5-HT(1A), SST(1), and SST(2) receptors mediate inhibitory postsynaptic potentials in the submucous plexus of the guinea pig ileum

Abstract

Vasoactive intestinal peptide (VIP) immunoreactive neurons are important secretomotor neurons in the submucous plexus. They are the only submucosal neurons to receive inhibitory inputs and exhibit both noradrenergic and nonadrenergic inhibitory synaptic potentials (IPSPs). The former are mediated by alpha(2)-adrenoceptors, but the receptors mediating the latter have not been identified. We used standard intracellular recording, RT-PCR, and confocal microscopy to test whether 5-HT(1A), SST(1), and/or SST(2) receptors mediate nonadrenergic IPSPs in VIP submucosal neurons in guinea pig ileum in vitro. The specific 5-HT(1A) receptor antagonist WAY 100135 (1 microM) reduced the amplitude of IPSPs, an effect that persisted in the presence of the alpha(2)-adrenoceptor antagonist idazoxan (2 microM), suggesting that 5-HT might mediate a component of the IPSPs. Confocal microscopy revealed that there were many 5-HT-immunoreactive varicosities in close contact with VIP neurons. The specific SSTR(2) antagonist CYN 154806 (100 nM) and a specific SSTR(1) antagonist SRA 880 (3 microM) each reduced the amplitude of nonadrenergic IPSPs and hyperpolarizations evoked by somatostatin. In contrast with the other antagonists, CYN 154806 also reduced the durations of nonadrenergic IPSPs. Effects of WAY 100135 and CYN 154806 were additive. RT-PCR revealed gene transcripts for 5-HT(1A), SST(1), and SST(2) receptors in stripped submucous plexus preparations consistent with the pharmacological data. Although the involvement of other neurotransmitters or receptors cannot be excluded, we conclude that 5-HT(1A), SST(1), and SST(2) receptors mediate nonadrenergic IPSPs in the noncholinergic (VIP) secretomotor neurons. This study thus provides the tools to identify functions of enteric neural pathways that inhibit secretomotor reflexes.

Fig. 1.

Effects of WAY 100135 (1 μM) on inhibitory postsynaptic potentials (IPSPs). Examples of 3-pulse (3p; 12 V, 30 Hz; A) and 15-pulse (15p; 30 Hz; B) evoked IPSPs and norepinephrine (NE; 1 mM; C) evoked hyperpolarizations in control (Con) and in the presence of WAY 100135 (WAY). D: quantified data for 3-pulse (n = 11) and 15-pulse (n = 11) IPSPs and NE hyperpolarization (n = 9). Amplitudes of all IPSPs were significantly reduced by WAY 100135 (2-tailed paired t-test, †P < 0.05, #P < 0.01). WAY 100135 had no effect on NE evoked hyperpolarizations.

Fig. 2.

Effects of WAY 100135 (1 μM) on nonadrenergic IPSPs. Examples of 3-pulse (12 V, 30 Hz; A) and 15-pulse (30 Hz; B) evoked IPSPs in idazoxan (Idaz or Ida; 2 μM), and in idazoxan together with WAY 100135 (IdaWAY). C: quantified data for 3-pulse (n = 10) and 15-pulse (n = 13) evoked IPSPs. Amplitudes of all nonadrenergic IPSPs were significantly reduced by WAY 100135 (2-tailed paired t-test; †P < 0.05, *P < 0.001).

Fig. 3.

Effects of 8-hydroxy-2-(di-n-propylamino)tertraline (8-OH-DPAT; 300 nM) and WAY 100135 (1 μM). A: quantified data for the amplitudes of fast excitatory postsynaptic potentials (fEPSPs) in myenteric neurons in control, 8-OH-DPAT (n = 5) or 8-OH-DPAT together with WAY 100135 (n = 5) and after washout. 8-OH-DPAT reversibly reduced the amplitude of fast EPSPs (repeated-measures ANOVA, #P < 0.01). This reduction is blocked by WAY 100135. B: quantified data from submucosal neurons for amplitudes of 3-pulse (n = 8) and 15-pulse (n = 8) evoked IPSPs and NE hyperpolarizations (n = 5) in control, in the presence of 8-OH-DPAT and after washout, as well as somatostatin (Som) hyperpolarizations (n = 5) and fEPSPs (n = 9), in control and in the presence of 8-OH-DPAT. No significant changes were observed. C: example of a hyperpolarization evoked by pressure ejection of 8-OH-DPAT (10 μM) onto a VIP submucosal neuron.

