical, cell biological, neuronal, and endocrine mechanisms that produce sexually differentiated vocal communication signals.

20.2 Vocal Communication The genus Xenopus consists of approximately 25 species of anuran amphibians indigenous to sub-Saharan 693

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highest energy at 2kHz, whereas female clicks have most energy at 1kHz (Tobias et al., 1998b). Social interactions – between males and females and among males – include calling and clasping. With females, males produce advertisement and answer calls and, when clasping, the amplectant call (Figure 2(b)). With other males, all six call types are produced: the clasped male growls and ticks whereas the clasping male usually advertises, chirps, and produces answer calls and amplectant calls as well (Figure 2(a); Tobias et al., 2004). In response to male clasping and calling, females vocalize according to their sexual receptivity: ticking when they are unreceptive and rapping when receptive. Rapping is a powerful acoustic aphrodisiac (Figure 3); males attempt to mate with loudspeakers during rapping broadcasts. Ticking, in contrast, silences males (Tobias et al., 1998b). Female ticking and rapping differ only in click rate: the average ticking ICI is 219ms, whereas the average rapping ICI is 81ms. Broadcasts of ticking suppress male calling within 91s; males then habituate to ticking and resume calling (Elliott and Kelley, 2007). On the other hand, rapping stimulates male calling, particularly the answer call; habituation to rapping has not been observed (Tobias et al., 1998b). The click rate distribution of ticking and rapping (Figure 4) overlaps at intermediate ICIs (e.g., 120ms ICI, Figure 4; Elliott and Kelley, 2007);

Africa (Evans et al., 2004). Xenopus is completely aquatic throughout the life span and its habitats usually consist of silt-filled, murky ponds. Like most anurans (frogs and toads), Xenopus call at night. The male advertisement call functions both in female attraction and in male–male competitions (Tobias et al., 2004). Among all the species within the genus, X. laevis has the largest vocal repertoire, consisting of two distinct female calls and six different male calls (Tobias et al., 2004). These calls are distinguished by the social context in which they are produced as well as acoustic features of the calls themselves (Figure 1). Calls are made up of a series of clicks, generated during separation of cartilaginous disks in the larynx. Each call has distinctive click rates (also expressed as inter-click intervals or ICIs), temporal patterns, and modulations in intensity. For example, the advertisement call, the most complex call in the repertoire, consists of alternating fast and slow trills, and the fast trills become progressively louder (i.e., intensity modulated). The ICI for the fast trill portion is 18ms, whereas for the slow trill it is 34ms. These temporal characteristics of calls are used in identifying the sex of the caller, particularly by male listeners (Vignal and Kelley, 2007) and in distinguishing sexually receptive and unreceptive females (Elliott and Kelley, 2007). Male and female clicks also differ in spectral properties: the male clicks have the Male calls

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Figure 1 The vocal repertoire of Xenopus laevis. Clicks are illustrated as oscillograms; note difference in timescales. Modified from Kelley DB and Tobias ML (1999) The vocal repertoire of Xenopus laevis. In: Hauser M and Konishi M (eds.) The Design of Animal Communication, pp. 9–35. Cambridge, MA: MIT Press.

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Figure 2 Vocal signaling in males depends on the sex of the receiver. Modified from Tobias ML, O’Hagan R, Horng SH, and Kelley DB (2004) Vocal communication between male Xenopus laevis; behavioral context and sexual state. Animal Behaviour 67: 363–365, with permission from Elsevier.

these ICIs are ambiguous as gauged by the male response. Within the distinctive and nonoverlapping click rates, characteristics of ticking (e.g., 219 and 160 ms ICI) and rapping (e.g., 81 and 98ms ICI) male responses are clear-cut: rapping stimulates and ticking suppresses male calling. During male–male interactions, continuous and persistent advertisement calling by a male can suppress the other male’s vocalization. This vocal suppression is inducible acoustically without physical contact and stops once the males are separated (Tobias et al., 2004).

20.3 Mechanisms of Sound Production and the Hindbrain Circuit Underlying Vocal Production Sound is produced by the larynx, the vocal organ. The larynx is a cartilaginous box containing the two

tightly opposing arytenoid disks that are connected to bipennate laryngeal dilator muscles by a tendon. The glottis, through which the larynx connects to the buccal cavity, lies at the anterior extent of the larynx. Calling always occurs underwater; respiration does not accompany, nor is required for, sound production. Instead, the opening of the arytenoid disks is associated with an instantaneous burst of sound, a click (Yager, 1992). A click is the basic unit of Xenopus calls. As laryngeal muscles relax, the disks close. The properties of laryngeal muscles, such as twitch and relaxation rate, are important determining factors for the speed at which the calls are produced. This sound-production mechanism is conserved across all species of Xenopus examined to date. En passant recordings from laryngeal nerves reveal patterned activity that precisely matches click production within calls (Yamaguchi and Kelley, 2000). If the larynx is removed and laryngeal nerves stimulated at call rates, the larynx will sing in the dish (Tobias and

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Figure 3 Rapping stimulates and ticking suppresses male calling. Modified from Tobias ML, Viswanathan SS, and Kelley DB (1998b) Rapping, a female receptive call, initiates male–female duets in the South African clawed frog. Proceedings of the National Academy of Sciences of the United States of America 95: 1870–1875. Copyright (1998) National Academy of Sciences, USA.

