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Link to original content: http://pubmed.ncbi.nlm.nih.gov/29422840/
Mechanisms Underlying Serotonergic Excitation of Callosal Projection Neurons in the Mouse Medial Prefrontal Cortex - PubMed Skip to main page content
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. 2018 Jan 18:12:2.
doi: 10.3389/fncir.2018.00002. eCollection 2018.

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

Emily K Stephens  1 , Arielle L Baker  1 , Allan T Gulledge  1
Affiliations

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

Emily K Stephens et al. Front Neural Circuits. .

Erratum in

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.

Keywords: 5-HT2A receptor; Kv7 channels; M-current; afterdepolarization; calcium; cortex; pyramidal neuron; serotonin.

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Figures

FIGURE 1
FIGURE 1
Serotonergic excitation of COM neurons. (A) Voltage traces showing responses to focal application of 5-HT (100 μM, 1 s, ∼15 psi; green bar) during current-evoked action potential generation (above) or at the resting membrane potential of -74 mV (below). Lower set of traces shows the 5-HT responses at a faster timescale. (B) Plot of peak changes in instantaneous spike frequencies following 5-HT application vs. baseline firing rates for all neurons recorded with standard (i.e., non-BAPTA) pipette solution (see also, Tables 1, 2). Regression analysis revealed a negative correlation between baseline firing rate and the magnitude of serotonergic excitation.
FIGURE 2
FIGURE 2
Serotonergic and cholinergic excitation share signaling mechanisms. (A) Voltage traces showing responses to focal application of 5-HT (100 μM, 1 s, ∼15 psi; green bar) during periods of action potential generation in control conditions (left; +66 pA DC current injection) and after bath-application of the cholinergic agonist carbachol (right; no current injection in 50 μM carbachol). Lower traces show 5-HT responses in the two conditions at a faster timescale. (B) Plots of mean (±SEM) instantaneous spike frequencies in baseline conditions (red) and in the presence of 50–100 μM carbachol (black) for 9 neurons. (C) Comparisons of the percent change in firing (left) and response integrals (right) for serotonergic responses in control and carbachol conditions.
FIGURE 3
FIGURE 3
5-HT induces a calcium-dependent afterdepolarization (ADP) in COM neurons. (A) Voltage traces from a COM neuron experiencing a train of ten high-amplitude (3 nA, 2 ms, 25 Hz) current injections in baseline conditions (blue) and following focal application of 5-HT (red). Action potentials are truncated. Black arrows indicate current step applications. (B) Average voltage responses following spike trains paired with 5-HT, resampled to 50 Hz and plotted as mean ± SEM, for 16 neurons in baseline conditions (blue) and after 5-HT (red). Gray symbols show responses following 5-HT application in a different group of COM neurons filled with 10 mM BAPTA (n = 6).
FIGURE 4
FIGURE 4
Serotonergic excitation of COM neurons is not calcium-dependent. (A) Serotonergic responses in COM neurons recorded with standard intracellular solution (red) or with a solution containing 10 mM BAPTA (blue). Lower traces show the responses at a faster timescale. (B) Plots of mean (±SEM) instantaneous spike frequencies in control (red; n = 16) and BAPTA-containing (blue; n = 13) COM neurons. (C) Comparisons of the peak increase in instantaneous spike frequencies (left) and response integrals (right) for COM neurons recorded in control (red) or BAPTA (blue) conditions.
FIGURE 5
FIGURE 5
Serotonergic excitation involves a calcium conductance. (A) Electrically evoked EPSPs (indicated by arrows) in a COM neuron in baseline conditions (red), after replacement of extracellular calcium with magnesium (black), and after return to normal calcium conditions (red). (B) Serotonergic responses in a COM neuron in control conditions (red trace) and after replacement of extracellular calcium with magnesium (black trace). Green bars indicate duration of 5-HT application. (C) Plots of mean instantaneous spike frequencies in baseline (red) and zero-calcium (black) conditions for 9 COM neurons. (D) Comparisons of peak increases in instantaneous spike frequencies (left) and response integrals (right) for COM neurons before (red) and after (black) removal of extracellular calcium. (E) Responses to 5-HT (green bars) in COM neurons recorded with 10 mM BAPTA in the whole-cell pipette, before (blue) and after (black) removal of extracellular calcium. (F) Plots of mean (±SEM) instantaneous spike frequencies for 12 BAPTA-filled COM neuron in baseline (blue) and calcium-free (black) conditions. (G) Comparisons of 5-HT response amplitudes (left) and integrals (right) in BAPTA-filled COM neurons in baseline (blue) and calcium-free (black) conditions.
FIGURE 6
FIGURE 6
Suppression of KV7 channels contributes to serotonergic excitation of COM neurons. (A,C) Responses to 5-HT (green bars) in a control COM neuron (red, A) and a COM neuron patched with 10 mM BAPTA (blue, C) in baseline conditions and after blockade of KV7 channels with 10 μM XE991 (black). (B,D) Plots of mean instantaneous spike frequencies for 10 control (B) and 10 BAPTA-filled (D) COM neurons in baseline (red or blue) and XE991 (black) conditions. (E,F) Comparisons of peak increases in instantaneous spike frequencies (E) and response integrals (F) in baseline conditions (red or blue) and after addition of XE991 (black) for control and BAPTA-filled COM neurons, respectively.
FIGURE 7
FIGURE 7
Serotonergic excitation of COM neurons involves activation of non-selective cation conductances. (A,B) Responses to 5-HT (green bars) in baseline conditions and after reducing extracellular potassium to 0.5 mM (black traces) in control (red, A) or BAPTA-filled (10 mM, blue, B) COM neurons. (C) Plots of mean instantaneous spike frequencies for COM neurons in baseline (red or blue) and low [K+]o (black) conditions for neurons recorded with standard intracellular solution or with BAPTA included, respectively. (D) Comparisons of peak increases in instantaneous spike frequencies (left) and response integrals (right) in baseline conditions (red or blue) and after lowering [K+]o to 0.5 mM (black).
FIGURE 8
FIGURE 8
Ionic effectors contributing to serotonergic excitation of COM neurons. Activation of Gq-coupled 5-HT2A receptors in COM neurons targets a range of ionic effectors, including suppression of M-current, calcium-dependent activation of the ADP current, and activation of a calcium-sensitive, but calcium-permeable, non-specific cation conductance (NSCC).
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