Resilience to anhedonia-passive coping induced by early life experience is linked to a long-lasting reduction of Ih current in VTA dopaminergic neurons

Abstract

Exposure to aversive events during sensitive developmental periods can affect the preferential coping strategy adopted by individuals later in life, leading to either stress-related psychiatric disorders, including depression, or to well-adaptation to future adversity and sources of stress, a behavior phenotype termed "resilience". We have previously shown that interfering with the development of mother-pups bond with the Repeated Cross Fostering (RCF) stress protocol can induce resilience to depression-like phenotype in adult C57BL/6J female mice. Here, we used patch-clamp recording in midbrain slice combined with both in vivo and ex vivo pharmacology to test our hypothesis of a link between electrophysiological modifications of dopaminergic neurons in the intermediate Ventral Tegmental Area (VTA) of RCF animals and behavioral resilience. We found reduced hyperpolarization-activated (I_h) cation current amplitude and evoked firing in VTA dopaminergic neurons from both young and adult RCF female mice. In vivo, VTA-specific pharmacological manipulation of the I_h current reverted the pro-resilient phenotype in adult early-stressed mice or mimicked behavioral resilience in adult control animals. This is the first evidence showing how pro-resilience behavior induced by early events is linked to a long-lasting reduction of I_h current and excitability in VTA dopaminergic neurons.

Keywords: Early stress; Ih current; In vivo pharmacology; Resilience; VTA.

Fig. 1

Young RCF female mice show altered R_mand I_hin DA neurons of the iVTAA, Membrane resistance (R_m) 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 (C_m) 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 I_h 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’ (V_H) 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 V_H − 60 mV, CTRL −5.8 ± 0.8 pA, RCF −6.2 ± 0.8 pA, p = 1; at V_H − 80 mV, CTRL −35 ± 6 pA, RCF −25 ± 3 pA, p = 1; at V_H − 100 mV, CTRL – 80 ± 10 pA, RCF – 55 ± 6 pA, *p = 0.037; at V_H – 120 mV, CTRL – 109 ± 11 pA, RCF – 71 ± 8 pA, ***p = 1.71 × 10⁻⁴ (n = 20 for each condition). To the right, typical I_h 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).

Fig. 2

Adult RCF mice show altered R_m, I_hand evoked AP firing in DA neurons of iVTAA, Membrane resistance (R_m) of DA neurons in the iVTA from adult RCF mice (P60) is significantly reduced compared to Controls: CTRL 287 ± 15 MΩ and RCF 237 ± 15 MΩ (n = 28 and 19, respectively; Student's t-test, t = 2.38, df = 43.86, *p < 0.05). B, Similar membrane capacitance across conditions: CTRL 60 ± 3 pF and RCF 53 ± 3 pF (28 and 19 neurons, respectively; t = 1.63, df = 42.16, p = 0.11). C, Similar AP spontaneous firing frequency between Control and RCF mice: CTRL 2.5 ± 0.4 Hz and RCF 2.1 ± 0.3 Hz (13 and 19 cells, respectively; t = 0.62, df = 22.02, p = 0.54; inset: typical cell-attached recordings). Scale bars: upper 30 pA, 1 s; lower 5 pA, 1 s. D, to the left: I–V curves depicting significant reduction of I_h amplitude in VTA DA neurons from RCF mice. In details, the two-way repeated measure ANOVA analysis returned: for ‘in vivo treatment’ (RCF vs CTR) F_(1,15) = 10.49, **p = 0.0055; for membrane potential (V_H), F_(3,13) = 73.49, ***p = 2.08 × 10⁻⁸; and for the interaction treatment x membrane potential F_(3,13) = 3.016, *p = 0.0489. The Bonferroni's post-hoc analysis returned: at V_H – 60 mV, CTRL – 6.8 ± 0.7 pA, RCF – 6 ± 1 pA, p = 1; at V_H – 80 mV, CTRL – 69 ± 9 pA, RCF – 42 ± 5 pA, p = 1; at V_H – 100 mV, CTRL – 168 ± 17 pA, RCF – 104 ± 9 pA, **p = 0.0015; at V_H – 120 mV, CTRL – 233 ± 20 pA, RCF – 165 ± 14 pA, ***p = 4.85 × 10⁻⁴ (CTRL, n = 16; RCF, n = 27). To the right, example I_h current traces (four superimposed consecutive sweeps). Scale bars: 200 pA, 0.5 s. E, frequency – injected current amplitude (f – I) average relationship (left) for evoked APs in adult CTRL or RCF neurons depicting significantly reduced ability to elicit APs in RCF mice. In details: for in vivo treatment F_(1,8) = 6.49, *p = 0.034; interaction treatment x injected current F_(3,6) = 3.96, p = 0.071; for injected current F_(3,6) = 46.51, ***p = 1.5 × 10⁻⁴ (two-way repeated measure ANOVA). The Bonferroni's post-hoc analysis returned: with I_inj +50 pA: CTRL 3 ± 1 Hz, RCF, 1.8 ± 0.6 Hz, p = 1; I_inj +100 pA: CTRL, 8 ± 2 Hz and RCF, 4.6 ± 0.7 Hz, p = 0.77; I_inj +150 pA: CTRL, 11 ± 2 Hz and RCF, 6.6 ± 0.7 Hz, *p < 0.05; I_inj +200 pA, CTRL, 13 ± 2 Hz and RCF, 8.2 ± 0.6 Hz, *p < 0.05. To the right, typical current-clamp recordings showing evoked firing. Scale bars: 30 mV, 1 s.

