L-type Ca²⁺ channel blockade with antihypertensive medication disrupts VTA synaptic plasticity and drug-associated contextual memory

Abstract

Drug addiction is driven, in part, by powerful and enduring memories of sensory cues associated with drug intake. As such, relapse to drug use during abstinence is frequently triggered by an encounter with drug-associated cues, including the drug itself. L-type Ca(2+) channels (LTCCs) are known to regulate different forms of synaptic plasticity, the major neural substrate for learning and memory, in various brain areas. Long-term potentiation (LTP) of NMDA receptor (NMDAR)-mediated glutamatergic transmission in the ventral tegmental area (VTA) may contribute to the increased motivational valence of drug-associated cues triggering relapse. In this study, using rat brain slices, we found that isradipine, a general LTCC antagonist used as antihypertensive medication, not only blocks the induction of NMDAR LTP but also promotes the reversal of previously induced LTP in the VTA. In behaving rats, isradipine injected into the VTA suppressed the acquisition of cocaine-paired contextual cue memory assessed using a conditioned place preference (CPP) paradigm. Furthermore, administration of isradipine or a CaV1.3 subtype-selective LTCC antagonist (systemic or intra-VTA) before a single extinction or reinstatement session, while having no immediate effect at the time of administration, abolished previously acquired cocaine and alcohol (ethanol) CPP on subsequent days. Notably, CPP thus extinguished cannot be reinstated by drug re-exposure, even after 2 weeks of withdrawal. These results suggest that LTCC blockade during exposure to drug-associated cues may cause unlearning of the increased valence of those cues, presumably via reversal of glutamatergic synaptic plasticity in the VTA.

Figure 1

Isradipine blocks NMDAR LTP induction in the VTA. (a) Isradipine (2 μM) had no effect on NMDAR EPSCs (n = 6 cells; example EPSC traces before and after isradipine application). (b) Example traces (top; aspartate iontophoresis at arrows) and summary time graphs (bottom) illustrating that isradipine had no effect on the frequency/number of spikes within the burst (n = 5 cells) or tonic firing (n = 8 cells). (c) Example experiments (EPSC traces at the times indicated) and summary time graph showing that isradipine blocked the induction of NMDAR LTP. Graph at the bottom right depicts average EPSC amplitude during baseline and after LTP (F_1,25 = 21.89, p < 0.001, n = 12–15 cells/group; mixed two-way ANOVA). ***p < 0.001 vs. baseline; ^###p < 0.001 between groups (Bonferroni post hoc test). (d) Isradipine blocked LTP induction without affecting synaptic facilitation of I_K(Ca). Data are from the same cells shown in (c) (LTP: t₂₅ = 4.71, p < 0.001; I_K(Ca) facilitation: t₂₅ = 0.79, p = 0.43; unpaired t test). Example traces depict I_K(Ca) evoked by a single AP alone and with preceding synaptic stimulation. Bottom graph illustrates the relationship between LTP magnitude and the degree of I_K(Ca) facilitation obtained from each cell (dashed lines: linear fit to all data points in each group).

Figure 2

Isradipine in the VTA blocks cocaine CPP acquisition. (a and b) Timeline of experiments [top; systemic (i.p.) or intra-VTA injection of isradipine (ISR)/vehicle (VEH) made at arrows] and summary graphs depicting changes in the preference for the cocaine (COC)-paired compartment after three conditioning sessions [(a) systemic: F_1,30 = 41.08, p < 0.001, n = 16 rats/group; intra-VTA: F_1,12 = 26.71, p < 0.001, n = 6–8 rats/group; (b) F_1,12 = 17.92, p < 0.01, n = 7 rats/group; mixed two-way ANOVA). *p < 0.05, ***p < 0.001 vs. pretest; ^###p < 0.001 between groups (Bonferroni post hoc test).

