M3 muscarinic acetylcholine receptor expression confers differential cholinergic modulation to neurochemically distinct hippocampal basket cell subtypes

Abstract

Cholinergic neuromodulation of hippocampal circuitry promotes network oscillations and facilitates learning and memory through cellular actions on both excitatory and inhibitory circuits. Despite widespread recognition that neurochemical content discriminates between functionally distinct interneuron populations, there has been no systematic examination of whether neurochemically distinct interneuron classes undergo differential cholinergic neuromodulation in the hippocampus. Using GFP transgenic mice that enable the visualization of perisomatically targeting parvalbumin-positive (PV+) or cholecystokinin-positive (CCK+) basket cells (BCs), we tested the hypothesis that neurochemically distinct interneuron populations are differentially engaged by muscarinic acetylcholine receptor (mAChR) activation. Cholinergic fiber activation revealed that CCK BCs were more sensitive to synaptic release of ACh than PV BCs. In response to depolarizing current steps, mAChR activation of PV BCs and CCK BCs also elicited distinct cholinergic response profiles, differing in mAChR-induced changes in action potential (AP) waveform, firing frequency, and intrinsic excitability. In contrast to PV BCs, CCK BCs exhibited a mAChR-induced afterdepolarization (mADP) that was frequency and activity-dependent. Pharmacological, molecular, and loss-of-function data converged on the presence of M3 mAChRs in distinguishing CCK BCs from PV BCs. Firing frequency of CCK BCs was controlled through M3 mAChRs but PV BC excitability was altered solely through M1 mAChRs. Finally, upon mAChR activation, glutamatergic transmission enhanced cellular excitability preferentially in CCK BCs but not in PV BCs. Our findings demonstrate that cell type-specific cholinergic specializations are present on neurochemically distinct interneuron subtypes in the hippocampus, revealing an organizing principle that cholinergic neuromodulation depends critically on neurochemical identity.

Figure 1.

Electrophysiological and neurochemical differences between PV BCs and CCK BCs. A, A two-dimensional (2D) flat projection from a tiled three-dimensional confocal image stack of streptavidin-Alexa 555 directed against intracellularly labeled biocytin, converted to grayscale. The PV-GFP cell was morphologically identified as a BC, as indicated by localization of the cell body in SP, vertically oriented dendrites, and axonal arborization in SP. B, Voltage responses of the cell in A to 1-s-long, +300, +100, and −100 pA steps. The cell exhibits a “fast spiking” phenotype (blue traces). C, 2D flat projection as in A indicating a morphologically identified CCK BC. The soma was in superficial SR and arborized predominantly in SP. D, Voltage responses of the cell in C to 1-s-long, +300, +100, and −100 pA steps, indicating a strongly accommodating, “regular spiking” phenotype (black traces). Dotted lines in B and D indicate a potential of −60 mV. E, F, Neurochemical profiles of representative subgroups of PV BCs (n = 8) (E) and CCK BCs (n = 17) (F). The representative cells in A and B and in C and D correspond to the representative scPCR bands in E and F, respectively. G, Mean input resistance, as measured from the peak voltage deflection to a 1-s-long, −30 pA current step from −60 mV. H, Relationship between current step and firing frequency for CCK BCs and PV BCs. I, Adaptation coefficient, defined by first ISI divided by the average of the last two ISIs. Blue symbols in G–I denote PV BCs; black symbols denote CCK BCs.

Figure 2.

PV and CCK BCs are sensitive to acetylcholine synaptic release. A, B, Images of 2D flat projections of three-dimensional confocal image stacks of vAChT-immunoreactive terminal labeling (red) in the vicinity of GAD65-GFP (A) or PV+ (B) cells (green). SO, SP, and SR are denoted by o, p, and r, respectively. C, Schematic showing protocol for cholinergic fiber stimulation, 5 pulses at 20 Hz, flanked by a “pre” (black) and “post” (red) 1 s depolarizing test pulse. D, E, Representative traces during prestimulation (black or blue) and post (red)-20 Hz stimulation epochs performed in a CCK BC (D) or PV BC (E). Action potentials have been truncated in D and E for clarity. F, G, Representative traces during prestimulation (black) and post (red)-20 Hz stimulation epochs performed in a CCK BC (F) or PV BC (G) in the presence of 20 μm tacrine. Insets in D–G illustrate expanded regions at the end of the current step to illustrate changes in AP frequency during the poststimulation epoch. H, Summary plot of the frequency ratio (poststimulation/prestimulation) in control, tacrine, and atropine conditions in PV and CCK BCs. I, Summary plot of the ADF before and after the stimulation in PV and CCK BCs. The dotted line at 0 mV indicates the initial baseline voltage before the current step.

Figure 3.