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Figure 4 Interclick intervals in rapping and ticking. Modified from Elliott TM and Kelley DB (2007) Male discrimination of receptive and unreceptive female calls by temporal features. Journal of Experimental Biology 210: 2836–2842.

Kelley, 1987). The isolated brain produces patterned neural activity recorded from the laryngeal nerve that matches amplectant calling and glottal activity during breathing (Zornik and Kelley, 2008) and the advertisement calling pattern (Rhodes et al., 2007). The ability

of isolated preparations to produce different fictive call patterns has facilitated understanding of the underlying neural circuitry. Neurons that reside in cranial nucleus (n.) IX–X (homologous to the mammalian nucleus ambiguus) innervate the larynx, their axons synapse on either glottal or laryngeal muscles (Brahic and Kelley, 2003; Zornik and Kelley, 2007). The connectivity and synaptic connections of the hindbrain vocal circuit have been mapped in some detail (Zornik and Kelley, 2007, 2008; Figure 5). Laryngeal motoneurons (LMNs) and glottal motoneurons (GMNs) are segregated in n. IX–X: GMNs occupy the anterolateral aspect of the nucleus, whereas LMNs are in posterior n. IX–X. This anatomical segregation between glottal and laryngeal motoneurons may indicate separation and specialization of neuronal mechanisms underlying respiration and vocalization, and is consistent with the behavioral and physiological observations that respiration and vocalization are mutually exclusive. The pretrigeminal nucleus of the dorsal tegmental area of medulla (DTAM), a small nucleus of the rostral hindbrain just ventral to the cerebellum (the counterpart of the mammalian parabrachial nucleus),

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20.4 Hormonal Control of Vocal Behaviors in Adult Males

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(b) Figure 5 Hindbrain vocal circuitry in Xenopus laevis. DTAM, dorsal tegmental nucleus of the medulla; DTAMIX–X, interneurons in DTAM projecting to n. IX–X; E, excitatory; GMN, glottal motoneurons; I, inhibitory; IIN, inhibitory interneurons; LMN, laryngeal motoneurons; TBD, to be determined; IX–XDTAM, interneurons in n. IX–X projecting to DTAM; IX–XIX–X commissural interneurons. Modified from Zornik E and Kelley DB (2007) Breathing and calling: Neuronal networks in the Xenopus laevis hindbrain. Journal of Comparative Neurology 501: 303–315.

provides monosynaptic, excitatory, glutamatergic input to both LMNs and GMNs. In addition, DTAM provides GABAergic inhibitory input to GMNs. In addition to motoneurons, n. IX–X contains at least two populations of interneurons that project beyond the nucleus. Neurons that project to contralateral n. IX–X provide monosynaptic, excitatory glutamatergic input to LMNs. These neurons are located in anteromedial n. IX–X and are mixed with another population that projects to DTAM. DTAM neurons do not show topographical segregation based on their projections to different class of motoneurons. There is a third class of DTAM neurons that projects both ipsi- and contralateral n. IX–X. All these three classes of DTAM neurons receive inputs from interneurons of n. IX–X. Both DTAM and n. IX–X receive input from a small nucleus of the raphe, the rRpd (Brahic and Kelley, 2003); terminals are serotonergic (Rhodes et al., 2007). The other major source of input to DTAM is a forebrain nucleus, the ventral striatum (Brahic and Kelley, 2003).

Calling occurs during the breeding season and is associated with specific reproductive behaviors (Tobias et al., 2004). Studies of the hormonal control of calling in Xenopus have focused on the advertisement call of males and the induction of rapping in females. Testis transplants into gonadectomized males induce advertisement calling (Watson and Kelley, 1992). Replacement with exogenous androgens (testosterone or dihydrotestosterone) also restores advertisement calling in gonadectomized males but the amount of calling is less than that induced in intact males by gonadotropin (GT) administration (Wetzel and Kelley, 1983; Watson and Kelley, 1992). Androgen is produced predominantly by male gonads in response to GT released by the pituitary, which in turn is stimulated by gonadotropin-releasing hormone (GnRH) from the hypothalamus. Thus, the action of GT in intact males is due, at least in part, to androgen secretion. Not all male calls are androgen-regulated; growling, for example, is unaffected by castration (Horng and Kelley, unpublished). Both juvenile and adult females can be induced to produce the advertisement call by testicular transplants or exogenous androgen (Hannigan and Kelley, 1986; Watson and Kelley, 1992; Potter et al., 2005). In addition to its effects on androgen secretion, GT itself may have a direct influence on vocalization. When given to castrated males, GT does not increase advertisement calling. However, in androgen-treated castrated males, GT can triple the amount of calling, suggesting a synergistic action between androgen and GT (Wetzel and Kelley, 1983). This particular study supplied GT systemically, and therefore indicates only that the locus of GT action is not the gonads. Yang et al. (2007) recently demonstrated that GT can influence calling by directly working on the brain, a mechanism separate from its role in stimulating the release of androgen from the gonads. Intracerebroventricular injections of GT significantly increased advertisement calling in androgen-replaced gonadectomized males at less than one-hundredth of the effective systemic dose. This CNS-mediated GT action is androgen dependent; intraventricular GT infusion in gonadectomized males does not increase advertisement calling. The action of GT occurs via binding to its functional cognate receptor, luteinizing hormone receptor (LHR), a receptor expressed particularly in the forebrain regions (the ventral striatum) implicated in vocal control. We cloned Xenopus

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LHR mRNA and mapped expression in the brain. Activation of a downstream transcription target of LHR occurs in receptor expressing neurons in the ventral striatum (VST), indicating that LHRs in this region are functional. Thus, the functional LHRs expressed in VST may mediate GT effects on vocalization.

