Differential composition of 5-hydroxytryptamine3 receptors synthesized in the rat CNS and peripheral nervous system

Abstract

The type 3 serotonin (5-HT3) receptor is the only ligand-gated ion channel receptor for serotonin in vertebrates. Two 5-HT3 receptor subunits have been cloned, subunit A (5-HT3A) and subunit B (5-HT3B). We used in situ hybridization histochemistry and reverse transcriptase-PCR amplification to demonstrate that 5-HT3A subunit transcripts are expressed in central and peripheral neurons. In contrast, 5-HT3B subunit transcripts are restricted to peripheral neurons. Thus, the prevalent form of 5-HT3 receptor synthesized within the CNS lacks the 5-HT3B subunit. Because coexpression of 5-HT3A and 5-HT3B subunits produces heteromeric 5-HT3A/3B receptors with properties that differ from those of 5-HT3A homomeric receptors, we investigated possible coexpression of both subunits at the cellular level. We found that near to 90% of all 5-HT3B expressing neurons coexpress the 5-HT3A subunit in superior cervical and nodose ganglia (NG). In addition, there is a cellular population that expresses only the 5-HT3A subunit. Therefore, peripheral neurons have the capacity to synthesize two different 5-HT3 receptors, 5-HT3A+/3B- and 5-HT3A+/3B+ receptors. We also determined that neurons of NG projecting to the nucleus tractus solitarium and those of dorsal root ganglia projecting to superficial layers of the spinal cord express 5-HT3A or 5-HT3A/3B subunits. Thus, presynaptic 5-HT3 receptors containing the 5-HT3B subunit might be present in these target brain areas. The compartmentalized structural composition of the 5-HT3 receptor may be the basis of functional diversity within this receptor. This raises the possibility that 5-HT3 receptors participating in sympathetic, parasympathetic and sensory functions may be functionally different from those involved in cognition and emotional behavior.

Fig. 1.

Phosphoimages comparing regional expression of mRNA encoding 5-HT_3A and 5-HT_3B subunits in the rat brain. A–D, Widespread expression of 5-HT_3A subunit throughout the rat brain.A′–D′, Lack of detection of 5-HT_3B subunit transcripts in adjacent brain sections. Acb, Nucleus accumbens; ARH, arcuate nucleus of the hypothalamus;CA1, field CA1 of the hippocampus; CA3, field CA3 of the hippocampus; Cg, cingulate cortex;COA, cortical nucleus of the amygdala;CPu, caudate putamen; DEn, dorsal endopiriform nucleus; DG, dentate gyrus;DTT, dorsal tecnia tecta; FS, fundus of the striatum; M, motor cortex; MH, medial habenular nucleus; Pir, piriform nucleus;PrL, prelimbic cortex; S, septum;SS, somatosensory cortex; Tu, olfactory tubercle; VMH, ventromedial hypothalamic nucleus. Scale bar, 1.4 mm.

Fig. 2.

Autoradiograms comparing cellular expression of mRNA encoding 5-HT_3A and 5-HT_3B subunits in sagittal sections of the rat brain. Bright-field (A–F) and dark-field (A′–F′) microscopy are shown. A, A′, High expression of 5-HT_3A mRNA was detected in neurons of the subiculum (S; small arrows) and basket cells (large arrows) in the subgranular layer of the dentate gyrus (large arrows). B, B′, C, C′, Prominent expression of 5-HT_3A mRNA was found in cortical neurons (arrows) distributed primarily in layer II. B, B′, Visual cortex. C, C′, Entorhinal cortex. Lack of expression of 5-HT_3B mRNA in the hippocampal formation (D, D′), visual cortex (E, E′), and entorhinal cortex (F, F′) is shown.CPu, Caudate putamen; DG, dentate gyrus;Ent, entorhinal cortex; Pir, piriform cortex; S, subiculum; SS, somatosensory cortex; VC, visual cortex. Scale bar: bright-field sagittal sections, 750 μm; A–F, A′–F′, 75 μm.

Fig. 3.

