Intrinsic membrane properties of laryngeal motoneurons that control sexually differentiated vocal behavior in African clawed frogs, Xenopus laevis.

Abstract

Males and females behave differently during reproduction. Although sexually differentiated patterns of behavior in vertebrates are clearly regulated by the action of gonadal steroids, the neural mechanisms underlying the expression of sex-specific behavior are largely unknown. Male and female African clawed frogs (Xenopus laevis) produce sexually distinct vocalizations composed of a series of clicks. The fundamental difference between male and female calls is the rate at which the clicks are repeated (reviewed in 1); male calls cover a wide range of click repetition rates (8 to 80 Hz), whereas female calls contain only slow repetition rates (2 to 20 Hz). This behavioral difference can conveniently be reduced to the sexual difference in contraction rate of laryngeal muscle (2), which, in turn, is determined by the sexually distinct firing patterns of laryngeal motoneurons (3). Thus, there is a direct correspondence between the sexually dimorphic patterns of vocalization and the activity of motoneurons. How, then, do male and female laryngeal motoneurons produce sex-specific patterned activity? While the overall pattern is probably produced by a pattern generator upstream of the motoneurons, the motoneurons themselves may have intrinsic membrane properties that differ between male and female Xenopus. Testing this possibility was the goal of this study. Whole-cell patch clamp recordings were used to characterize the membrane properties of laryngeal motoneurons in n.IX-X of adult male and female Xenopus. A thick brain slice preparation of Xenopus hindbrain was developed, and the neurons were visualized by IR/DIC microscopy. To facilitate identification, the motoneurons were retrogradely labeled with fluorescent dye (AlexaFluor 594 biocytin, Molecular Probes, Eugene, OR) before the brain was sliced. Responses to hyperpolarizing and depolarizing current steps (200 ms long) by 6 male and 10 female motoneurons (3 male and 6 female frogs), were recorded in current clamp mode. The resting membrane potential, threshold, spike amplitude, and spike half-width were directly measured from voltage traces. The membrane time constant was determined by fitting single exponential curves to hyperpolarizing voltage responses. Input resistance was calculated from the steady-state membrane potential in response to different hyperpolarizing current pulses. The capacitance of the cell was calculated from input resistance and time constant. The peak firing rate was determined by measuring the interval between the first two action potentials in response to the largest depolarizing current (1.5–2nA) applied to each neuron. Membrane properties of male and female motoneurons are summarized in Table 1. All the properties measured are statistically similar in the two sexes except for the input resistance and the cell capacitance; input resistance is significantly lower, and the cell capacitance is significantly higher in male motoneurons than in female motoneurons. These differences predict that male motoneurons are larger than female motoneurons. A previous study has shown that the dendrites of male n.IX-X neurons are longer than those of female n.IX-X neurons, although the somal size is similar in the two sexes (4). The difference in the dendritic arborization may account for the differences in input resistance and cell capacitance in the two sexes. Functionally, sexual differences in the input resistance imply that the male and female motoneurons exhibit different responsiveness to synaptic input. At depolarized membrane potentials, all the motoneurons showed repeated action potential firing that accommodated over a time course of 100 ms. The peak firing rates, determined by the first two spikes in response to depolarizing currents, were well over 100 Hz in both sexes. To determine whether the neurons could maintain this rapid firing frequency for more than two spikes, four female and two male motoneurons were stimulated with trains of depolarizing pulses at various rates (0.5 ms, 7–10 V, 10 to 300 Hz). Both male and female motoneurons could follow the depolarizing pulses at frequencies of at least 90 Hz. Although the maximum click rate of female vocalizations is 20 Hz, and that of male calls is 80 Hz, the motoneurons of both sexes can fire at a much higher frequency than is required for call production. Taken together, the results suggest that the laryngeal motoneurons of Xenopus do not limit the click repetition rate, and that the motoneurons may be sexually differentiated in their responsiveness to synaptic input.