20.5 Sex Differences and the Roles of Sex Steroid Hormones 20.5.1

Laryngeal Muscle

There is a strong sexual dimorphism in the vocal organ, regulated through a developmental trajectory controlled by gonadal sex steroids. Male laryngeal muscle fibers are larger in diameter and more numerous than female fibers. In addition, male muscles show high oxidative capacity and are composed of homogenous cells that exhibit acid-sensitive ATPase activity, indicating a fast-twitch fiber type (Sassoon et al., 1987). High oxidative capacity and fast-twitch fibers facilitate rapid contractions repeatedly over long bouts of male calling. In particular, the advertisement call requires ~70 Hz clicks for the fast trill: muscle contraction and relaxation cycles at 70 Hz. On the other hand, female muscles are composed of heterogenous fibers most of which exhibit acidinsensitive ATPase activity, indicating slow-twitch characteristics. The threshold for maintained muscle tension at male-like high rates of stimulation differs dramatically in the sexes. In the isolated larynx, nerve stimulation can produce tension transients up to 77 Hz in males and up to 33 Hz in females without tetanus (Tobias and Kelley, 1987). When tension is maintained, however, the arytenoid disks cannot close; no more clicks are produced until the muscle relaxes. This sex difference in fiber types parallels differences in call click rates: fast-twitch fiber types support fast male calls, whereas slow-twitch fiber types support slower female calls. Expression of a specific myosin heavy chain (MYHC) isoform, laryngeal myosin (LM), underlies sexually dimorphic fiber types. MYHC expression is a major determinant of the contractile properties of individual muscle fibers, and differences in fiber types directly correlate with differences in MYHC gene expression. All the fibers in male laryngeal muscle express LM mRNA (Catz et al., 1992) and the LM protein and only around 20% of female laryngeal muscle fibers express LM (Nasipak, 2007). LM is a member of a genomic array of

MYHC isoforms, whose expression is otherwise limited to larval and embryonic muscles (Nasipak and Kelley, 2008). Sexually dimorphic expression of LM, fiber-type composition, development of tension transients upon nerve stimulation, and morphology are the result of the sex-steroid milieu in which male and female Xenopus develop. At the end of metamorphosis, male and female larynges do not differ in fiber type (Tobias et al., 1991), in the number of laryngeal muscle fibers, and in the rate at which cell proliferation occurs (Sassoon and Kelley, 1986; Marin et al., 1990). Over the next 6 months males experience a sevenfold increase in the amount of cell proliferation relative to the female larynx (Sassoon and Kelley, 1986). Paradoxically, during this time androgen levels are low in both sexes (Kang et al., 1995). However, there are higher levels of androgen receptor expression in male than in the female larynx, which may explain the greater response to androgen in males (Kelley et al., 1989). The number of muscle fibers in the male larynx grows from approximately 3500 fibers at the end of metamorphosis to 30 000 at 6 months later, in comparison to an increase to 10000 fibers in the female larynx (Marin et al., 1990). During this time, LM mRNA expression, fiber-type switching to fast-twitch type, muscle fiber addition, and the development of adult malelike tension transients are androgen dependent. All characteristics are induced by exogenous androgen treatment and blocked by gonadectomy (Marin et al., 1990; Tobias et al., 1991). In contrast, neither withdrawal of androgen nor androgen treatment has an effect in adult males (Watson et al., 1993). During juvenile development, exogenous androgen treatment induces cell proliferation, particularly in satellite cells, a subclass of myoblasts, which are normally recruited for muscle fiber repair and replenishment; they can fuse with each other or be incorporated into differentiated myocytes (Sassoon et al., 1986). To examine the relation between myoblast proliferation and LM expression, Nasipak (2007) examined the direct effect of androgen on these cells in explant layngeal cultures. In response to androgen treatments, LM mRNA and protein are upregulated to levels comparable to those measured during juvenile stages in vivo. Both in vivo and in vitro, myogenesis occurs before androgen-induced LM induction. When cell proliferation was pharmacologically blocked, androgen-induced LM upregulation was inhibited. These results suggest that myogenesis and cell proliferation are required for androgen

Hormones and the Regulation of Vocal Patterns in Amphibians

action on LM induction, a larynx-specific molecular marker for switching from slow- to fast-twitch muscle fiber types. 20.5.2

