Serotonin 5-HT(3) receptors in rat CA1 hippocampal interneurons: functional and molecular characterization

Abstract

The molecular makeup of the serotonin 5-HT(3) receptor (5-HT(3)R) channel was investigated in rat hippocampal CA1 interneurons in slices using single-cell RT-PCR and patch-clamp recording techniques. We tested for the expression of the 5-HT(3A) (both short and long splice variants) and 5-HT(3B) subunits, as well as the expression of the alpha4 subunit of the neuronal nicotinic ACh receptors (nAChRs), the latter of which has been shown to co-assemble with the 5-HT(3A) subunit in heterologous expression systems. Both the 5-HT(3A)-short and alpha4-nAChR subunits were expressed in these interneurons, but we could not detect any expression of either the 5-HT(3B) or the 5-HT(3A)-long subunits. Furthermore, there was a strong tendency for the 5-HT(3A)-short and alpha4-nAChR subunits to be co-expressed in individual interneurons. To assess whether there was any functional evidence for co-assembly between the 5-HT(3A)-short and alpha4-nAChR subunits, we used the sulphydryl agent 2-aminoethyl methanethiosulphonate (MTSEA), which has previously been shown to modulate expressed 5-HT(3)Rs that contain the alpha4-nAChR subunit. In half of the interneurons examined, MTSEA significantly enhanced the amplitude of the 5-HT(3)R-mediated responses, which is consistent with the notion that the alpha4-nAChR subunit co-assembles with the 5-HT(3A) subunit to form a native heteromeric 5-HT(3)R channel in rat CA1 hippocampal interneurons in vivo. In addition, the single-channel properties of the 5-HT(3)R were investigated in outside-out patches. No resolvable single-channel currents were observed. Using non-stationary fluctuation analysis, we obtained an estimate of the single-channel conductance of 4 pS, which is well below that expected for channels containing both the 5-HT(3A) and 5-HT(3B) subunits.

Figure 1. 5-HT₃ receptor (5-HT₃R)-mediated currents and single-cell RT-PCR analysis in rat CA1 hippocampal interneurons

A, the pressure application of 5-HT (100 μm for 500 ms; see Methods) induced an inward current response (at a holding potential of -60 mV) that was blocked by the bath application of the specific 5-HT₃R antagonist, MDL-72222 (100 nm). B, example agarose gels of the single-cell RT-PCR products. The lanes marked α4 and 5-HT_3A are from the same individual hippocampal interneuron (located at the strata radiatum/lacunosum border) and indicate the co-expression of the α4 subunit of the neuronal nicotinic ACh receptor (α4-nAChR) and 5-HT_3A subunit mRNA from this cell. Marker lanes (M) show a 100 base pairs (bp) ladder, and the expected band sizes for the α4 (356 bp) and 5-HT_3A (205 bp) subunits are indicated with arrows. The fainter band apparent on the α4 gel at 205 bp is most likely a carryover PCR product from the 5-HT_3A primers that was also present in the initial 25 cycles of multiplex PCR. The middle lanes (two on the left gel for the α4-nAChR subunit mRNA and one on the right gel for 5-HT_3A subunit mRNA) are from different interneurons where the PCR product was not detected. C, the proportion of individual cells where we detected each particular mRNA examined. The experimentally determined detection limits are reported in Methods. Results were obtained from 50 individual interneurons.

Figure 2. Correlation between the amplitude of 5-HT₃R-mediated currents and the detection of 5-HT_3A subunit mRNA

A, two superimposed recordings from interneurons, one with a large 5-HT-induced response, and the other with a barely detectable response, in response to the application of 5-HT (100 μm for 100 ms). B, the gel shows four lanes plus the marker. The two outside lanes (1 and 4) correspond to the cells recorded from (marked by stars), and the expected band size for the 5-HT_3A subunit is shown by the arrows (205 bp). Marker lanes (M) show a 100 bp ladder. Lane 2 is another interneuron (which was negative for the 5-HT_3A subunit) and lane 3 is a negative control for the 5-HT_3A subunit. All three neurons were positive for β-actin, and the control was negative.

Figure 3. 2-Aminoethyl methanethiosulphonate (MTSEA) potentiates 5-HT₃R-mediated responses in Xenopus oocytes only when co-expressed with the α4-nAChR subunit

The bath application of MTSEA (1 mm for 4 min; see Methods) had no effect on 5-HT-gated currents in oocytes expressing homomeric 5-HT_3ARs (A), but it potentiated currents (by 15 % for this particular oocyte) in oocytes where the α4-nAChR subunit was co-expressed with the 5-HT_3AR subunit (B).

Figure 4. MTSEA potentiates 5-HT₃R-mediated responses in rat hippocampal interneurons

The bath application of MTSEA (1 mm for 4 min) potentiated the amplitude of the 5-HT-gated current, by 97 % for this particular neuron. The cell was held at a potential of -70 mV, and the concentration of 5-HT was 100 μm.

Figure 5. Non-stationary fluctuation analysis of 5-HT₃R-mediated ion currents

A, six superimposed 5-HT₃R-mediated ion currents (grey traces) and the mean of those currents (black trace), recorded from an outside-out patch. B, plot of the variance against the mean of the current. The continuous line represents the fit according to σ² = iI - I²/N (see Methods). γ, single channel conductance.

Figure 6. Heterogeneous spiking properties of 5-HT₃R-expressing interneurons

Examples of responses from a regular-spiking (A) and fast-spiking (B) interneuron. Both interneurons were located in the stratum radiatum and responded to 5-HT application with 5-HT₃R-mediated inward current responses. Action potentials were evoked by current injection (+150 pA and -100 pA for the upper and lower traces, respectively), and the resting membrane potentials were -71 mV (A) and -68 mV (B). The averaged action potential firing rate was 26 ± 5 Hz for regular-spiking interneurons (n = 8) and 58 ± 11 Hz for fast-spiking interneurons (n = 3).