Endogenous Acetylcholine Release Enhances Granule Cell Excitability by Lowering the Action Potential Threshold
(Ai) Hippocampal slice preparation schematic and example image of granule cell. Scale bar, 50 μm.
(Aii) Representative slow excitatory synaptic potentials before and after atropine (3 μM). The first burst is shown on an expanded timescale.
(Bi and Ci) Typical traces obtained in response to 400 ms current steps before (control), immediately after stimulation (stim), and 25 min post-stimulation (25 min PS) in the absence and presence of atropine, respectively. The scale applies to all traces.
(Bii and Cii) Mean action potential numbers (AP. No.) before and after cholinergic afferent stimulation with and without atropine.
(Biii and Ciii) Typical action potentials and phase plane plots before, immediately after, or 25 min post-stimulation when atropine was absent or present.
(Biv and Civ) Individual (open square) and mean (filled squares) spike threshold before and after stimulation without and with atropine.
(Bv) The spike threshold change time course after stimulation in a subset of control neurons.
(Bvi) Representative 50 Hz glutamatergic EPSP trains and alpha EPSPs before and after cholinergic stimulation. Also shown are the average spike numbers produced prior to and post cholinergic stimulation.
(Bvii) Example records and average frequency of spontaneous action potentials before and after HF cholinergic stimulation is shown on the right. In all graphs, the numbers of observations are indicated in parenthesis and asterisks denote significant (p < 0.05) differences.

The Muscarinic Receptor-Induced Action Potential Threshold Long-Term Plasticity Is Axon Dependent
(Ai, Bi, Ci, Di, and Ei) Representative traces under control conditions, after either global or local application of Oxo-M (1 μM) or stimulation of cholinergic fibers targeting distal granule cell (GC) axons and following 25 min Oxo-M washout or post-stimulation in intact axon (long axon [LA]) neurons or axonless neurons. The RMP is indicated adjacent to the traces. The inset shows the first trace segment on an expanded timescale. The average action potential number (AP No.) under the different conditions is shown on the right. The scales apply to all traces within the panels.
(Aii, Bii, Cii, Dii, and Eii) Typical single action potentials and phase plane plots under control conditions (black), after Oxo-M application or cholinergic fiber stimulation (red) and 25 min following Oxo-M washout or termination of the stimulation paradigm (blue).
(Fi) The mean spike threshold before, during, and 25 min after either Oxo-M treatment or cholinergic afferent stimulation.
(Fii) The threshold change time course during Oxo-M treatment and following washout of Oxo-M.
(Gi) Example electron micrographs showing immunoreactivity for muscarinic M1 receptor subunit. ax, axon; den, dendrites; AIS, axon initial segment. Scale bars, 0.2 μm.
(Gii) Quantification of gold particles labeling M1 receptor subunits in dentate gyrus. The numbers of observations are in parenthesis. n.s., nonsignificant.

Axonal K_V7 Channels Regulate the Action Potential Threshold
(Ai and Ci) Example records from long axon (LA) and axonless granule cells in the absence and presence of XE991 (3 μM). The RMP values are adjacent to the traces. The scale shown applies to both traces in the panel.
(Aii and Cii) Typical spikes and phase plane plots before (black) and after (red) XE991 treatment in LA and axonless neurons.
(B and D) Average spike numbers (AP No.) in response to 400 ms current pulses and mean spike threshold under control conditions and following XE991 treatment in LA and axonless neurons, respectively.
(Ei and Fi) Representative traces and RMP values (adjacent to the traces) when either ABP (8 mM) or scrambled ABP (sABP, 8 mM) were included in the patch pipette for 2 min (control) and 25 min. XE991 was subsequently applied. The scale shown applies to all traces in the panel.
(Eii and Fii) Action potentials at 2 min, 25 min, and after XE991 treatment when ABP and sABP were incorporated in the intracellular solution, respectively.
(G and Gii) Average spike numbers generated with varying 400 ms current pulses 2 min and 25 min after ABP/sABP dialysis and following XE991 treatment.
(Hi) Average spike threshold 2 min, 25 min, and after XE991 with ABP or sABP.
(Hii) The action potential threshold time course with ABP or sABP.

Suppressed K_V7 Currents Underlie Muscarinic Receptor-Mediated Spike Threshold Decrease
(Ai and Aiii) Typical responses from neurons pre-treated with XE991 under control conditions, after HF stimulation or treatment with Oxo-M for 10 min and 25 min following stimulation or washout of Oxo-M. The insets in (Aiii) show the first 1 s of the trace on an expanded timescale. The scales apply to all traces.
(Aii and Aiv) Example single action potentials and phase plane plots before, after treatment (high-frequency stimulation or Oxo-M), and 25 min post-treatment.
(Av and Avi) Average action potential numbers (AP No.) and action potential threshold generated in pre-treated XE991 neurons before, during, and 25 min after high-frequency stimulation or Oxo-M treatment.
(Bi) Raw current traces obtained when the de-activation protocol was applied under control conditions, after 10 min 1 μM Oxo-M, washout, and after XE991 application. The records in the presence of XE991 were subtracted from those in the absence to obtain the XE991-subtracted K_V7/M currents (traces obtained with hyperpolarization to −50 mV in red). The scales apply to all traces.
(Bii) The average XE991-subtracted current at −50 mV prior to, during Oxo-M, and after 5–20 min Oxo-M washout.

