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. 2020 Jul 20;9:e56920. doi: 10.7554/eLife.56920

Figure 1. Changes in ChI spontaneous firing frequency induced by DA depletion followed by chronic L-DOPA treatment.

(A) DA lesion and chronic L-DOPA treatment paradigm. 3–4 weeks after unilateral 6-OHDA lesion, mice were randomly divided into two groups to receive either saline or L-DOPA. Experimental groups included sham: mice with vehicle injection into the MFB, 6-OHDA: mice with MFB lesions injected with daily IP saline, and chronic LD: MFB-lesioned mice treated with 3 mg/kg L-DOPA IP once daily. Electrophysiological slice recordings were carried out 3–11 weeks after the initiation of L-DOPA or saline injections. (B) Representative cell-attached recordings and distributions of average (per cell) instantaneous spontaneous action potential frequency (sAP) of ChIs from sham-lesioned (n = 83 neurons/26 mice), 6-OHDA-lesioned (n = 81 neurons/29 mice), and 6-OHDA-lesioned mice treated with chronic LD (n = 87 neurons/25 mice). Scale bars are 1 s and 50 pA. (C) Dot plots of spontaneous cell activity in the absence (same as in B) and the presence of synaptic blockers CNQX (10 µM), APV (25 µM) and picrotoxin (PCX) (25 µM). Number of recordings with synaptic blockers: sham n = 13 neurons/3 mice, 6-OHDA n = 11 neurons/3 mice, chronic-LD 13 neurons/3 mice. (D) Coefficient of variation (CV) of instantaneous sAP frequencies in sham, 6-OHDA, and chronic LD groups (same N as in B). For C and D, line denotes median, error bars show interquartile range, p<0.05 (*), p<0.01 (**), p<0.001 (***), or p<0.0001 (****) by Kruskal-Wallis test with Dunn’s multiple comparison test; (E) The number of evoked action potentials following current injection was decreased in ChIs from 6-OHDA lesioned mice but restored to sham levels after chronic L-DOPA treatment. p<0.01 (**), 6-OHDA vs. the two other groups by two-way ANOVA with Tukey’s post-hoc test; sham n = 11 neurons/4 mice, 6-OHDA n = 16 neurons/5 mice, chronic-LD n = 18 neurons/5 mice. (F) Averaged perforated-patch recordings of sAP in ChIs following 30 µM DA perfusion in the presence of synaptic blockers. Sham n = 7 neurons/3 mice, 6-OHDA n = 7 neurons/2 mice, chronic-LD n = 10 neurons/3 mice. (G) Changes in average sAP frequencies in individual cells before and after DA exposure. (same N as in F). p<0.01 (**) by paired t-test.

Figure 1—source data 1. Individual neuron data and statistics for all panels and figure supplements.
elife-56920-fig1-data1.xlsx (196.8KB, xlsx)

Figure 1.

Figure 1—figure supplement 1. Expression of L-DOPA-induced dyskinesia (LID) in 6-OHDA-lesioned mice.

Figure 1—figure supplement 1.

(A) All 6-OHDA-lesioned mice developed contralateral front paw stepping deficits, p<0.0001 vs. 50% for both groups, Wilcoxon signed rank test. There was no difference in stepping deficit between the mice assigned to the 6-OHDA-only or chronic L-DOPA groups, p=0.5467 by Mann-Whitney test; 6-OHDA n = 21 mice, chronic-LD n = 30 mice. (B) Time course of LID expression following single injection of L-DOPA (3 mg/kg) either as the first dose or after chronic administration of L-DOPA. First dose n = 11 mice, chronic-LD n = 23 mice; p<0.0001 for time x treatment interaction by two-way ANOVA, (*) p<0.05 between first dose vs. chronic administration at the same time point after L-DOPA injection by Bonferroni post-hoc test. (C) Total LID was not different between the groups, p=0.2946 by Mann-Whitney test. (D) LID expression occurred sooner after L-DOPA injection in mice that received chronic L-DOPA, p=0.0002, Mann-Whitney test.
Figure 1—figure supplement 2. Burst-pause activity in ChIs.

Figure 1—figure supplement 2.

The number and duration of bursts and pauses were quantitated using the robust Gaussian surprise method. (A–F) Firing patterns of ChIs from sham (n = 83 neurons/26 mice), 6-OHDA (n = 81 neurons/29 mice, and chronic-LD (n = 87 neurons/25 mice) groups. (G–L) Firing patterns of ChIs in untreated mouse brain slices (n = 17 neurons/6 mice), those treated with 1 μM ZD7288 and 1 nM apamin (n = 8 neurons/3 mice) or the same concentration of apamin alone (n = 10 neurons/3 mice). p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****) by Kruskal-Wallis test with Dunn’s multiple comparison. Burst duration (C and I) and pause duration (F and L) were analyzed only in neurons exhibiting bursts and pauses: sham n = 66, 75; 6-OHDA n = 52, 64; chronic-LD n = 80, 85; untreated n = 17, 16; ZD+apamin n = 6, 8; apamin n = 10, 10 (bursts, pauses). Blockade of HCN and SK currents largely reproduced changes in firing patterns observed in the 6-OHDA and chronic LD groups. Also note that number of bursts and pauses was not different between any groups when bursts or pauses were normalized to the number of action potentials (B,H,E,K), suggesting that the expression of bursts and pauses is related to the change in the firing rate.
Figure 1—figure supplement 3. Basic electrophysiological characteristics in ChIs from control, 6-OHDA, and chronic LD mice.

Figure 1—figure supplement 3.

(A) Voltage-current dependence, (B) resting membrane potential, (C) input resistance, (D) membrane capacitance and (F) rheobase were unchanged in 6-OHDA and chronic-LD groups. (E) Ramp protocol for rheobase determination. Recordings in A-D were recorded in whole-cell mode in the presence of 1 µM TTX (n = 12–34 neurons/4–9 mice per group). (G) Representative and (H) averaged traces and shape characteristics (I) of sAPs from the three experimental groups (n = 21–27 neurons/8–10 mice per group). (J) Current protocol (lower) and representative trace (upper) of action potentials evoked by 100 pA current injection. (K, L) Shape characteristics of action potentials evoked by 100 pA current injection (n = 19–28 neurons/8–11 mice per group). p<0.05 (*) or p<0.01 (**) by Kruskal-Wallis test with Dunn’s multiple comparison analysis. Exact n for each figure are listed in Figure 1—source data 1.