Disruption of alcohol-related memories by mTORC1 inhibition prevents relapse

Abstract

Relapse to alcohol abuse is an important clinical issue that is frequently caused by cue-induced drug craving. Therefore, disruption of the memory for the cue-alcohol association is expected to prevent relapse. It is increasingly accepted that memories become labile and erasable soon after their reactivation through retrieval during a memory reconsolidation process that depends on protein synthesis. Here we show that reconsolidation of alcohol-related memories triggered by the sensory properties of alcohol itself (odor and taste) activates mammalian target of rapamycin complex 1 (mTORC1) in select amygdalar and cortical regions in rats, resulting in increased levels of several synaptic proteins. Furthermore, systemic or central amygdalar inhibition of mTORC1 during reconsolidation disrupts alcohol-associated memories, leading to a long-lasting suppression of relapse. Our findings provide evidence that the mTORC1 pathway and its downstream substrates are crucial in alcohol-related memory reconsolidation and highlight this pathway as a therapeutic target to prevent relapse.

Figure 1. The mTORC1 signaling pathway is activated in the central amygdala, medial prefrontal and orbitofrontal cortices following reactivation of alcohol-associated memories

A-C. Immunohistochemical staining of S6 phosphorylation 30 min after reactivation of alcohol-associated memory. Shown is dual-channel immunofluorescence images of phosphoS6 (pS6, red), NeuN (a marker for neurons, green), and overlay (yellow), in the basolateral (BLA) and central (CeA) nuclei of the amygdala (A), the prelimbic (PrL) region of the medial prefrontal cortex (B), and the orbitofrontal cortex (OFC; C). Images are representative of results from 4 rats (3-4 sections/region/rat). Scale bar, left: 100 μm; right: 20 μm. Quantification of the immunohistochemical staining of pS6-positive cells normalized by the total area in 3 slices per brain region from each rat. Data are mean ± SEM (t’s(6)>4.17; **p<0.01, n=4). D. Quantification of the immunohistochemical staining of S6 phosphorylation. Data are mean ± SEM expressed as percentage of no reactivation controls (t’s(6)>4.17; **p<0.01, n=4). IL=infralimbic cortex, OFC-orbitofrontal cortex, PrL=prelimbic cortex, NAc=nucleus accumbens, Hipp=dorsal hippocampus, CeA=central nucleus of the amygdala, BLA=basolateral amygdala E. Western blot analyses of 4E-BP, S6 kinase (S6K) and S6 phosphorylation in the amygdala (Amyg), medial prefrontal cortex (mPFC) and OFC. Immunoreactivity of 4E-BP, S6K and S6 phosphorylation was normalized to the total protein and expressed as percentage of control (no reactivation). Data are mean ± SEM, (t’s(6)>2.50; *p<0.05, **p<0.01, ***p<0.005, #p=0.08, n=4 per group).

Figure 2. Reactivation of alcohol-associated memories increases levels of synaptic proteins

A-C. Immunoblotting of mTORC1-regulated proteins in the amygdala (Amyg; A), mPFC (B) and OFC (C), 60 min after reactivation of alcohol-associated memory. A-C (left pane). The levels of Arc, GluR1, PSD-95 and NR were determined by western blot analysis and normalized to GAPDH. A-C (right pane). The memory reactivation-induced increase in Arc immunoreactivity was blocked by rapamycin (20 mg/kg, i.p) administered immediately after memory reactivation. Data are mean ± SEM and expressed as percentage of control. (A-C, left pane: t test; t’s(6)>2.50; *p<0.05, **p<0.01, A-C, right pane, Two-way ANOVA; Reactivation X Treatment interaction, [F(1, 12)>4.90, p<0.05], post-hoc comparisons **p<0.01; n=4 per group).