The Male Neuromuscular Synapse

Laryngeal motoneurons located in posterior n. IX–X make synaptic contacts on laryngeal muscle fibers. Using the vox in vitro preparation (the larynx with its innervating motor nerve intact in physiological saline), this neuromuscular (NM) synapse has been examined in detail. The male NM synapse is weak; trains of stimuli are required for action potential production by muscle fibers and transmission is not reliable (Tobias and Kelley, 1987; Tobias et al., 1995). With repeated stimulation, the amplitude of the postsynaptic response increases until an action potential is produced, indicating facilitation of synaptic transmission. In contrast, female synapses are strong; each nerve stimulation results in an action potential in muscle fibers. Analysis of synaptic failures indicates that the female neuromuscular synapses have greater quantal contents (Tobias et al., 1995). Because miniature endplate potentials, measuring postsynaptic depolarization due to spontaneous release of a vesicle, were the same in males and females, the quantal content results suggest that sex differences in NM synaptic transmission are presynaptic in origin. During trains of nerve stimulation, simulating calling (Yamaguchi and Kelley, 2000), the amplitude of the electromyograms (EMGs) in the male larynx increases progressively, accompanying increases in click loudness. Thus, the facilitation of weak synapses in the male larynx contributes to the progressive increase in click intensity in the male call. 20.5.3

Vocal Nerve Activity

What is the relation between the pattern of activity of laryngeal motoneurons and actual calling? Yamaguchi and Kelley (2000) recorded en passant from the laryngeal nerve of male and female X. laevis during singing. They observed that vocal patterns closely match nerve compound action potentials (CAPs) both for advertisement calling and for ticking. The nerve contains axons of laryngeal motoneurons and thus the pattern of vocal activity originates within the CNS. During rapid calls (e.g., the fast trills of advertisement calling), CAP durations are short; the action potentials of individual laryngeal motoneurons are highly synchronized. This synchrony is important when laryngeal muscles have to contract and relax

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fully in the short intervals between clicks, a characteristic of rapid trills. With slower calls like amplectant calls and ticking however, CAPs are longer and less synchronous for both sexes. Progressive increases in CAP amplitudes during the fast trill accompany progressively louder clicks (intensity modulation). Increases in EMG responses to a train of nerve stimulation are also observed in the vox in vitro preparation. Thus, intensity modulation is accomplished at two levels: progressive recruitment of laryngeal motoneurons and progressive facilitation of laryngeal NM synapses onto laryngeal muscle fibers. 20.5.4

Laryngeal Motoneurons

The rhythmic patterns of different calls are relayed to the vocal organ by laryngeal motoneurons, resident in nucleus IX–X of the hindbrain. The biophysical properties of motoneurons in n. IX–X differ between the sexes: in firing patterns, passive membrane properties, and ionic currents (Yamaguchi et al., 2003). Male vocal motoneurons have smaller input resistances and larger membrane capacitances associated with their larger size. Unlike the tonically firing and weakly adapting female neurons, male motoneurons are strongly adapting and fire phasically. They express characteristic hyperpolarization-activated cationic currents (IH) and low-threshold potassium currents (IKL); IH values are exclusively expressed in males. Modeling results suggest that IKL play an important role in expression of strongly adapting firing patterns. Together with short-latency spike onset of male motoneurons, these biophysical characteristics support precision in following synaptic inputs. These currents also support synchronized firing of populations of vocal motoneurons, required for producing the fast spike trains that generate the rapid and precisely timed clicks of male advertisement call. Weakly adapting firing patterns with less precise time coding in vocal motoneurons support slow clicks with variable ICIs, such as the slow trills in male advertisement or the female calls rapping and ticking. Males have approximately twice as many neurons in n. IX–X as females (Kelley and Dennison, 1990). This difference is due to androgen-induced rescue of neurons from ontogenetic cell death (Kay et al., 1999). Both cell proliferation in the larynx and rescue of neurons in n. IX–X from cell death by androgen are initiated by secretion of thyroxine during metamorphosis (Cohen and Kelley, 1996). The ability of androgen to induce LM expression is regulated by prolactin (Edwards et al., 1999).

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20.5.5 Sexually Differentiated Vocal Patterns and Vocal Circuitry in X. laevis 20.5.5.1 Masculinization

To what extent do gonadal hormone-induced sex-specific features account for differences in vocal circuitry? Testis transplants given to ovariectomized females masculinize laryngeal fiber number and type at every developmental stage (Watson et al., 1993). The larynx of females with testis transplants contains entirely fast-twitch fibers with completely masculinized tension transients at rates of stimulation equivalent to the male fast trill. Axon numbers in the laryngeal nerve increase as well, suggesting either that more neurons may have been added or that axons have added branches due to the testis transplant. If the testis is transplanted at juvenile stages, all the females were able to produce male advertisement calls as adults with durations and trill rates indistinguishable from those of males (Watson and Kelley, 1992). When exogenous androgen was administered instead of a transplant, fiber numbers did not increase in adult females (Watson et al., 1993) and vocal behaviors were only partially masculinized (Hannigan and Kelley, 1986). More recently, Potter et al. (2005) re-examined androgen effects in ovariectomized females. Over the course of several weeks, females that had previously produced only normal female release calls (2 Hz and single clicks) began to also produce double and triple clicks (as in Hannigan and Kelley, 1986) as well as rapid clicks with random interclick intervals and intensity modulation. At 8 weeks, androgen treatment induced male-like advertisement calling in most of the treated females. This male-like advertisement calling contains alternating fast and slow trill portions and was only observed when treated females were exposed to another female, suggesting that social context is important. By week 13 of androgen treatment, all females produced male-like advertisement calling. In the advertisement calls produced by these females, both the fast and the slow trill portions were significantly slower than those of male calls. An organizational role of androgen during development may account for this incomplete masculinization. The volume of laryngeal motoneuron somata, visualized by a backfill from the terminals in laryngeal muscle, is sexually dimorphic with females having smaller somata than males (Potter et al., 2005), although the cross-sectional areas when measured with Golgi staining methods are similar between