Fig. 3

Adult RCF mice intra-VTA infused with Lamotrigine rescue Control behavior and show potentiated I_hcurrentA, pooled data for Forced Swim Test (FST; left), Tail Suspension Test (TST; middle) and Saccharin Preference Test (SPT, right). RCF mice intra-VTA infused with Lamotrigine (RCF-LTG) show increased immobility behavior in both FST (*p < 0.05, t -test = −2.4, 8 RCF-Veh and 6 RCF-LTG mice) and TST (***p < 0.001, t -test = −5.04, 5 RCF-Veh and 6 RCF-LTG mice) as well as reduced percentage of saccharin intake in comparison with Vehicle-treated animals (RCF-Veh; *p < 0.01). B, I–V curves depicting significant potentiation of I_h current density in DA neurons in the iVTA from RCF-LTG mice compared to RCF-Veh: two-way ANOVA with repeated measures showed statistical difference for ‘treatment’ (i.e. LTG infusion; *p = 0.0359), weak interaction between I_h amplitude and V_H (p = 0.0621) and predictably significant effect of V_H (***p < 0.0001). Post-hoc Bonferroni's multiple comparisons test showed that I_h current density recorded at V_H – 120 mV was significantly larger in RCF-LTG neurons (*p < 0.05; 13 RCF-Veh and 18 RCF-LTG neurons). To the right, typical I_h currents. Scale bars: 100 pA, 0.3 s. C, f – I relationships (left) and typical current-clamp recordings (right) depicting lack of difference in frequency of APs evoked in RCF-LTG and RCF-Veh neurons: two-way ANOVA with repeated measures showed no difference for ‘treatment’ (i.e. LTG infusion; p = 0.2190) at the I_inj shown (13 RCF-Veh and 17 RCF-LTG neurons). Note the increased latency of the first evoked spike in RCF-LTG neurons. Scale bars: 30 mV, 0.3 s.