Fig. 4.

Example confocal micrographs of 5-HT immunoreactive varicosities apposing a VIP-IR submucosal neuron. A: recording of a 15-pulse IPSP in a submucosal neuron that was filled with biocytin (B, green fluorescence). C: 5-HT immunoreactivity (red fluorescence). D: green and red immunofluorescence superimposed showing 5-HT-immunoreactive varicosities closely apposed to the soma and individual processes of the biocytin-filled neuron. This image is comprised of multiple focal planes. E: Z-series of the submucosal neuron and surrounding 5-HT terminals (apposed varicosities identified by the absence of any pixel between the terminal and the biocytin staining are indicated by white arrowheads; note several of these contacts are at some distance from the cell body; scale bar represents 20 μm).

Fig. 5.

Effects of CYN 154806 (CYN; 100 nM) on nonadrenergic IPSPs. Examples of 3-pulse (A; 12 V 30 Hz) and 15-pulse (B; 30 Hz) evoked IPSPs and Som (C; 100 μM) evoked hyperpolarizations in idazoxan (2 μM), and in idazoxan together with CYN 154806 (IdaCYN). D: quantified data for the amplitudes of 3-pulse (n = 10) and 15-pulse (n = 10) evoked IPSPs and Som (100 μM) evoked hyperpolarizations (n = 5). Amplitudes of all nonadrenergic IPSPs and Som hyperpolarizations were significantly reduced by CYN 154806. E: quantified data for the durations of 3-pulse (n = 10) and 15-pulse (n = 10) evoked IPSPs. CYN 154806 produced a greater reduction on the durations of all the nonadrenergic IPSPs compared with their amplitude (1-tailed paired t-test, †P < 0.05, #P < 0.01, *P < 0.001).

Fig. 6.

Effects of idazoxan (2 μM) and WAY 100135 (1 μM) together with CYN 154806 (100 nM) on nonadrenergic IPSPs. Examples of 15-pulse (A; 12 V, 30 Hz) evoked IPSPs in idazoxan, idazoxan with WAY 100135, idazoxan, and WAY 100135 together with CYN 154806 (IdaWAYCYN). B: quantified data for the amplitudes of 3-pulse (n = 3) and 15-pulse (n = 4) evoked IPSPs. Amplitudes of the 15-pulse evoked nonadrenergic IPSPs were reduced more in the presence of both WAY 100135 and CYN 154806 than with either antagonist alone (repeated-measures ANOVA, #P < 0.01, *P < 0.001).

Fig. 7.

Effects of SRA 880 (SRA; 3 μM) on nonadrenergic IPSPs. Examples of 3-pulse (12 V, 30 Hz; A) and 15-pulse (30 Hz; B) evoked IPSPs and Som (100 μM; C) evoked hyperpolarizations in idazoxan (2 μM), and in idazoxan together with SRA 880. D: quantified data for the amplitudes of 3-pulse (n = 5) and 15-pulse (n = 6) evoked IPSPs and Som hyperpolarizations (n = 6). Amplitudes of all nonadrenergic IPSPs and Som hyperpolarizations were significantly reduced by SRA 880 (1-tailed paired t-test, †P < 0.05, #P < 0.01).

Fig. 8.

RT-PCR analysis of guinea pig 5-HT_1A, SST₂, and SST₁ receptor gene expression in stripped submucous plexus (SMP) and the myenteric plexus (MP) with its attached longitudinal muscle. The ribosomal 18S shows the equivalent quality of cDNA between samples. Negative control, water; RT−/−, no reverse transcriptase in cDNA synthesis reaction. Marker = 100 bp DNA ladder.