Fig 2: Insulin-dependent regulation of striatal DA release requires ACh from ChIs.(a) ChI filled with biocytin, then immunolabelled for ChAT, and InsR (representative of 4/4 biocytin-filled ChIs); merged image shows co-localization; scale bar, 10 µm. (b–e) Response of striatal ChIs to a series of depolarizing current pulses (3-s duration; 200, 300 and 400 pA; 120-s intervals) before and after insulin (30 nM). (b) Spike frequency adaptation in a ChI (upper) is seen in loss of action potential (AP) discharge during current injection, whereas spiking persists throughout the current pulse in insulin (lower); complete data set shown in d. (c) Representative time course of the insulin-induced increase in AP number with each current step for the ChI in b. (d) Summary of AP number during current pulses delivered before and at the peak effect of insulin exposure (n=21 paired stimulations, 7 neurons, 5 rats) (e) Mean responses showing the effect of insulin (+Ins) under control conditions (Con; n=21 paired stimulations, 7 neurons, ***P<0.001, paired two-tailed t-test), in the presence of HNMPA (5 µM) (n=12 paired stimulations, 4 neurons, 4 rats, P>0.05, paired two-tailed t-test), and in the presence of PPP (1 µM) (n=18 paired stimulations, 6 neurons, 6 rats, **P<0.01, Wilcoxon matched pair signed rank test). (f) Average single-pulse-evoked [DA]o in NAc core before and after insulin (30 nM) in mecamylamine (Mec; 5 µM) or DHßE (1 µM) normalized to 100% peak control (n=20–40 sites per subregion per condition from 3–4 rats, P>0.05 versus control, unpaired t-test). (g) Average single-pulse-evoked [DA]o in forebrain slices from heterozygous control (Het) and ChAT KO mice before and after insulin (30 nM), normalized to 100% peak control. Insulin increased evoked [DA]o in heterozygous mice by 190±23% in NAc shell, 140±8% in NAc core and 137±12% in CPu (n=15–25 sites per subregion from 3–4 mice per genotype, **P<0.01, ***P<0.001 versus control unpaired t-test), but had no effect on evoked [DA]o in any striatal subregion of ChAT KO mice (P>0.1).