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.
Highlights
Our results demonstrate that serotonin 2A receptors engage multiple ionic effectors, including the M-current, a calcium-sensitive but calcium-permeable nonspecific cation conductance, and the calcium-dependent nonspecific cation conductance underlying the ADP, to promote action potential generation in COM neurons
Across all COM neurons recorded with our normal whole-cell pipette solution (n = 54), the magnitude of serotonergic excitation, as measured by the peak increase in instantaneous spike frequency (ISF) following 5-HT application, was negatively correlated with baseline firing rate (p = 0.004, R = −0.39, Figure 1B), suggesting a ceiling effect of serotonergic excitation at high baseline firing rates
While response integrals in these BAPTA-filled COM neurons (249 ± 56 Hzs) were not significantly larger than those in control neurons (176 ± 26 Hzs; p = 0.22; Figure 4C), in three additional independent experimental groups we observed significant enhancement of both amplitudes and integrals of 5-HT responses in BAPTA-filled neurons relative to controls. These findings demonstrate that serotonergic excitation, as a whole, is not calcium-dependent, and suggest that the calcium-dependent ADP may not represent the major ionic effector mediating 5-HT responses in COM neurons
Summary
In the prefrontal cortex (PFC), serotonin (5-HT) acts as a neurotransmitter to regulate diverse cognitive processes, including working memory (Williams et al, 2002; Liy-Salmeron and Meneses, 2008), cognitive flexibility (Clarke et al, 2004, 2005), impulsivity (Harrison et al, 1997; Winstanley et al, 2003), attention (Winstanley et al, 2003), and fear (Almada et al, 2015; Marinho et al, 2015; Leon et al, 2017). 5-HT regulates prefrontal circuits by acting on a variety of receptor subtypes differentially expressed in subpopulations of cortical neurons. The ionic mechanisms responsible for 2A-dependent excitation of pyramidal neurons have been less well studied (Araneda and Andrade, 1991; Spain, 1994; Villalobos et al, 2005, 2011), largely due to the fact that the 2A receptor is not universally expressed in adult neurons. Gq-coupled excitation in pyramidal neurons is associated with suppression of potassium conductances, including those mediating the “M” (muscarine-suppressed) current and those responsible for afterhyperpolarizations (AHPs) in pyramidal neurons following bouts of action potential generation (Krnjevic et al, 1971; Schwindt et al, 1988; McCormick and Williamson, 1989; Araneda and Andrade, 1991; Wang and McCormick, 1993; Villalobos et al, 2005, 2011). Our results demonstrate that serotonin 2A receptors engage multiple ionic effectors, including the M-current, a calcium-sensitive but calcium-permeable nonspecific cation conductance, and the calcium-dependent nonspecific cation conductance underlying the ADP, to promote action potential generation in COM neurons
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