Endogenous serotonin acts on 5-HT2C-like receptors in key vocal areas of the brain stem to initiate vocalizations in Xenopus laevis

Abstract

Serotonin initiates various rhythmic behaviors in vertebrates. Previously we have shown that serotonergic neurons innervate the central vocal pathway in the African clawed frog (Xenopus laevis). We also discovered that exogenous serotonin applied to isolated brains in vitro activates fictive vocalizations by activating 5-HT(2C)-like receptors. In this study, we examined the location of 5-HT(2C)-like receptors and determined whether endogenously released serotonin also initiates vocalizations by activating 5-HT(2C)-like receptors in male Xenopus brains. To this end, we first identified the specific location of 5-HT(2C)-like receptors using immunohistochemistry. We next examined which of the populations of neurons that express 5-HT(2C)-like receptors are functionally relevant for initiating fictive vocalizations by applying a 5-HT(2C) receptor agonist to brains transected at various levels. Of four populations of immunopositive neurons, we showed that 5-HT(2C)-like receptors located in two areas of the brain stem vocal circuit, the raphe nucleus and motor nucleus IX-X, initiate fictive vocalizations. We next showed that endogenous serotonin can also activate fictive vocalizations by increasing the extracellular concentration of endogenous serotonin using a selective serotonin reuptake inhibitor (SSRI). The SSRI-induced vocal initiation is also mediated by activation of 5-HT(2C)-like receptors because blockade of these receptors prevents fictive vocalization. The results suggest that in vivo release of serotonin initiates male vocalizations by activating 5-HT(2C)-like receptors in the brain stem vocal nuclei.

Fig. 1.

Isolated Xenopus brain and the fictive vocal behavior observed in the presence of serotonin. A: simultaneous nerve (top) and sound (bottom, sound spectrogram) recordings of advertisement calls produced by a male Xenopus in vivo, using a recording method described in Yamaguchi and Kelley (2000). A bout of advertisement call is typically made of fast and slow trills, but it can be variable within individual; the 1st and the 3rd bout do not contain slow trills, whereas the 2nd and the 4th trills include slow trills. B: isolated Xenopus brains in vitro are typically silent (left) until serotonin (middle) is administered to the brain. Fictive vocal behavior continues until serotonin is washed out of the bath (right). An example trace obtained from a male brain. C: isolated brain photo (left), dorsal view with suction electrode placed on the left laryngeal nerve. Brain schematic (right) demonstrating reciprocal connections exist between major vocal nuclei (DTAM and n.IX-X) in Xenopus brain stem. The raphe nucleus also sends projections to both DTAM and n.IX-X, the major vocal nuclei.

Fig. 2.

The size of Xenopus 5-HT_2C-like receptor is similar to 5-HT_2C receptor of human and rat. A: BLAST alignment of Xenopus laevis, human, and rat 5-HT_2C receptor. Sequence is the C-terminus of the 5-HT_2C receptor protein. C-termini of X. tropicalis 5-HT_2A and 5-HT_2B sequences are also provided for comparison. Boxed regions indicate the same amino acids. B: Western blot of 5-HT_2C receptor. Whole-brain extracts of Xenopus (middle) and rat (right) demonstrate a band of the appropriate size (∼52 kDa) but not in Xenopus stomach tissue (left). C: actin served as loading control for the presence of protein in all lanes.

Fig. 3.

Immunohistochemistry for 5-HT_2C-like receptors in Xenopus brain. 5-HT_2C-like receptors are located in 4 distinct regions of the Xenopus brain. A–C: line drawings of horizontal sections of a Xenopus brain showing the locations of the receptor populations (left to right is dorsal to ventral; top to bottom is rostral to caudal). D: schematic shows the relative plane of section for schematics drawn in A–C. E, H, and I: Nissl-stained tissue sections representing the location of each 5-HT_2C receptor population. Boxed area corresponds with photomicrographs shown in F, and 2, G, 1 and 2, H, 1 and 2, and I, 1 and 2. N.IX-X, nerve IX-X; iRF, inferior reticular formation; Tel., telencephalon; Dien., diencephalon;.N.V, nerve V. Scale for Nissl stained images = 800 μm. F1: antero-medial portion of n.IX-X (n.IX-Xam). F3: higher magnification of area enclosed in F1. Black arrows point to immunopositive somata, black arrowheads point to immunopositive puncta. G1: rostral reticular formation of spinal cord (sRF). H1: ventral tegmental area (VTeg) of the midbrain. I1: rostral raphe nucleus (rRpd). I3: higher magnification of area enclosed in I1. Black arrows point to immunopositive somata, black arrowheads point to immunopositive puncta. Scale for low- and high-magnification images = 50 μm. F2–I2: adjacent control tissue sections of F1–I1 (respectively) preadsorbed with immunizing peptide lacked 5-HT_2C-like receptor staining. White arrows,e tissue midline; black arrows, lateral edge of tissue. Scale 50 μm.

Fig. 4.

5-HT_2C-like receptors in the brain stem initiate fictive vocalizations in the sexes. A, 1 and 2: progressive transection reveals that agonists to 5-HT_2C receptors can initiate fictive vocal behavior even in the absence of telencephalon and diencephalon. B, 1 and 2: further transections to remove the midbrain and rostral spinal cord reveal that 5-HT_2C-like receptors contained in the brain stem can still initiate fictive vocal behavior in isolated male and female brains.

Fig. 5.

Application of citalopram initiates male fictive advertisement call. A: timeline of experiment. N.IX-X recording indicates the duration of time during which a number of fictive calls were recorded from control and experimental brains. B: example traces from a citalopram-treated brain (left) and a saline-treated brain (right). Scale for both traces is 2 s. C: enlarged view of the 1st bout from the citalopram-treated brain shown in B, left. Scale 1 s. Both fast and slow trills are present in citalopram-induced fictive advertisement call. D: a box plot showing the number of advertisement call bouts generated in the presence (n = 7) and in the absence (saline treatment, n = 5) of citalopram during 1 h posttreatment. Brains treated with citalopram produced a significantly larger number of call bouts compared with brains treated with saline alone.

Fig. 6.

5-HT_2C receptor antagonist blocks citalopram-induced fictive vocalizations. A: timeline of the experiment. N.IX-X recording indicates the duration of the time during which a number of fictive call bouts were counted and compared in control and experimental brains. B: example traces from a citalopram-treated brain with DMSO control (left) and a citalopram-treated brain with 5-HT_2C receptor antagonist (right). Scale for both traces is 2 s. C: enlarged view of the 5th bout from the citalopram plus DMSO-treated brain shown in B, left. Scale is 0.5 s. D: a box plot showing the number of advertisement call bouts generated in the presence of 5-HT_2C receptor antagonist (n = 5), and in the presence of vehicle (DMSO) alone (n = 5). Brains treated with vehicle alone generated significantly more bouts of advertisement calls compared with brains treated with 5-HT_2C receptor antagonist.