Figure 3. Inhibition of mTORC1 after reactivation of alcohol-associated memories attenuates relapse measured as instrumental responding for alcohol

A. Schematic representation of the experimental procedure. B, C & E. Data are mean ± SEM of active lever presses before abstinence (baseline), and during retention test and reacquisition stages. B. Effects of rapamycin (20 mg/kg ,i.p.) or vehicle given immediately after memory reactivation using presentation of context as well as odor-taste cue on lever presses during test and reacquisition. (Two-way ANOVA; Stage X Treatment interaction [F(2,22)=6.38, p<0.01]; post-hoc comparisons **p<0.01, n=12). C. Active and inactive lever presses during the test stage (Two-way ANOVA; Stage X Lever [F(1,22)=27.57, p<0.001]; post-hoc comparisons, active vs. inactive lever presses, **p<0.01 ***p<0.0001, n=12). D. Correlation plot of the number of lever presses during the reactivation session and the percentage of rapamycin-induced suppression in lever presses during the test (calculated as (presses in test / presses in baseline) X 100 in the rapamycin group). E. Effects of rapamycin (20 mg/kg, i.p) or vehicle, given 24 h before test without a reactivation session, on lever presses during test and reacquisition. (Two-way ANOVA; Stage X Treatment interaction [F(2,18)=0.53, p=0.59]; n=10).

Figure 4. Infusion of rapamycin or anisomycin into the CeA after reactivation of alcohol-associated memories attenuates relapse

A&B. Effects of rapamycin (A; 50 μg/side) or anisomycin (B; 62.5 μg/side) or vehicle infused into the CeA immediately after memory reactivation on lever presses during test and reacquisition. Data are mean ± SEM of active lever presses before abstinence (baseline), and during retention test and reacquisition stages. (A, rapamycin: Two-way ANOVA; Stage X Treatment interaction [F(2,14)=10.95, p<0.005]; post-hoc comparisons **p<0.01, n=8; B, anisomycin: Two-way ANOVA; Stage X Treatment interaction [F(2,11)=8.59, p<0.005]; post-hoc comparisons *p<0.5, **p<0.01, n=6-7).

Figure 5. Inhibition of mTORC1 after reactivation of alcohol-associated memories in the home cage induces a potent, long-term suppression of relapse

A. Effects of rapamycin (20 mg/kg, i.p) or vehicle, given immediately after memory reactivation using an alcohol odor-taste cue in the home cage, on active lever presses during test and reacquisition (Left pane; Two-way ANOVA; Stage X Treatment interaction [F(2,26)=14.51, p<0.0001]; post-hoc comparisons *p<0.005, **p<0.001, n=8) and on active and inactive lever presses during the test stage (Right pane; Two-way ANOVA; Stage X Lever [F(1,13)=132.27, p<0.0001]; post-hoc comparisons, active vs. inactive lever presses, ***p<0.0001, n=8). Data are mean ± SEM of lever presses. B. Effects of rapamycin (20 mg/kg ,i.p.) or vehicle, given after memory reactivation on relapse to alcohol drinking in 2-bottle choice procedure. Data are mean ± SEM of alcohol intake (g/kg/24 h) during a 24 h 2-bottle choice session, in rapamycin- or vehicle-treated rats before abstinence (baseline), 24 h after reactivation, 14 d after reactivation, in the absence of reactivation, and 24 h after reactivation with a delayed (5 h) administration of rapamycin. (Two-way ANOVA, Condition X Treatment interaction [F(4, 106)=7.12, p<0.0001], post-hoc comparisons **p<0.001, n=8-12).

Figure 6. Rapamycin does not induce place aversion

A. Design and schedule of the rapamycin place aversion experiment: rats of the rapamycin condition were systemically administered with rapamycin (20 mg/kg) and vehicle 3 h before the 30-min conditioning paired and unpaired sessions, respectively. Rats of the vehicle condition received vehicle only. B. Place preference/aversion for rapamycin is expressed as the ratio ± SEM of the time spent in the rapamycin-paired compartment divided by time spent in paired+unpaired compartments. (Two-way ANOVA, Treatment X Conditioning interaction [F(1, 16)=1.50, p=0.24], n=9).