PV BCs and CCK BCs exhibit distinct cholinergic response profiles upon mAChR activation. A, Voltage response from −60 mV in (blue and black) control and (red) after bath application of 10 μm muscarine for PV BCs and CCK BCs, respectively. Bottom, Expanded, overlaid traces showing increased AP frequency and changes in ADF in the presence of muscarine. B, C, AP frequency (B) and adaptation coefficient (C), normalized to the first minute in control conditions. D, E, ADFs (D) in a 100 ms window 200 ms after the current offset and changes in holding current (E) at −60 mV relative to control conditions. In B–E, muscarine was bath applied at time 0 as indicated by the bar in B. CCK BCs (n = 29) and PV BCs (n = 15) are denoted by closed and open symbols, respectively.

Figure 4.

mAChR-induced modulation of AP waveform in PV BCs and CCK BCs. A, B, Representative phase plots of dV/dt vs Voltage of the first 26 consecutive APs for PV BC (blue) (A) and CCK BCs (B) in control conditions (black) and after bath application of 10 μm muscarine (red). Insets illustrate the APs overlaid. C, D, Phase plots for 26 consecutive APs for PV BCs (C) and CCK BCs (D). C, Average AP waveform during the first (i) or last (ii) 100 ms of the 1 s, 300 pA current step in control (blue and black) and 10 μm muscarine (red) conditions for PV BCs (C) and CCK BCs (D). E, AP half-width, binned in 100 ms intervals during the 1 s current step for a population of 6 PV BCs (open symbols) and 15 CCK BCs (closed symbols), respectively, in control (blue and black) and 10 μm muscarine (red) conditions. F, Similar to E, but plotted for dV/dt negative peak.

Figure 5.

AP frequency dependence of the mAChR-induced ADP (1–100 Hz in 1 s) in PV BC and CCK BC subtypes. A, B, Representative traces at 2, 20, and 50 Hz are shown for PV (A) and CCK (B) BCs. C, E, Cumulative integral after the offset of the last action potential for PV (C) and CCK (E) BCs. D, F, Plots of ADF amplitude vs frequency for PV (n = 6) (D) and CCK BCs (n = 4) (F). CCK BCs exhibited a peak ADP at 20 Hz; in contrast, the ADP of PV BCs showed no frequency dependence. Vertical lines denote the area where ADF was measured. Asterisks denote p < 0.05.

Figure 6.

Activity dependence of mAChR-induced ADPs. To examine the AP dependence of the mAChR-induced ADP, we varied the AP number (1–100 APs at 20 Hz) in CCK and PV BC subtypes. A, B, PV and CCK BCs representative traces at 1, 10, and 100 APs are shown, aligned at the offset of the last AP. C, E, Cumulative integral after the offset of the last AP for PV (C) and CCK (E) BCs. D, F, Plots of ADF amplitude vs AP number for PV (D) and CCK (F) BCs. CCK BCs exhibited a peak ADP at 2–20 APs; in contrast, the ADF of PV BCs displayed no AP dependence.

Figure 7.

M3 muscarine receptor distinguishes cholinergic phenotype between PV and CCK BCs. A–C, Representative scRT-PCR gel and mAChR profile for PV BCs (A), CCK BCs (B), and M3 KO CCK BCs (C). Each row indicates a different cell. D, Representative traces for a PV BC from a M1 KO mouse in control conditions (blue) and in the presence of 10 μm muscarine (red). E, Normalized AP frequency plot for WT PV BCs (blue circles) and PV BCs in M1 KO mice (open circles). F, ADF plot for WT PV BCs (blue circles) and PV BCs in M1KO mice (open circles). G, Representative traces for a CCK BC from a M1 KO mouse in control conditions (black) and in the presence of 10 μm muscarine (red). H, I, Normalized AP frequency (H) and ADF plot (I) for WT CCK BCs (black circles), CCK BCs in M1 KO mice (open circles), CCK BCs in M3KO mice (open squares), and CCK BCs in M1/M3 double KO mice (open triangles).

Figure 8.

Glutamatergic excitation of CCK BCs is enhanced by mAChR activation. A, B, Representative trace of a single EPSP (A) or a train of 5 EPSPs (B) evoked onto a PV BC under control conditions (blue) or after bath application of 10 μm muscarine (red). Black vertical lines in A and B indicate onset of synaptic stimulation. C, Expanded region delineated by dashed box in B illustrating the EPSP train. D–F, Representative traces of a single EPSP (D) or a train of 5 EPSPs (E, F) evoked onto a CCK BC under the same conditions. G, ADF population plot for PV BCs (black open circles) and CCK BCs (black filled circles). H, Integral population plot for PV BCs (black open circles) and CCK BCs (black filled circles).