the sexes (Kelley et al., 1988). With as little as 1 week of androgen treatment, female motoneuron cell bodies enlarge to the size of male neurons. When female laryngeal muscle is stimulated via the nerve, the muscle produces the maintained tension at low stimulation rates (20 Hz) only in comparison to 100 Hz in male muscles. After 4 weeks of androgen treatment, the profile of tension transients is similar to that of male muscle, although the frequency at which maintained tension occurs is still lower in female muscles. Single twitch tension of female laryngeal muscle (slope of tension onset and halfrelaxation time) becomes indistinguishable from that of male muscles. Since androgen treatment in adulthood does not induce laryngeal myogenesis (Sassoon and Kelley, 1986; Sassoon et al., 1986; Watson et al., 1993), these changes are most likely due to androgendriven alteration of existing muscle fibers. Interestingly, the most common click rates in the male-like vocalizations produced by androgentreated females and the stimulation rate at which androgen-treated female laryngeal muscle begins to produce maintained tension are very similar, though lower than the male vocalization and threshold. No further changes are produced by up to 16 weeks of androgen treatment, suggesting that neither the level of androgen nor the duration of treatment are responsible for incomplete masculinization. Developmentally driven androgen effects are more likely. 20.5.5.2 Generating female vocalizations

Females produce two distinct calls, rapping and ticking (Figure 1), distinguishable by the click rate (Figure 4). Rapping is evoked by male calling and produced during a very narrow reproductive window, between 9 and 18 h after GT injection, presumably coincident with the passage of eggs through the oviduct (Wu et al., 2001). Ovulation and oviposition are associated with increasing titers of estrogen and progesterone. Estrogen controls synaptic strength: ovariectomy weakens the strong synapses of adult females, and estrogen strengthens the weak synapses of male and female juveniles (Tobias et al., 1998a). The effects of ovariectomy on decreasing quantal contents in adult females and of estrogen treatment of juveniles on increasing quantal contents (Wu et al., 2001) are slow (2–4weeks). GT weakens laryngeal synapses and effects are much more rapid (12h). The weakening action of GT is mimicked by estrogen but not progesterone. Thus, activation of the hypothalamic–pituitary–gonadal axis in females has two phases: an acute phase in which synapses are

Hormones and the Regulation of Vocal Patterns in Amphibians

weakened under the influence of GT-evoked estrogen secretion and a chronic phase in which laryngeal synapses are strengthened by estrogen. Rapping is produced during the acute phase while ticking occurs in both phases. The weak, facilitating synapses of males contribute to the intensity modulation of clicks in the advertisement call (Yamaguchi and Kelley, 2000). Rapping is not intensity modulated. Studies of male and female vox in vitro preparations using paired pulse facilitation as well as trains (Ruel et al., 1998) reveal no sex difference in the ability to facilitate at male-like interclick intervals (15 and 30 ms). However, we would not expect facilitation to occur at the much slower ICIs that characterize rapping (80–100 ms) and ticking (160–220 ms). We have speculated that the strengthening effects of estrogen insure vocal function during the acute weakening produced by GT-evoked estrogen secretion. Because postsynaptic responses to nerve stimulation are the same in the sexes we believe that the locus for sex differences in synaptic strength is presynaptic (Tobias et al., 1995). However, laryngeal motoneurons do not express estrogen receptor (ER). In contrast to LMNs, laryngeal muscle does express a classical 66 kDa ER (Wu et al., 2003). In juveniles, estrogen treatment upregulates ER expression concomitantly with increases in synaptic strength (Wu et al., 2003). Estrogen could be acting via retrograde signaling from vocal muscles. At the molecular level,

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we identified two distinct ERa genes; the primary ER expressed in laryngeal muscle is the novel gene xlERa2; xlERa1 is the gene primarily expressed in forebrain, oviduct, and liver. The complementary spatial expression of these two genes is consistent with the subfunctionalization model for evolution after gene duplication (Wu et al., 2003). Unlike the effects of androgen on muscle fiber type, the effects of estrogen are not permanent. Because around 20% of all male NM synapses are strong, factors other than sex differences in the level circulating estrogen must be involved in determining synaptic strength.

20.6 Neuromodulators and the Vocal Circuit The isolated brain preparation can generate the patterned activity that produces different calls (Rhodes et al., 2007; Zornik and Kelley, 2008). In males, spontaneous activity that resembles amplectant calling and the glottal bursting that accompanies breathing can be recorded from the laryngeal nerve. Application of 5-HT to the isolated brain preparation induces activity that resembles male advertisement calls and CAPs recorded from the laryngeal nerve of a singing male Xenopus in temporal pattern, sequence of slow and fast trills, interclick intervals, and intensity modulation (Figure 6). Other patterns

