5-HT2C-like receptors in the brain of Xenopus laevis initiate sex-typical fictive vocalizations

Abstract

Vocalizations of male and female African clawed frogs (Xenopus laevis) are generated by brain stem central pattern generators. Serotonin (5-HT) is likely important for vocal initiation because, when applied in vitro, sex-typical fictive vocalizations are evoked from isolated brains. To explore the mechanisms underlying vocal initiation, we identified the types of serotonin receptors mediating vocal activation pharmacologically using a whole brain, fictive preparation. The results showed that 5-HT(2C)-like receptors are important for activation of fictive vocalizations in the sexes. 5-HT(2C) receptor agonists elicited fictive vocalizations, and 5-HT(2C) receptor antagonists blocked 5-HT-induced fictive vocalizations, whereas agonists and antagonists of 5-HT(2A) and 5-HT(2B) receptors failed to initiate or block 5-HT-induced fictive vocalizations in the sexes. The results indicate that serotonin initiates fictive vocalizations by binding to 5-HT(2C)-like receptors located either within or upstream of the vocal central pattern generator in both sexes. We conclude that the basic mechanism of vocal initiation is shared by the sexes despite the differences in the actual vocalizations between males and females. Sex-typical vocalizations, therefore, most likely arise from activation of different populations of 5-HT(2C) receptor expressing cells or from differential activation of downstream pattern generating neurons.

FIG. 1.

Variability in vocalizations in vivo and induction of fictive vocalizations in vitro. A: simultaneous sound (bottom trace, sound spectrogram) and nerve (top trace) recordings obtained in vivo from a male Xenopus show that advertisement calls may consist of fast and slow trills (as in the 2nd and 4th bouts) or only fast trills (as in the 1st and 3rd bouts). See Yamaguchi and Kelley (2000) for the recording methods. B: isolated brains of Xenopus in vitro are typically silent (left 1-h trace) until serotonin (30 μΜ) is introduced to the recording chamber (middle 5-min trace). Fictive vocal behavior continues until serotonin is washed out of the recording chamber (right 1-h trace).

FIG. 2.

Quantitative characterization of fictive vocalizations of Xenopus. A: an example frequency histogram (bin size 1 Hz) of instantaneous compound action potential (CAP) rate calculated from 10 bouts of fictive advertisement calls of a male brain. The histogram was fit with bimodal Gaussian curves. Higher and lower peaks (μ₂ and μ₁) are used as estimates of mean fast and slow trill rates. A1: example trace of fictive advertisement call from which the histogram in A was plotted. Slow and fast trills are labeled. B: an example frequency histogram (bin size 1 Hz) of instantaneous CAP rates calculated from fictive ticking obtained from 10 females. A large unimodal peak (μ) is used as an estimate for mean ticking rate. B1: 2 traces each from 2 different females, showing inter- and intraindividual variation in the rate of CAPs.

FIG. 3.

5-HT₂–like receptors are important for the initiation of fictive vocalizations in males and females. A: 5-HT induces fictive advertisement call before (left) and after washout (right) but not during (middle) 5-HT₂ receptor antagonist (LY 53857; 100 μΜ, 10 min) application in a male brain. B: 5-HT induces fictive ticking before (left) and after washout (right) but not during (middle) 5-HT₂ receptor antagonist (altanserin; 100 μΜ, 10 min) application in a female brain. C: fictive advertisement calls were recorded from a male brain only when a 5-HT₂ receptor agonist (α-Me-5-HT; 30 μΜ; 5 min) was applied (left) and not before the treatment (right). D: before treatment with agonist, brains do not produce fictive ticking (left). Application of a 5-HT₂ receptor agonist (α-Me-5-HT; 30 μΜ; 5 min) initiates fictive ticking in a female brain (right).

FIG. 4.

