5-HT3 receptors mediate serotonergic fast synaptic excitation of neocortical vasoactive intestinal peptide/cholecystokinin interneurons

Abstract

Neocortical neurons expressing the serotonin 5-HT3 receptor (5-HT3R) were characterized in rat acute slices by using patch-clamp recordings combined with single-cell RT-PCR and histochemical labeling. The 5-HT3A receptor subunit was expressed selectively in a subset of GABAergic interneurons coexpressing cholecystokinin (CCK) and vasoactive intestinal peptide (VIP). The 5-HT3B subunit was never detected, indicating that 5-HT3Rs expressed by neocortical interneurons did not contain this subunit. In 5-HT3A-expressing VIP/CCK interneurons, serotonin induced fast membrane potential depolarizations by activating an inward current that was blocked by the selective 5-HT3R antagonist tropisetron. Furthermore, we observed close appositions between serotonergic fibers and the dendrites and somata of 5-HT3R-expressing neurons, suggestive of possible synaptic contacts. Indeed, in interneurons exhibiting rapid excitation by serotonin, local electrical stimulations evoked fast EPSCs of large amplitude that were blocked by tropisetron. Finally, 5-HT3R-expressing neurons were also excited by a nicotinic agonist, indicating that serotonergic and cholinergic fast synaptic transmission could converge onto VIP/CCK interneurons. Our results establish a clear correlation between the presence of the 5-HT3A receptor subunit in neocortical VIP/CCK GABAergic interneurons, its functional expression, and its synaptic activation by serotonergic afferent fibers from the brainstem raphe nuclei.

Fig. 1.

5-HT₃R is expressed selectively in a subset of GABAergic interneurons coexpressing VIP and CCK. Single-cell RT-mPCR analyses of 107 neocortical neurons, including 13 pyramidal cells and 94 interneurons, revealed 5-HT_3A expression in five IS and 14 RSNP interneurons. The histogram in Aillustrates the percentage of 5-HT_3A-expressing IS (dark gray) and RSNP (light gray) neurons that expressed each of the biochemical markers. The high occurrence of GAD 65, GAD 67, VIP, and CCK (95, 89, 95, and 100%, respectively) indicated that 5-HT₃R expression was restricted to a subset of neocortical GABAergic interneurons coexpressing VIP and CCK. 5-HT₃-expressing interneurons were also characterized by a low occurrence of PV, SOM, and NPY (5, 16, and 21%, respectively) and the relatively frequent expression of CB and CR (37 and 53%, respectively). B, C, Two histograms illustrating the molecular profiles of IS interneurons and VIP/CCK RSNP cells. The 5-HT_3A mRNA was detected in 42% of IS and 38% of RSNP VIP/CCK interneurons. No significant difference was observed between 5-HT_3A-expressing and 5-HT_3A-nonexpressing IS and RSNP VIP/CCK neurons. The 5-HT_3B subunit was never detected (56 neurons tested).

Fig. 2.

Functional 5-HT₃R expression by VIP/CCK interneurons. A, In current-clamp mode the RSNP and IS neurons were identified by their firing properties after depolarizing current injections (100 pA; holding potential, −71 mV). The RSNP neuron exhibited a 50.9% reduction of firing frequency along the discharge (A1), and the IS neuron showed an initial burst of action potentials followed by irregularly emitted action potentials (A2). B, Local pressure applications of 5-HT (200 μm, 50 msec) induced fast membrane potential depolarizations in these neurons (B1, B2). Note the action potential discharge in B1.C, In voltage clamp (holding potential, −71 mV) the 5-HT applications induced inward currents (C1, −68.5 pA; C2, −67.5 pA) that were blocked by bath application of tropisetron (10 nm). D, Agarose gel analysis of PCR products obtained from these two 5-HT₃-responsive neurons (D1, D2) revealed the expression of the 5-HT_3A subunit together with GAD65 (and GAD67 in D1), VIP, and CCK.

Fig. 3.

5-HT-containing fibers form close appositions with 5-HT₃-responsive neurons. Confocal reconstruction shows two mCPBG-responsive neurons from layers V (A2) and II (B2) labeled by biocytin (green) and immunostained 5-HT-containing fibers (red). Responses to local pressure applications of mCPBG (100 μm, 50 msec), a selective 5-HT₃R agonist, consisted of membrane potential depolarizations surmounted by action potentials (A1, B1). Confocal reconstructions shown inA2 and B2 (scale bars, 20 μm) consisted of a z-series of 48 and 28 images, respectively, projected in one layer via the maximum intensity method (the spacing of successive z-images was 1 μm). mCPBG-responsive neurons exhibited a bipolar morphology, typical of VIP/CCK-expressing interneurons. 5-HT immunostaining showed thin varicose fibers coursing in the vicinity of these neurons. As seen at higher magnification (6×) on unitary z-images, 5-HT-containing varicosities were in close apposition to the soma (A4, A5, B3) or dendrites (A3, A6, A7, B4) of mCPBG-responsive neurons.

Fig. 4.

Serotonergic synaptic transmission in 5-HT₃-responsive cells. A 5-HT-responsive RSNP interneuron located in layer II was recorded in voltage-clamp mode at a holding potential of −78 mV (corresponding to the reversal potential of GABA_A-mediated events). Electrical stimulation (0.2 mA; 0.067 Hz) applied in layer I, ∼100 μm above the soma of this neuron, evoked d-AP-5/CNQX-resistant EPSCs. The example of evoked d-AP-5/CNQX-resistant EPSCs shown inA had an amplitude of −310 pA and a latency to peak of 2.25 msec. For this EPSC the rise time was 0.29 msec (10–90%), and the decay was fit with two exponentials with time constants of 0.65 msec (87%) and 2.63 msec (13%). The same EPSC shown inB at a different time scale was followed by a slowly inactivating outward current (17 pA; arrowhead). Both the EPSC and the outward current were blocked completely by the 5-HT₃R antagonist tropisetron (A, B).C shows the steady amplitude of the evoked EPSCs (mean, −287 ± 13 pA for 10 successive events) that were blocked completely by tropisetron 2.5 min after the beginning of the bath application.

Fig. 5.

5-HT₃R expression is restricted to a subset of DMPP-sensitive interneurons. On 86 recorded neurons we successively applied mCPBG (local pressure application, 100 μm; 50 msec) and the nicotinic agonist DMPP (bath application, 100 μm). All of the mCPBG-responsive neurons (n = 7) also responded to DMPP (A). However, 42 neurons responded only to DMPP (B), and 37 neurons did not show any response (data not shown). DMPP responses were not affected by tropisetron (1 nm; data not shown).