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resembling slow trills alone, amplectant calls, and ticking were also observed in response to 5-HT. In the isolated female brain, 5-HT can induce ticking. Isolated brains from ovariectomized females treated with androgen for 8 weeks can produce fictive activity similar to male advertisement calling in response to 5-HT treatment (Figure 7). Is there endogenous source of 5-HT that activates the hindbrain vocal circuit? A small nucleus of the raphe (the rostral raphe pars dorsalis (rRpd)) projects to the entire vocal circuit, including DTAM and n. IX–X in both sexes (Brahic and Kelley, 2003). Tryptophan hydroxylase (a rate-limiting enzyme in 5-HT synthesis) immunoreactive neurons are located in the rRpd and axon projections and varicosities are present in n. IX–X, reticular formation, and DTAM (Rhodes et al., 2007). When WGA-HRP was injected into n. IX–X, dorsal raphe neurons were labeled, suggesting that they may provide direct input to motoneurons in n. IX–X. 5-HT-evoked fictive advertisement calling in males and ticking in females are abolished by transection just caudal – but not rostral – to DTAM (Rhodes et al., 2007). The hindbrain pattern generators for both male- and female-specific vocalizations thus

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reside in the hindbrain and both can be activated by 5-HT. Calling in Xenopus is regulated by social context, particularly the acoustic cues provided by another frog’s song. Females rap when receptive and in response to male advertisement calling. While ticking is normally evoked by male clasping, after several bouts some females produce ticking in response to advertisement calling alone. Males respond to ticking with vocal suppression and to rapping with enhanced calling (Tobias et al., 1998b). Though normally associated with clasping, advertisement calling by a dominant male can suppress calling in a subordinate even without physical contact (Tobias et al., 2004). How does auditory information gain access to the hindbrain neural circuitry that generates different vocal patterns? Different call types are identified by their characteristic click rates (Vignal and Kelley, 2007). Within the central auditory pathway, click-rate sensitivity emerges in the midbrain, specifically in the laminar nucleus of the torus semicircularis (LTOR, Elliott and Kelley, 2007; Elliot, 2007). Nucleus LTOR projects to the central nucleus of the thalamus (Edwards and Kelley, 2001) which in turn projects to the VST (Kim and Kelley, unpublished). The VST is the major source of input to DTAM (Brahic and Kelley, 2003), expresses very high levels of GT receptors (Morrell et al., 1975; Yang et al., 2007) and also ER (Morrell et al., 1975). The VST also, like all components of the neural circuitry for vocalization, is reciprocally connected with the rRpd (Brahic and Kelley, 2003), the endogenous source of serotonergic input to the hindbrain vocal pattern generator (Rhodes et al., 2007). Thus either through direct projections to DTAM and/or through its connections with the rRpd, the VST is a strong candidate for the acoustic modulation of vocal signaling so characteristic of social interactions between male and female X. laevis. Whether the neural circuitry that conveys acoustic information functions differently in the sexes and, if so, how sexually differentiated functions are produced, remain to be determined.

20.7 Singing and Breathing 0.5 s

Figure 7 Androgen treatment of adult females results in the ability of serotonin to induce fictive advertisement calling; without treatment 5-HT induces ticking. Modified from Rhodes HJ, Yu HJ, and Yamaguchi A (2007) Xenopus vocalizations are controlled by a sexually differentiated hindbrain central pattern generator. Journal of Neuroscience 27: 1485–1497.

While, unlike terrestrial frogs, vocal production in X. laevis does not require breathing, the neural pathways for vocalization and respiration are still coordinated. The glottis – a component of the respiratory system – opens to allow air to flow into and out of the lungs. Contraction of glottal muscles is controlled by

Hormones and the Regulation of Vocal Patterns in Amphibians

a specific pool of motoneurons located in anterolateral n. IX–X, segregated from the more posterior laryngeal motoneurons in the same nucleus. To examine the interplay of activity in these motor pools, Zornik and Kelley (2008) developed an isolated vocal system, an isolated Xenopus brain with an intact nerve n. IX–X connection to the isolated larynx. In this preparation, the intracellular activities of laryngeal and glottal motoneurons, activities of axons in the laryngeal nerve, and the EMGs produced by laryngeal and glottal muscles can be recorded simultaneously, precisely correlating the activity patterns of all three components. Spontaneous, high-frequency bursts that correlate with activity in glottal motoneurons and muscles can be recorded from the laryngeal nerve. These bursts resemble activity recorded from the laryngeal nerve when a frog rises to the surface and the nares are exposed to air (Rhodes et al., 2007). Glottal bursts recorded from the nerve do not overlap with activity produced by laryngeal motoneurons in vivo (Yamaguchi and Kelly, 2008) or in vitro (Zornik and Kelley, 2008), suggesting the presence of neural circuitry that insures mutually exclusive activity. How are glottal bursts controlled? The same nucleus of the rostral hindbrain, DTAM, that drives the activity of laryngeal motoneurons also affects glottal bursting. When DTAM is stimulated, laryngeal motoneurons fire. After they cease firing, glottal bursting is recorded from the nerve. DTAM projections both excite and inhibit glottal motoneuron activity. Intracellular recording of glottal motoneurons during DTAM stimulation reveals short-latency EPSPs truncated by IPSPs; DTAM stimulation in the middle of a glottal burst results in a brief pause in activity. Inhibition of glottal activity can be blocked by bicuculline indicating that transmission is GABAergic (Zornik and Kelley, 2008). Thus, inhibitory input from DTAM blocks glottal activity during calling and insures that breathing and singing do not occur simultaneously (a disadvantage for underwater song).

