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. 2021 May 24;10:e63708. doi: 10.7554/eLife.63708

Figure 3. DA application or phasic stimulation of midbrain inputs hyperpolarizes a fraction of dm-ITCs.

(A, D) Representative traces of dorsomedial-intercalated cells (dm-ITCs) recorded at resting membrane potential in current clamp mode from young (postnatal day 20–28) and adult (8–10 weeks) mice. Dopamine (DA, 15–30 µM) was applied during the time indicated. Scale bars for (A): 3 mV, 30 s; and for D: 2 mV, 30 s. DA hyperpolarized some cells (responding), but not others (non-responding). (B, E) Distribution of recorded cells according to how DA affects their membrane potential represented as z-scores (x-axis) and their initial membrane potential (y-axis). Responding cells (z-score cut-off at −3) are shown in pink (young) or red (adult), non-responding cells in grey. Insets: fraction of DA-responsive neurons in young mice (n=18 cells from seven animals) and adult mice (n=19 cells from 10 animals). (C, F) Left: Absolute and relative changes in membrane potential in DA-responsive neurons recorded from young (C) and adult mice (F). DA significantly hyperpolarized responsive dm-ITCs in slices from young (n=9 cells from five animals, repeated-measures ANOVA: F(2, 16)=16.23, p<0.001; Bonferroni post-hoc test: Bsl vs. DA **p=0.001, DA vs. Wash **p=0.004) and adult animals (n=11 cells from eight animals, repeated-measures ANOVA: F(2, 20)=7.91, p=0.003; Bonferroni post-hoc test: Bsl vs. DA *p=0.016, DA vs. Wash *p=0.019). DA-induced hyperpolarization amounted to –3.18 ± 0.48 mV and –2.94 ± 0.83 mV in neurons from young and adult mice, respectively. (G) Representative traces of dm-ITCs recorded in current clamp mode from adult mice. Trains of 10 pulses at 30 Hz optogenetic stimulation of DA midbrain afferents were applied during the time indicated. Scale bars: 0.5 mV, 1 s. (H) Distribution of recorded cells according to 30 Hz stimulation of DA afferents affects their membrane potential represented as z-scores (x-axis) and their initial membrane potential (y-axis). Responding cells (z-score cut-off at −3) are shown in blue, non-responding cells in grey. Insets: Fraction of stimulation-responsive neurons (n=16 cells from three animals). (I) Absolute and relative changes in membrane potential in responsive neurons. 30 Hz stimulation significantly hyperpolarized responsive dm-ITCs (n=11 cells from two animals, repeated-measures ANOVA: F(2, 20)=49.01, p<0.001; Bonferroni post-hoc test: Bsl vs. Stim *p<0.001, Stim vs. Recovery *p=0.001). 30 Hz stimulation-induced hyperpolarization amounted to –2.84 ± 0.35 mV.

Figure 3—source data 1. Data Figure 3B, C, E, F and H-I, and Figure 3—figure supplement 1.

Figure 3.

Figure 3—figure supplement 1. 30 Hz stimulation hyperpolarizes dm-ITCs in the presence of a GABAB receptor blocker.

Figure 3—figure supplement 1.

Left: Representative traces of dorsomedial-intercalated cells (dm-ITCs) recorded in current clamp mode from adult mice. Trains of 10 pulses at 30 Hz optogenetic stimulation of dopaminergic (DA) midbrain afferents were applied in the absence (top, Figure 3G) or in the presence of GABAB receptor antagonist CGP55845 (10 µM) with and without light stimulation (bottom) during the time indicated. Scale bars 0.5 mV, 1 s. Right: The relative changes in membrane potential in responsive neurons did not differ between neurons recorded in the absence (–2.77 ± 0.22 mV, n=6 cells from two animals) or presence of CGP55845 (–2.92 ± 0.76 mV, n=5 cells from one animal), unpaired t-test, p=0.836.
Figure 3—figure supplement 2. Distribution of dm-ITCs according to their response to DA and 30 Hz stimulation.

Figure 3—figure supplement 2.

(A, B) Distribution of recorded dorsomedial-intercalated cells (dm-ITCs) at different bregma levels in young (A) and adult mice (B) color-coded according to their response to dopamine (DA) application. (C) Distribution of recorded dm-ITCs at different bregma levels in adult mice color-coded according to their response to 30 Hz afferent stimulation. Bregma levels are indicated at the bottom.