Fig. 1.
Young RCF female mice show altered Rmand Ihin DA neurons of the iVTAA, Membrane resistance (Rm) of DA neurons in the iVTA from RCF mice (P16-22) is significantly lower compared to Control mice in presence of intracellular K+ (left panel), but not with intracellular Cs+ (right panel). With K+i, CTRL 277 ± 16 MΩ and RCF 217 ± 11 MΩ (both n = 20; Student t-test, t = 3.14, df = 33.04,**p < 0.01); with Cs+i, CTRL 485 ± 38 MΩ and RCF 535 ± 38 MΩ (42 and 30 cells, respectively; t = −0.925, df = 68.2, p = 0.358). B, Lack of difference in membrane capacitance (Cm) across conditions: CTRL 70 ± 4 pF and RCF 76 ± 3 pF (35 and 29 cells, respectively; Student's t -test: t = −1.38, df = 61.9, p = 0.17). C, Lack of difference across conditions in spontaneous AP firing: CTRL 1.0 ± 0.2 Hz and RCF 0.9 ± 0.1 Hz (9 and 18 cells, respectively; t = 0.28, df = 14.18, p = 0.77; inset: example cell-attached recordings). Scale bars: upper 10 pA, 4 s; lower 5 pA, 4 s. D, Current (pA) – to – Voltage (mV) relationship depicting significant reduction of Ih amplitude in VTA neurons from RCF mice (left). For the I–V relationship the two-way repeated measure ANOVA analysis returned: for ‘in vivo treatment’ (i.e. RCF vs CTR) F(1,19) = 8.71, **p = 0.0082; and for ‘membrane potential’ (VH) F(3,17) = 56.5, ***p = 4.73 × 10-9; with the interaction treatment x membrane potential F(3,17) = 3.016, p = 0.0586. The Bonferroni's post-hoc analysis returned: at VH − 60 mV, CTRL −5.8 ± 0.8 pA, RCF −6.2 ± 0.8 pA, p = 1; at VH − 80 mV, CTRL −35 ± 6 pA, RCF −25 ± 3 pA, p = 1; at VH − 100 mV, CTRL – 80 ± 10 pA, RCF – 55 ± 6 pA, *p = 0.037; at VH – 120 mV, CTRL – 109 ± 11 pA, RCF – 71 ± 8 pA, ***p = 1.71 × 10−4 (n = 20 for each condition). To the right, typical Ih currents recorded from CTRL or RCF neurons in response to command voltage steps (four consecutive sweeps superimposed). Scale bars: 100 pA, 0.5 s. E, Lack of difference across conditions for the AMPA/NMDA ratio of evoked EPSCs from iVTA DA neurons (left, bar plots of pooled data; right, typical traces): CTRL 0.30 ± 0.04 and RCF 0.31 ± 0.05 (8 and 9 cells, respectively; p = 0.91). Scale bars: 100 pA, 50 ms. F, Rectification index of synaptic AMPARs is not altered in iVTA DA neurons from RCF mice respect to CTRL, as depicted in bar plots of pooled data (left) and representative traces (right): CTRL 0.94 ± 0.08 and RCF 1.07 ± 0.09 (13 and 7 cells, respectively; p = 0.32). Scale bars: 100 pA, 50 ms. G, Effect of the GluN2B antagonist ifenprodil on evoked NMDAR-mediated EPSCs showing similar sensitivity across conditions. Left, time-course of evoked NMDAR-mediated EPSCs (amplitudes were normalized to the baseline) before and during bath application of ifenprodil (3 μM). Middle, representative NMDAR-EPSC currents before (1) and 25–30 min after (2) ifenprodil application. Scale bars: 100 pA, 50 ms. Right, bar plots of residual NMDAR-EPSCs amplitude (% of baseline) showing similar sensitivity to ifenprodil in CTRL and RCF iVTA DA neurons: CTR 70 ± 5% and RCF 69 ± 8% (8 and 9 cells, respectively; p = 0.88).