20.8 Generating Patterned Vocal Output 20.8.1

Xenopus laevis

Laryngeal muscles must contract simultaneously to produce a click. Laryngeal motoneuron axons do not cross the midline so the job of coordination must fall to the interneurons in the hindbrain vocal circuit. There are three major sources of synaptic input to

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laryngeal motoneurons: neurons in DTAM (both ipsilateral and contralateral), neurons in anteromedial n. IX–X, and neurons in the rRpd (Figure 5). All of these connections are reciprocal (Brahic and Kelley, 2003; Zornik and Kelley, 2007); DTAM also projects to the contralaterally projecting interneurons in anteromedial n. IX–X. When rostral hindbrain is transected in the sagittal plane (disrupting DTAM/DTAM and contralateral DTAM/n. IX–X projections), while patterned activity on the laryngeal nerve is still present, the amplitude of the CAPs is severely diminished (Rhodes et al., 2007). This result suggests that while these projections support robust activity of laryngeal motoneurons, they do not per se generate the vocal pattern. Sexually differentiated passive and active membrane properties allow male and female motoneurons to follow patterned input and produce firing patterns that support different click rates and intensity modulation. Androgen treatment masculinizes adult female motoneurons to produce male-like rapid clicks by changing the size of the somata and their firing properties (Potter et al., 2005), further supporting an important functional role of biophysical properties in translating synaptic inputs into CAPs with precise timing. How are vocal patterns generated? Motoneurons in n. IX–X do not show patterned output in response to step depolarizations used to induce spikes in in vitro whole cell recordings (Yamaguchi et al., 2003); the pattern must originate upstream of laryngeal motoneurons. A good candidate for the source of patterned inputs is the major afferent to n. IX–X, DTAM, a small nucleus of the rostral hindbrain, just ventral to the cerebellum composed of approximately 300 neurons. DTAM stimulation elicits one-to-one CAPs in the laryngeal nerve with consistent latencies around 7–7.3 ms and very small jitters at varying stimulation rates (20, 40, and 60 Hz). DTAM activity drives laryngeal motoneurons monosynaptically. In laryngeal motoneurons, an EPSP or a single spike is induced by each pulse of DTAM stimulation (Zornik and Kelley, 2008). The production of fictive advertisement calling by exogenous 5-HT application requires an intact connection between DTAM and n. IX–X (Rhodes et al., 2007). When DTAM was electrically stimulated at 20–40Hz, two out of eight brains produced male-like fictive advertisement calling. The pattern of fast and slow trill CAPs is similar to that observed in 5-HTinduced fictive calling. A transection immediately posterior to DTAM blocks fictive advertisement calling in response to 5-HT treatment. In contrast,

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Hormones and the Regulation of Vocal Patterns in Amphibians

DTAM removal does not influence glottal bursting. When the forebrain was removed, the fast trill portion of the fictive advertisement call was significantly elongated; elongation of the fast trill is a hallmark of the answer call (Figure 1) and may indicate a role for the forebrain nucleus VST (and its gonadotropin receptors) in vocal responsiveness. The fast and slow trill portions of the advertisement call may have separate generators. Transections ventral to DTAM that cut commisural fibers selectively abolish the fast trill portion of fictive advertisement calling, without influencing the slow trill portion or release calls. This result suggests that bilateral coordination between DTAM is required not only for robust calling but also for fast trill production. 20.8.2 Vocal Pattern Generation in Terrestrial Amphibians In the terrestrial frog, Rana pipiens, pulsed sounds are produced during expiration by contraction of glottal muscles (Schmidt, 1972). When bilateral connections at the level of the posterior hindbrain (n. IX–X) were severed (Schmidt, 1992), the synchrony of inspiratory neural correlates was lost, while when the midline transactions were done at the level of DTAM, bilateral coordination of fictive vocalizations was reduced (Schmidt, 1992). These results suggest that both DTAM and n. IX–X are important for the coordination of bilateral motor output for vocalization in Rana. Using isolated brainstem preparations, Schmidt recorded fictive advertisement calls and associated neural activity. He also recorded from DTAM and n. IX–X after stimulating the ventral forebrain. Forebrain stimulation induced the production of advertisement calls in male frogs. DTAM stimulation also resulted in correlated activity in n. IX–X and fictive advertisement calling. Vocalization in terrestrial species requires both pulmonary and buccal respiration, correlates of which can be recorded simultaneously from the laryngeal nerve while a Rana male is calling. Based on stimulation and lesion studies, Schmidt proposed a model of two independent generators for calling: DTAM and a pulmonary respiration generator (PPG). The PPG is coextensive with n. IX–X, and comprised of an expiratory generator in the anterior, and an inspiratory generator in the posterior, half. Lesions of the posterior extent of n. IX–X remove only pulmonary–respiratory correlates leaving vocal correlates intact. Vocal correlates in anterior n. IX–X are observed by stimulating ventral forebrain and DTAM. Without DTAM, the ventral forebrain still