Figure 3

Isradipine and compound 8 promote reversal of previously induced NMDAR LTP. (a) After LTP induction and its development, LTP reversal protocol consisting of 1) sustained synaptic stimulation alone or 2) synaptic stimulation paired with a single AP was repeatedly delivered (30 times; arrowhead). In the third and fourth groups, the latter protocol (“SS+AP-30×”) was delivered in the presence of isradipine (ISR) or compound 8 (C8). (b) The “SS+AP-30×” protocol was delivered in control solution or in isradipine without prior LTP induction. (c) LTP reversal protocol consisting of synaptic stimulation alone was repeatedly delivered (10 times) in control solution, in isradipine, or in isradipine and AP5 (5 μM; produced 83 ± 4% peak inhibition of potentiated EPSCs, n = 6 cells). Summary time graphs of these experiments are shown on the left, while graphs depicting average EPSC amplitude during baseline, after LTP [except for (b)], and following delivery of LTP reversal protocol are shown on the right [(a) F_6,40 = 4.85, p < 0.001, n = 5–7 cells/group; (b) F_1,10 = 0.01, p = 0.94, n = 6 cells/group; (c) F_4,32 = 10.49, p < 0.001, n = 6–7 cells/group; mixed two-way ANOVA). Example traces for the experiments indicated are shown in the middle. Synaptic stimulation intensity was adjusted to evoke ∼100 pA baseline EPSCs in each cell; thus the degree of synaptic facilitation of I_K(Ca) was similar in different groups (Supplementary Figures S8d-f). **p < 0.01, ***p < 0.001 between two LTP stages; ^###p < 0.001 between groups (Bonferroni post hoc test).

Figure 4

Isradipine and compound 8 promote extinction of cocaine/ethanol CPP and prevent future reinstatement. (a and b) Summary graphs depicting the effects of systemic isradipine administration (i.p.) on the expression and extinction of CPP previously induced with cocaine (a) or ethanol (b) (three conditioning sessions for both). A single injection of isradipine (1.2 mg/kg) or vehicle [1 ml/kg of 16% ethanol (0.13 g/kg)] was made prior to the second posttest, while cocaine/ethanol injections were made immediately before the fifth and seventh posttests to trigger reinstatement (2-week interval between fifth and sixth posttests) [(a) F_7,126 = 3.40, p < 0.001, n = 10 rats/group; (b) F_7,91 = 3.21, p < 0.01, n = 7–8 rats/group; mixed two-way ANOVA). (c and d) Summary graphs showing the effects of intra-VTA injection of isradipine (c) or compound 8 (d) made before the second posttest following cocaine CPP acquisition. Cocaine-induced reinstatement was tested on the fifth posttest [(c) F_5,80 = 13.82, p < 0.001, n = 9 rats/group; (d) F_5,70 = 9.62, p < 0.001, n = 7–9 rats/group; mixed two-way ANOVA). (e) Intra-VTA isradipine injection had no effect when done immediately after the second posttest (F_3,42 = 0.45, p = 0.72, n = 7–9 rats/group; mixed two-way ANOVA). (f) Systemic isradipine injection (i.p.) followed by intra-VTA injection of AP5 or vehicle was made before the second posttest (F_3,30 = 9.71, p < 0.001, n = 6 rats/group; mixed two-way ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001 vs. pretest; ⁺p < 0.05, ⁺⁺⁺p < 0.001 between two successive posttests; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 between groups (Bonferroni post hoc test).

Figure 5

Isradipine prevents future reinstatement when administered before the posttest following complete extinction. (a) Average time course of cocaine CPP extinction during repeated posttests over 9 days (F_9,288 = 77.30, p < 0.001, n = 33 rats; repeated measures one-way ANOVA). (b and c) A single systemic injection of isradipine or vehicle was made before the tenth posttest performed with (b) or without (c) cocaine injection (orange arrow), while the eleventh posttest was always done with cocaine injection [(b) F_2,32 = 16.54, p < 0.001, n = 9 rats/group; (c) F_2,26 = 30.33, p < 0.001, n = 7–8 rats/group; mixed two-way ANOVA]. **p < 0.01, ***p < 0.001 vs. pretest; ⁺⁺⁺p < 0.001 between two successive posttests; ^###p < 0.001 between groups (Bonferroni post hoc test).