Fig. 4

Adult Control mice intra-VTA infused with ZD7288 show RCF behavior and reduced I_hcurrentA, pooled data for Forced Swim Test (FST; left), Tail Suspension Test (TST; middle) and Saccharin Preference Test (SPT, right). Control mice intra-VTA infused with ZD7288 (CTRL-ZD) show reduced immobility behavior in both FST (***p < 0.001, t -test = 4.69, 7 CTRL-ZD and 6 CTRL-Veh mice) and TST (*p < 0.05, t -test = 2.7, 7 CTRL-ZD and 6 CTRL-Veh) as well as increased percentage of saccharin intake in comparison with Vehicle-treated animals (CTRL-Veh mice; p < 0.05). B, to the left, I–V curves depicting significant reduction of I_h current density in DA neurons in the iVTA from CTRL-ZD mice compared to CTRL-Veh. Two-way repeated measures ANOVA: in vivo treatment, F_(1,31) = 8.58, **p = 0.0063; in vivo treatment x membrane potential, F_(3,29) = 7.09, **p = 0.0010; membrane potential, F_(3,29) = 48.21, ***p = 2.17 × 10⁻¹¹. For CTRL-Veh vs CTRL-ZD with Bonferroni's post-hoc analysis: at V_h −60 mV, CTRL-Veh 0 ± 1 pA, CTRL-ZD – 2 ± 1 pA, p = 1; at V_h −80 mV, CTRL-Veh – 37 ± 5 pA, CTRL-ZD – 17 ± 3 pA, p = 1; at V_h −100 mV, CTRL-Veh – 138 ± 15 pA, CTRL-ZD – 76 ± 10 pA, **p = 0.0029; at V_h – 120 mV, CTRL-Veh – 204 ± 23 pA, CTRL-ZD – 137 ± 15 pA, ***p = 8.12 × 10⁻⁴ (CTRL-Veh, n = 40; CTRL-ZD, n = 32). To the right, example I_h currents. Scale bars: 200 pA, 0.5 s. C, f – I relationships (left) and typical current-clamp recordings (right) depicting lack of difference in frequency of APs evoked in CTRL-ZD and CTRL-Veh neurons, as revealed by two-way repeated measures ANOVA: treatment, F_(1,28) = 3.4, p = 0.075; treatment x I_Inj F_(3,26) = 0.099, p = 0.95; (CTRL-Veh n = 39, CTRL-ZD n = 30). Scale bars: 30 mV, 1 s.

Supplementary Fig. 1

Potassium currents mediated by voltage-dependent, DR channels are unaltered by RCF in adult mice. I–V curves for the ‘instantaneous’ (peak; left) and ‘steady-state’ (middle) component of currents mediated by voltage-dependent Delayed Rectifier (DR) K⁺ channels showing lack of differences in RCF or CTRL DA neurons of the iVTA from adult mice. In details, at V_H + 20 mV, for I_peak: CTRL 3.5 ± 0.4 nA and RCF 3.0 ± 0.3 nA (18 and 11 cells, respectively; two-way repeated measure ANOVA, F_(1,10) = 7.91 × 10⁻⁴, treatment p = 0.97); and for I_s-s: CTRL 2.3 ± 0.2 nA and RCF 2.2 ± 0.3 nA (same cells; two-way repeated measure ANOVA, treatment, F_(1,10) = 1.34 × 10⁻⁴, p = 0.99). The voltage-clamp step protocol used to activate the currents is shown (upper right) with typical outward K⁺ current elicited (lower; scale bars 500 pA, 1 s). Left and right arrows indicate the time point where the amplitude of peak and steady-state currents were estimated.

Supplementary Fig. 2

HCN2 protein is unaltered by RCF in VTA punches from adult mice. Lack of difference between RCF and CTRL mice (both n = 7) on HCN2 total protein levels. Detection of actin was used as loading control.

Supplementary Fig. 3

Intra-VTA infusion of LTG does not affect naïve (CTRL) female mice. A, lack of alteration in Forced Swimming Test (FST, left; ns), Tail Suspension Test (TST, middle; ns) and Saccharin Preference Test (SPT, right; ns) in naïve (CTRL) adult females following intra-VTA infusion of Lamotrigine (CTRL-LTG) compared to mice infused with vehicle (CTRL-Veh). B,C lack of alteration of I–V relationship (Panel B; ns) for Ih current density and f - I relationship (Panel C; ns) for evoked firing from DA iVTA neurons of CTRL-LTG compared to CTRL-Veh mice.