Muscarinic Receptor-Induced Spike Threshold Long-Lasting Plasticity Is Calcium Dependent
(Ai and Bi) Example traces showing the effects of diC8-PIP₂ (125 μM) and cadmium chloride (CdCl₂; 200 μM) in the absence and presence of Oxo-M (1 μM). diC8-PIP₂ controls were obtained 2 min after achieving whole cell. The RMP values are indicated adjacent to the traces. The scales apply to all traces.
(Aii and Bii) Typical action potentials and associated phase plane plots when diC8-PIP₂ was included in the patch pipette or CdCl₂ applied.
(Aiii, Aiv, Biii, Biv, Bv, and Bvi) The average action potential number (AP No.) and mean spike threshold values before, during, and after Oxo-M treatment when diC8-PIP₂ or BAPTA (20 mM) was included in the patch pipette or CdCl₂ applied.

Enhanced T-Type Ca²⁺ Channel Activity Is Required for Muscarinic Receptor-Mediated Persistent Spike Threshold Reduction and K_V7/M Current Suppression
(Ai and Avi) Example traces under control conditions, in the presence of the TTA-P2 (500 nM), either co-application of TTA-P2 and Oxo-M (1 μM) or following HF stimulation in the presence of TTA-P2 and 25 min post-treatment. The RMP values are next to the traces. The insets show 1 s of each trace on an expanded scale. The scales apply to all traces.
(Aii, Aiii, Aiv, Av, and Avii) The action potentials numbers (AP No.) under control conditions, in the presence of a voltage-gated Ca²⁺ channel inhibitors (VGCCI), in the presence of the VCCI either following 10 min Oxo-M treatment or HF stimulation and 25 min post-treatment.
(Bi) Representative single spikes and phase plane plots before and after Oxo-M treatment when TTA-P2 was present.
(Bii) The average spike threshold under control conditions, in the presence of the VCCI and pre- and post-treatment with the VCCI.
(Ci) Example perforated-patch voltage-clamp traces obtained when the de-activation protocol was applied under control conditions, in the presence of TTA-P2, following a 10 min co-application of TTA-P2 and Oxo-M and subsequent application of XE991 (3 μM).
(Cii) The traces obtained in the presence of XE991 were subtracted from all other traces to obtain the K_V7/M currents.
(Ciii) The average K_V7/M current (I_M) amplitude obtained by hyperpolarizing to −50 mV from −20 mV.
(Civ) The apparent activation curve for the K_V7/M currents under control conditions, in the presence of TTA-P2 and during co-application of TTA-P2 and Oxo-M.

Muscarinic Receptor Activation Induces Sustained Ca²⁺ Entry via Axonal T-Type Ca²⁺ Channels
(Ai) Electron micrographs showing immunogold particles (as indicated by arrows) for Ca_V3.2 subunits. Scale bar, 0.2 μm.
(Aii) Immunogold particle quantification in dentate gyrus.
(Bi) A two-photon image of a granule cell filled with Alexa Fluor 594 for 30 min. Cross-section line scans were obtained approximately 25 μm from the cell body as shown by the yellow bar.
(Bii and Biii) Average line scan images obtained using Alexa Fluor 594 and Oregon green Bapta-1 (OGB-1) under control conditions, after 10 min Oxo-M (1 μM), and following 40 min washout of Oxo-M in the absence and presence of TTA-P2 (500 nM), respectively.
(Biv and Bv) Individual (gray open squares) and average (black filled squares) ΔG/R measurements before, after Oxo-M treatment, and following Oxo-M washout in the absence and presence of TTA-P2.
(Ci) Superimposed example single action potentials and associated axonal [Ca²⁺]_i signals before, during, and after 25 min washout of Oxo-M.
(Cii) Average ΔG/R measurements for action potential-associated [Ca²⁺]_I signals under control conditions, after 10 min Oxo-M application, and following 25 min washout of Oxo-M.

T-Type Ca²⁺ Channel Antagonists Restore Muscarinic Receptor-Induced Spike Threshold Alterations
(Ai) Typical traces under control conditions, following Oxo-M treatment, and after Oxo-M washout during which TTA-P2 (500 nM) was included. The RMP values are adjacent to the traces. The insets show the traces on an expanded timescale. The scale bars apply to all traces.
(Aii) The average action potential number (AP. No.) under control conditions, in the presence of Oxo-M and with TTA-P2 included in the external solution during the washout of Oxo-M.
(Bi) Example single action potentials and phase plane plots before, during, and after Oxo-M treatment. TTA-P2 was included in the external solution during the washout.
(Bii) Individual (open gray squares) and mean (black filled squares) spike threshold values before, during, and after Oxo-M treatment when TTA-P2 was included in the solution.
(Biii) Time course to show the recovery of the action potential threshold following Oxo-M treatment when TTA-P2 is included in the external solution during washout.