Mechanisms Underlying Serotonergic Excitation of Callosal Projection Neurons in the Mouse Medial Prefrontal Cortex

Abstract

Serotonin (5-HT) selectively excites subpopulations of pyramidal neurons in the neocortex via activation of 5-HT2A (2A) receptors coupled to Gq subtype G-protein alpha subunits. Gq-mediated excitatory responses have been attributed primarily to suppression of potassium conductances, including those mediated by KV7 potassium channels (i.e., the M-current), or activation of non-specific cation conductances that underlie calcium-dependent afterdepolarizations (ADPs). However, 2A-dependent excitation of cortical neurons has not been extensively studied, and no consensus exists regarding the underlying ionic effector(s) involved. In layer 5 of the mouse medial prefrontal cortex, we tested potential mechanisms of serotonergic excitation in commissural/callosal (COM) projection neurons, a subpopulation of pyramidal neurons that exhibits 2A-dependent excitation in response to 5-HT. In baseline conditions, 5-HT enhanced the rate of action potential generation in COM neurons experiencing suprathreshold somatic current injection. This serotonergic excitation was occluded by activation of muscarinic acetylcholine (ACh) receptors, confirming that 5-HT acts via the same Gq-signaling cascades engaged by ACh. Like ACh, 5-HT promoted the generation of calcium-dependent ADPs following spike trains. However, calcium was not necessary for serotonergic excitation, as responses to 5-HT were enhanced (by >100%), rather than reduced, by chelation of intracellular calcium with 10 mM BAPTA. This suggests intracellular calcium negatively regulates additional ionic conductances gated by 2A receptors. Removal of extracellular calcium had no effect when intracellular calcium signaling was intact, but suppressed 5-HT response amplitudes, by about 50%, when BAPTA was included in patch pipettes. This suggests that 2A excitation involves activation of a non-specific cation conductance that is both calcium-sensitive and calcium-permeable. M-current suppression was found to be a third ionic effector, as blockade of KV7 channels with XE991 (10 μM) reduced serotonergic excitation by ∼50% in control conditions, and by ∼30% with intracellular BAPTA present. Together, these findings demonstrate a role for at least three distinct ionic effectors, including KV7 channels, a calcium-sensitive and calcium-permeable non-specific cation conductance, and the calcium-dependent ADP conductance, in mediating serotonergic excitation of COM neurons.

Preferential cholinergic excitation of corticopontine neurons

“…), but while 5‐HT triggers excitatory responses in COM neurons that are similar in magnitude to those evoked by ACh, and appear to be mediated by identical ionic mechanisms (Stephens et al . ), serotonergic SK‐mediated inhibition is absent (Avesar & Gulledge, ). Similarly, while expression of melanopsin, a light‐activated G q ‐coupled receptor, allows for robust light‐evoked SK‐mediated inhibition and long‐lasting excitation in pyramidal neurons, targeted expression of chimeric melanopsin to cellular subdomains can greatly reduce light‐evoked excitation (McGregor et al .…”

“…Indeed, the localization of G q -coupled receptors appears to be critical for gating various downstream effectors. For instance, mouse COM neurons express G q -coupled 5-HT 2A receptors in addition to mAChRs (Avesar & Gulledge, 2012;Stephens et al 2014), but while 5-HT triggers excitatory responses in COM neurons that are similar in magnitude to those evoked by ACh, and appear to be mediated by identical ionic mechanisms (Stephens et al 2018), serotonergic SK-mediated inhibition is absent (Avesar & Gulledge, 2012). Similarly, while expression of melanopsin, a light-activated G q -coupled receptor, allows for robust light-evoked SK-mediated inhibition and long-lasting excitation in pyramidal neurons, targeted expression of chimeric melanopsin to cellular subdomains can greatly reduce light-evoked excitation (McGregor et al 2016).…”

“…For example, serotonin (5‐HT) inhibits CPn neurons via G i/o ‐coupled 5‐HT1A receptors, while selectively exciting COM neurons via activation of G q ‐coupled 5‐HT 2A receptors (Avesar & Gulledge, ; Stephens et al . , ). On the other hand, CPn neurons are preferentially excited by α2‐adrenergic (Dembrow et al .…”

“…In addition to having distinct morphological and physiological properties (Morishima & Kawaguchi, 2006;Dembrow et al 2010), CPn and COM neurons differ in their responses to neuromodulatory input (Dembrow & Johnston, 2014). For example, serotonin (5-HT) inhibits CPn neurons via G i/o -coupled 5-HT1A receptors, while selectively exciting COM neurons via activation of G q -coupled 5-HT 2A receptors (Avesar & Gulledge, 2012;Stephens et al 2014Stephens et al , 2018. On the other hand, CPn neurons are preferentially excited by α2-adrenergic (Dembrow et al 2010) and dopamine D2 (Gee et al 2012) receptors, while COM neurons are preferentially excited via activation of dopamine D1 receptors (Seong & Carter, 2012).…”

Serotonergic regulation of corticoamygdalar neurons in the mouse prelimbic cortex

“…In COM neurons, serotonergic excitation is mediated by Gq-coupled 2A receptors that engage three postsynaptic ionic effectors to enhance cortical neuron excitability: inhibition of KV7 potassium channels and activation of two nonspecific cation conductances (Stephens et al, 2018). Interestingly, these same ionic mechanisms underlie cholinergic excitation of CPn neurons in the prelimbic cortex , suggesting that they have a conserved role in Gq-mediated excitation across cortical neuron subtypes and neurotransmitter systems.…”