Vocal initiation is not governed by the activation of 5-HT_2A–like and 5-HT_2B–like receptors. A: 5-HT induces male fictive advertisement call before (left) and during (right) 5-HT_2A receptor antagonist (ketanserin, 40 μΜ, 5 min) administration. B: 5-HT induces fictive ticking before (left) and during (right) 5-HT_2A receptor antagonist administration. C: 5-HT_2A/C receptor agonist (DOI, 50 μΜ, 10 min) evokes fictive advertisement call (right) but not in the presence of a 5-HT_2C receptor antagonist (RS102221, 50 μΜ, 15 min; left). D: 5-HT_2A/C receptor agonist (DOI, 50 μΜ, 10 min) evokes fictive ticking (right) but not in the presence of a 5-HT_2C receptor antagonist (RS102221, 50 μΜ, 15 min). E: 5-HT induces male fictive advertisement call before (left) and during (right) the application of 5-HT_2B receptor antagonist (SB 204741, 100 μΜ, 20 min). F: 5-HT induces female fictive ticking before (left) and during (right) the application of 5-HT_2B receptor antagonist (SB 204741, 100 μΜ, 20 min). G: 5-HT_2B receptor agonists (BW 723C86, 100 μΜ, 20 min) do not elicit fictive advertisement call from male brains (left), yet subsequent application of 5-HT to the same brains initiates fictive advertisement calls (right). H: 5-HT_2B receptor agonists do not activate fictive ticking from female brains (left), although subsequent application of 5-HT to the same brains initiates fictive ticking (right). Scale = 1 s for all traces.

FIG. 5.

Blocking 5-HT_2C–like receptors prevents 5-HT–induced fictive vocal behavior in both males and females but does not block respiration. A: 5-HT initiates male fictive advertisement calls before (left) and after the wash out (right) but not during 5-HT_2C receptor antagonist (RS 102221, 50 μΜ, 10 min) administration. B: 5-HT initiates female fictive ticking before (left) and after the wash out (right) but not during (middle) 5-HT_2C receptor antagonist (RS 102221, 50 μΜ, 10 min) administration. C: 5-HT induces male fictive advertisement calls before (left) and after the wash out (right) but not during an alternative 5-HT_2C receptor antagonist (SB242084, 100 μΜ, 10 min) administration. D: 5-HT induces female fictive ticking before (left) and after the wash out (right) but not during (middle) administration of an alternative 5-HT_2C receptor antagonist (SB242084, 100 μΜ, 10 min). E: 5-HT_2C receptor antagonists do not block fictive breathing. An example trace from a male brain showing episodes of fictive breathing before (left trace) and during (right trace) application of a 5-HT_2C receptor antagonist (RS 102221; 50 μΜ, 15 min). The trace is truncated at the time of antagonist application indicated by an arrow. Asterisks indicate episodes of fictive breathing.

FIG. 6.

5-HT_2C receptor agonists initiate fictive vocalizations. A and B: a 5-HT_2C receptor agonist (MK-212, 50 μΜ, 10 min) initiates fictive advertisement calls from male brains and fictive ticking from female brains (right traces), whereas before application of the agonist the brains were silent (left traces). C: an alternative 5-HT_2C receptor agonist, Ro 60–0175 (50 μΜ, 10 min) initiates fictive advertisement calls in male brains (right), whereas before application of the agonist the brains were silent (left). D: the same 5-HT_2C receptor agonist, Ro 60–0175 (50 μΜ, 10 min) fails to initiate fictive ticking in female brains (middle). This was not caused by ill health of the brains because all brains were capable of ticking in response to 5-HT (right). E: the application of 5-HT_2A/C receptor agonist (DOI; 50 μΜ, 10 min) to male brains preincubated with 5-HT_2A receptor antagonist (ketanserin, 40 μΜ, 5 min, n = 3) initiates fictive advertisement calls (right), as in application of DOI alone (middle). Before treatment, all brains were silent (left). F: the application of 5-HT_2A/C receptor agonist (DOI; 50 μΜ, 10 min) to female brains preincubated with 5-HT_2A receptor antagonist (ketanserin, 40 μΜ, 5 min, n = 3) initiates fictive ticking (right), as in application of DOI alone (middle). Before treatment, all brains were silent (left).

FIG. 7.

Vocalizations initiated in the presence of agonists and antagonists are similar to those induced by 5-HT alone. A: box plots of mean instantaneous CAP rate for slow trills (μ₁) in males divided by treatment; H = 12.86, P = 0.07. B: box plots of mean instantaneous CAP rate for fast trills (μ₂) in males by treatment; H = 2.99, P = 0.93. C: box plots of maximum sustained CAP rate for male advertisement call; H = 9.18, P = 0.33. D: box plots of number of bouts of advertisement call across treatment; H = 8.76, P = 0.36. E: box plots of mean instantaneous CAP rate for ticking (μ) in females by treatment; H = 9.27, P = 0.23. F: box plots of maximum sustained CAP rate for female ticking by treatment; H = 10.74, P = 0.15. All comparisons were done using the Kruskal-Wallis test. Lines within boxes indicate median, box bounds are 25th and 75th percentiles, whiskers are 10th and 90th percentiles, and dots indicate data <10th percentile or >90th percentile.