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. 2019 Apr 29;8:e44954. doi: 10.7554/eLife.44954

Figure 1. Delayed increase in intrinsic excitability following muscarinic receptor activation by endogenous acetylcholine.

(A) Diagram of experiment protocol; orange bar indicates timing of light pulses that trigger ACh release via ChR2. (B) Example step responses in control conditions (black trace) and when ACh was released 2 s before and during the step response (orange trace). Blue arrowhead indicates timing of first AP in the control step response that was accelerated when ACh was released (Quantified in G). (C) Blockade of muscarinic cholinergic receptors with 10 μM atropine abolished the increase in excitability triggered by ACh release. Black traces acquired in control conditions; orange traces illustrate responses with coincident light stimulation to the same depolarizing step. (D) Summary of the average number of APs evoked by depolarizing steps in five experiments similar to C. *p=0.0106, T = 4.526; n.s., p=0.847, T = 0.206. paired t-test. (E) Example trace showing that ACh release in the absence of a depolarizing step does not modulate the membrane potential. Inset, in the same cell, pairing ACh release with depolarizing step increases the number of APs triggered. (F) Comparison of mean membrane potential in control conditions (black bar) and after different durations of light pulse trains only (orange bars; p>0.05; paired t-test). Experiments conducted on neurons in which coincident light trains increased the number of APs triggered by depolarizing steps. (G) Summary plot of the average time during similar step responses before an AP was accelerated (shorter latency for the Nth AP when ACh was released, compare B for example traces). (H) Example illustrating different light/step timing protocols. Response shown (orange trace) is from ‘post’ protocol. (I) Coincident ACh release is required to increase neuronal excitability. Plot of the number of APs evoked the same depolarizing step in control conditions (black bar) and when a 6 s light pulse train was applied 7 s before (Pre), 2 s before (Coinc) or 2 s after (Post) the depolarizing step. **p=0.00334, T = 5.8373; paired t-test, Bonferroni corrected. (J) Coincident ACh release and depolarizing step stimuli reveals an afterdepolarization potential (ADP; red arrowhead) that is abolished by 10 μM atropine. Action potentials truncated. (K) Summary plot of the dependence of the ADP on coincident ACh release. Same light train/depolarizing step timing as G. *p=0.0219, T = 3.977; paired t-test, Bonferroni corrected. Plots are mean ± SEM unless noted throughout study.

Figure 1.

Figure 1—figure supplement 1. Control experiments for ChR2 stimulation.

Figure 1—figure supplement 1.

(A) Superimposed traces showing step and step + light (top) and light-only (bottom) from one neuron. (B) By itself, ChR2 stimulation did not affect input resistance. (C) Demonstration rapid reversal of ChR2 enhancement of excitability (trial 3, second control episode). Subsequent ChR2 stim produced a similar enhancement in excitability (trial 4). (D) Increase in excitability and ADP responses were not affected by blockade of ionotropic glutamate and GABA-A/B receptors. (E) Blockade of mAChRs with atropine (10 uM) abolished afterdepolarization normally observed following pairing step + stimulation. *p<0.05.
Figure 1—figure supplement 2. Minimal effect of changing light pulse frequency used to stimulate ChR2.

Figure 1—figure supplement 2.

(A) Plot of number of APs in step only (black) and step + light conditions (orange) from the same neurons as light pulse frequency was varied from 5 to 40 Hz. (B) Plot of increase in number of APs (step plus light - step only) in the six experiments. (C) Plot of ADP amplitude versus light pulse frequencies in the same experiments.
Figure 1—figure supplement 3. Calibration of stereotaxic coordinates used for AAV injections in NB.

Figure 1—figure supplement 3.

(A) Raw low-power brightfield stereomicroscopic image of coronal brain section (P21 rat) with DiI (1 mM) injection site (interaural 7.28 mm; bregma −1.72 mm in Paxinos and Watson (2006). Contrasted-enhanced version of image shown in A with superposition of corresponding landmark annotations. (C) Enlargements of the boxed region in B centered on the NB (circled region in A). Anatomical landmark abbreviations defined to the left of panel C. Calibration bars are 1 mm in each image.