Autoradiograms comparing cellular expression of mRNA encoding 5-HT_3A and 5-HT_3B subunits in the spinal cord. Bright-field (A, B) and dark-field (A′, B′) microscopy is shown. A′, Expression of 5-HT_3A subunits in neurons distributed in the dorsal (small arrows) and ventral (large arrows) horn of the spinal cord. B′, Lack of detection of 5-HT_3B subunit transcripts in an adjacent section. Scale bar, 290 μm.

Fig. 4.

Expression of 5-HT_3A and 5-HT_3B subunits in peripheral ganglia. Bright-field (A–D) and dark-field (A′–D′) microscopy are shown. A, A′, B, B′, SCG. C, C′, D, D′, NG. Expression of 5-HT_3A subunit was detected in neurons of the SCG (A′) and NG (C′). Neuronal expression of 5-HT_3B subunit was found in the SCG (B′) and NG (D′). Scale bar, 175 μm.

Fig. 5.

Amplification of 5-HT_3A and 5-HT_3B subunit transcripts from RNA of the CNS and PNS. RT-PCR amplification of 5-HT_3A and 5-HT_3Bsubunit transcripts using RNA isolated from the entire brain, anterior olfactory nucleus (AON), frontal cortex (FC), striatum, hippocampus (Hipp), TG, SCG, NG, and DRG is shown. PCR products of the predicted size (425 bp) for 5-HT_3A transcripts (3A) were obtained from RNA of central and peripheral origin. However, PCR products of predicted size (354 bp) for 5-HT_3B transcripts (3B) were amplified only from peripheral RNA.

Fig. 6.

Frequency histogram showing percentage of neurons expressing either 5-HT_3A or 5-HT_3B subunits in the total neuronal population of TG, SCG, and NG. The data are presented as mean ± SEM; n = number of cells.

Fig. 7.

Coexpression of 5-HT_3A and 5-HT_3B subunits in the NG. Pairs of micrographs of adjacent serial sections show expression of 5-HT_3A (A, A′) and 5-HT_3B (B, B′) subunits. Bright-field (A, B) and dark-field (A′, B′) microscopy is shown. Arrows indicate examples of neurons coexpressing 5-HT_3A and 5-HT_3B subunits. Scale bar, 50 μm.

Fig. 8.

Detection of 5-HT_3A and 5-HT_3B transcripts in fluorogold retrogradely-labeled neurons in the NG. A, B, Fluorogold-labeled neurons.A′, B′, Dark-field microscopy indicating detection of transcripts encoding 5-HT_3A (A′) or 5-HT_3B (B′) subunits. Arrowsindicate examples of retrogradely labeled neurons coexpressing 5-HT_3A and 5-HT_3B transcripts. Scale bar, 60 μm.

Fig. 9.

The compartmentalized structural composition of the 5-HT₃ receptors may be the basis of pharmacological and electrophysiological diversity within this receptor. The 5-HT_3A but not 5-HT_3B subunit mRNA (5-HT_3A+/3B−) is expressed in central neurons [for example, neurons of the dorsal horn (DH) and ventral horn (VH) of the spinal cord and neurons of the hypoglossal nucleus (12)]. However, two major subpopulations of neurons are present in the periphery: one that coexpresses 5-HT_3A and 5-HT_3B subunits [(5-HT_3A+/3B+); see neurons 1, 3, and 4 and 1′, 3′, and 4′] and another expressing only 5-HT_3A subunit [(5-HT_3A+/3B−); see neurons 2 and 2′]. Both types of neurons are present in DRG (A; 1–4) and NG (B; 1′–4′) and might have central and peripheral targets. These results suggest that central nerve endings of peripheral neurons might contain homomeric (5-HT_3A) and heteromeric (5-HT_3A/3B) receptors. Thus, despite the lack of detection of 5-HT_3B subunit mRNA in the CNS, 5-HT_3B subunit protein might be present in central areas innervated by DRG (i.e., superficial layers of the spinal cord) and NG (i.e., the NTS). Central and peripheral compartmentalization of receptors containing or lacking 5-HT_3B subunit might occur in neurons endowed with both subunits (see neurons 1, 1′, 4, and 4′). We cannot discount the possibility that additional as yet unidentified subunits (5-HT_3x) might be present in central and peripheral neurons. AP, Area postrema;gr, gracile fasciculus; 10, dorsal motor nucleus of the vagus.