Serotonergic Modulation of Glutamate Release in Layer II of Rat Somatosensory Cortex: Mechanisms and Network Specificity

Abstract

As axons from the raphe nuclei densely innervate the somatosensory cortex, the modulation of transmitter release by serotonin (5-HT) was investigated in pyramidal cells in layer II of rat barrel cortex. The idea was that 5-HT, via presynaptic 5-HT2 receptors (5-HT2R) coupled to Gq proteins, activates Ca2+ release from stores to increase spontaneous transmitter release. Addition of 10 µM 5-HT, in the presence of TTX and gabazine, caused a waxing and waning of the instantaneous mEPSC frequency. Specifically, 5-HT increased the frequency by 28 ± 7% within 5 minutes (phase 1). Later, within 5 – 12 minutes, it dropped to below control (-15 ± 3%; phase 2). Thereafter, it resurged back to 27 ± 7% (phase 3). Concomitantly, the mEPSC amplitude remained unaffected. These changes in spontaneous release were mediated by 5-HT2CR and 5-HT2AR, with the former providing a larger contribution. The downstream signalling was verified by blocking PLCβ, IP3R, and Ca2+ release from stores. The findings were consistent with the activation of a classical Gq cascade. Inhibiting PKC by Gö 6983 rendered the increase sustained, suggesting that a phosphorylation caused the reduction in frequency after reaching an initial peak. These findings were restricted to a subset of cells (47%), which were subsequently termed responders. No change occurred in non-responders. The two groups differed by the size of the reduction in input resistance (Rin) and the change in holding current. For responders, the former was large, but the latter small. In contrast, for non-responders, the former was small, but the latter large. In connected pairs of pyramidal cells in this layer, 5-HT depressed the EPSC amplitude by 49 ± 3% without significantly altering the paired-pulse ratio. This depression occurred downstream of 5-HT2R activation. It was caused by Gβγ, because it was blocked by Gβγ-binding peptides (mSIRK/ct-SNAP-25). As Gβγ most likely inhibited voltage-dependent Ca2+ channels, limited influx caused the EPSC depression. Because 5-HT depressed EPSCs in most pairs, specificity in the connectivity between responders and non-responders must exist. In fact, responders were typically post-, whereas non-responders presynaptic. Consistent with this idea, the mEPSC frequency only increased in post-, but not presynaptic cells. Furthermore, postsynaptic cells showed a large drop in Rin associated with a small outward current. Conversely, presynaptic cells showed a small drop in Rin together with a considerable hyperpolarization. My results revealed that, in contrast to the classical tenet of Katz’ hypothesis of transmitter release, spontaneous transmitter release increased, whereas evoked release depressed downstream of 5-HT2R activation. The two mechanisms dissociated at the Gq protein level. Spontaneous release was increased by Ca2+ release from stores, whilst evoked release was depressed most likely via inhibition of VDCC by Gβγ. Because of their restriction to responders, these mechanisms would differentially affect the neocortical microcircuitry.