Title: Sex differences in the structure, function and regulation of vocal circuits in Xenopus
Description: Vertebrate motor behaviors vary widely both in form and complexity, and so do the brains that generate them. Despite this variability, there is a high degree of conservation across vertebrate taxa in the organization of the neural circuits which control the patterning and expression of motor behavior, which are usually distributed across multiple regions within the nervous system. Attempts to understand the principles of how nervous systems generate motor outputs are aided by taking a broad perspective, comparing how neural circuits at different levels of the brain interact and cooperate to produce behaviors with differing levels of complexity. We investigated the question of how variable motor patterns of a single class of behaviors are generated and expressed by motor control circuits in a relatively simple vertebrate model system, the vocalizations of the African clawed frog, Xenopus laevis. Female and male Xenopus make temporally stereotyped sex-specific calls using a single pair of muscles.

Calls vary in complexity, and female calls are considerably simpler than those of males in terms of temporal structure, but both sexes switch between different components of a sex- specific vocal repertoire in response to external stimuli and internal states. Sex differences in vocal behavior are regulated by gonadal hormones, and both the patterning and the expression of sex-specific call types can be modified by manipulating hormone levels in adulthood. We took advantage of this flexible control of otherwise stereotyped motor behavior to analyze how motor circuits pattern and express different vocal behaviors in Xenopus using an ex vivo isolated brain preparation. Fictive calling episodes closely matching the temporal structure of in vivo calls are readily induced ex vivo in both male and female Xenopus by bath application of serotonin (5- HT). We used 5-HT-elicited fictive calling episodes to probe how the activity of vocal circuits varies between male and female calling patterns, and investigated the mechanisms that generate these differences using female brains whose vocal circuits had been masculinized by treatment with exogenous androgen.

We show that vocal patterning circuits can be masculinized even where there is no expression of vocal behavior in vivo, that sex differences in vocal patterns are expressed at multiple levels of the vocal pattern generating circuit, and that individual characteristics that vary as a function of sex differ in their sensitivity to masculinization by exogenous androgen. Masculinization of ex vivo vocal patterns without masculinization of vocal behavior in vivo suggests that the circuits governing patterning are distinct from those governing action selection in this system. Using a combination of tracing and microstimulation techniques in the isolated brain, we outline a putative top down control circuit for vocal control in Xenopus. This circuit is centered on the anuran central amygdala nucleus (CeA), located in the ventral subpallium of the telencephalon. We show that this forebrain nucleus receives auditory input from a thalamic sensory nucleus, and projects directly and indirectly to vocal pattern generating circuits in the hindbrain.

Electrically stimulating CeA in the ex vivo preparation induces fictive calling episodes in the absence of exogenous 5-HT. Electrical stimulation is equally effective in a neighboring subpallial nucleus, the bed nucleus of the stria terminalis (BNST). BNST and CeA share several common targets within the diencephalon and isthmo-mesencephalic tegmentum, however BNST does not project directly to hindbrain vocal pattern generating nuclei. The fictive calling generated by these two subpallial nuclei is indistinguishable, indicating that the ability of microstimulation to drive activity in hindbrain vocal circuits is mediated through indirect connections. In female (but not male) brains, the temporal characteristics of fictive calling induced by microstimulation differ from those induced by 5-HT application, occurring at faster repetition rates that resemble the call made by receptive female Xenopus in response to auditory stimulation. We propose that fictive calling induced by stimulation of the telencephalon represents an ex vivo correlate of this behavioral pattern.

The reproductive state of the female frog at the time of brain isolation does not alter the response of the ex vivo preparation to either 5-HT or microstimulation. We hypothesize that the effects of behavioral state on neural circuits are mediated by sensory nuclei upstream of forebrain motor control circuits. Considered as a whole, the work presented in this thesis shows that variations in motor behaviors are expressed through multiple levels of motor control circuits throughout the central nervous system. These results emphasize the benefits of studying motor behaviors with a view to diversity and variation, both when considering the behaviors themselves and when analyzing the circuits that pattern and govern them.