generates complex slow waves in n. IX–X, while an intact connection with the PPG is required for vocal production. The PPG is spontaneously active, rhythmic, and slow (1 pulse per 2 s); DTAM is not rhythmically active. For the DTAM-induced vocal correlates, reciprocal bilateral connections between the PPGs are important for inhibition. All the vocal patterns that stem from this generator are shaped differently by sensory and endocrine inputs. Schmidt’s model fits well with the notion that vocalization is a specialized derivative of breathing activity. 20.8.3 The Evolution of Hindbrain Vocal Pattern Generators Bass and Baker (1997) proposed that the generation of rhythmic activity within the hindbrain can be attributed to the emergence of specialized interneurons. These interneurons are located in the caudal hindbrain, are derived from rhombomeres 7 and 8, and are rhythmically active. They are postulated to drive a variety of circuits, including those involved in eye movements, electric organ discharges, breathing, and vocalization. In Rana, the PPG interneurons are in the right location (caudal hindbrain) and have rhythmic activity. In rodents, cells in the pre-Botzinger complex, which is at the rostral tip of nucleus ambiguus, are spontaneously active and drive respiration (Feldman, 1995; Smith et al., 2000). These cells project to pre-motoneurons, which are coextensive with nucleus ambiguus and translate rhythmic inputs to various coordinated activities during inspiration and expiration. Subgroups of interneurons in n. IX–X of frogs may be the anatomical analogs of the specialized cells in the mammalian pre-Botzinger complex. Interneurons in the anteromedial portion of n. IX–X that projects to laryngeal motoneurons might have spontaneous oscillatory activity patterns. Because the fast trill portion of advertisement calls is produced by bilateral coordination between DTAMs (Rhodes et al., 2007), different patterns may be generated by pre-motoneurons in different locations. DTAM is homologous to the parabrachial nucleus of mammals (Marin et al., 1997), which plays a role in vocal production and respiration in bats, birds, cats, and squirrel monkey ( Jurgens, 2002; Smotherman et al., 2006). DTAM and the parabrachial nuclei both project to the nucleus ambiguus, which contains laryngeal motoneurons, and both participate in vocal production and respiration. The origins of hindbrain circuitry controlling vocalization thus appear to reside in a more ancient

Hormones and the Regulation of Vocal Patterns in Amphibians

system underlying respiration (Bass and Baker, 1997). Xenopus evolved from terrestrial frogs (Evans et al., 2004) in which vocalization accompanies expiration. As courtship song is present in almost all extant anurans we presume that this ability is ancestral and was modified in Xenopus for function in underwater sound communication. The production of clicks is supported by adaptive modifications of the vocal organ. The suppression of breathing during calling also appears to be supported by an adaptive switch (from excitation to inhibition) in the responsible brainstem circuitry.

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which hormone is involved, in the distribution of receptors, in the excitatory or inhibitory nature of specific synapses and so on, the basic building blocks for sexually differentiated vocal communication are probably very ancient. The conservation of essential functions across species that reflects common descent contributes (Thornton and Kelley, 1998) to the utility of the animal model systems that have proved so useful in our mechanistic understanding of how these systems work.

References 20.9 Evolution and the Sexual Differentiation of Neuroeffectors for Vocalization The overall scheme for the neural control of vocal behaviors used in social communication is remarkably similar for the diverse group of vertebrates examined thus far: sonic fish, aquatic frogs, songbirds, and squirrel monkeys. Each system includes a hindbrain vocal pattern generator consisting of motoneurons, pre-motoneurons, and rhythmically active neurons. This hindbrain circuit can be accessed by the forebrain, either directly or via midbrain nuclei, and vocal forebrain nuclei receive appropriate auditory information that guides the social context of vocal signaling. The state of the pattern generator is strongly influenced by neuromodulators. Though not yet explored in detail in mammalian systems, vocal circuitry is sexually differentiated in sonic fish, Xenopus, and songbirds. Some aspects of sexual differentiation are controlled during development by steroid hormones and some reflect differences associated with reproductive state in adulthood. Given the strong association of vocal signaling with reproduction, and attendant selection pressures, it is possible that the sexual differentiation of vocal circuitry arose very early in evolution, perhaps immediately following upon the emergence of the rhythmic interneurons in rhombomeres 7 and 8, believed to be the precursors for vocal pattern generation, among other functions (Bass and Baker, 1997). The steroid hormones and their receptors are quite ancient, evolutionarily, having emerged before the separation of the tetrapod lineages from the fish (Thornton and Kelley, 1998) and provide a powerful mechanism for coupling new powers of vocal signaling to the reproductive context in which signaling occurs. While there are many species-specific variations in exactly

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Biographical Sketch

Eun-Jin Yang, PhD, received her BA from Chung-Ang University in Seoul, South Korea – from which she holds the Presidential Merit Award – and her PhD from the University of Texas at Austin. Her postdoctoral training was in the Department of Biological Sciences at Columbia University. Dr. Yang is currently postdoctoral fellow in molecular and cellular biology at Harvard University. Her research interests center on the mechanisms of hormone action and social context on the neural substrates for aggressive and courtship behaviors.

Darcy B. Kelley, PhD, received her BA from Barnard College and her PhD from the Rockefeller University where she was also a postdoctoral fellow. Dr. Kelley was a faculty member in the Neuroscience Program at Princeton University before joining the Department of Biological Sciences at Columbia University where she is currently HHMI Professor. Her honors include a Javits award from the NIH (twice); she is editor of developmental neurobiology. Dr. Kelley’s research interests include the sexual differentiation of the neural circuits for courtship song and the mechanisms for matching hearing to utterance.