The Small GTPase Rac1 Contributes to Extinction of Aversive Memories of Drug Withdrawal by Facilitating GABAA Receptor Endocytosis in the vmPFC

Abstract

Extinction of aversive memories has been a major concern in neuropsychiatric disorders, such as anxiety disorders and drug addiction. However, the mechanisms underlying extinction of aversive memories are not fully understood. Here, we report that extinction of conditioned place aversion (CPA) to naloxone-precipitated opiate withdrawal in male rats activates Rho GTPase Rac1 in the ventromedial prefrontal cortex (vmPFC) in a BDNF-dependent manner, which determines GABA_A receptor (GABA_AR) endocytosis via triggering synaptic translocation of activity-regulated cytoskeleton-associated protein (Arc) through facilitating actin polymerization. Active Rac1 is essential and sufficient for GABA_AR endocytosis and CPA extinction. Knockdown of Rac1 expression within the vmPFC of rats using Rac1-shRNA suppressed GABA_AR endocytosis and CPA extinction, whereas expression of a constitutively active form of Rac1 accelerated GABA_AR endocytosis and CPA extinction. The crucial role of GABA_AR endocytosis in the LTP induction and CPA extinction is evinced by the findings that blockade of GABA_AR endocytosis by a dynamin function-blocking peptide (Myr-P4) abolishes LTP induction and CPA extinction. Thus, the present study provides first evidence that Rac1-dependent GABA_AR endocytosis plays a crucial role in extinction of aversive memories and reveals the sequence of molecular events that contribute to learning experience modulation of synaptic GABA_AR endocytosis.SIGNIFICANCE STATEMENT This study reveals that Rac1-dependent GABA_AR endocytosis plays a crucial role in extinction of aversive memories associated with drug withdrawal and identifies Arc as a downstream effector of Rac1 regulations of synaptic plasticity as well as learning and memory, thereby suggesting therapeutic targets to promote extinction of the unwanted memories.

Keywords: GABAA receptor; Rac1; aversive memory; drug withdrawal; endocytosis; extinction.

Figure 1.

Extinction training results in LTP induction and CPA extinction by dynamin-dependent GABA_AR endocytosis. A, Extinction training resulted in endocytosis of GABA_AR β3 subunits (Fig. 1-1). B, Endocytosis of GABA_AR β3 subunits was prevented by bilateral infusion of dynamin function-blocking peptide (Myr-P4, 30 pmol/0.5 μl/side) but not scramble inactive peptide (Myr-S) into the vmPFC 60 min before extinction training (Fig. 1-2). C, The 50 Hz stimulus induced reliable LTP in slices from rats that underwent extinction training but not in those that did not undergo extinction training. D, HFS at 50 Hz failed to induce LTP in slices from rats that were pretreated with bilateral infusion of Myr-P4 (30 pmol/0.5 μl/side) into the vmPFC 60 min before extinction training, but it normally induced reliable LTP in slices from rats that were pretreated with Myr-S before extinction training. E, In the presence of GABA_AR blocker picrotocxin (100 μm), HFS at 50 Hz was able to elicit LTP induction in slices from rats that did not undergo extinction training. F, The same picrotoxin application did not induce significant change in the facilitated LTP induction found in slices from rats that underwent extinction training. G, CPA extinction was impaired by intra-vmPFC infusions of Myr-P4 (30 pmol/0.5 μl/side) but not Myr-S before extinction training. H, Schematic representation of injection sites in the vmPFC for rats used in the experiments. A, B, Top, Representative blots of surface (S) and internalized (I) GABA_AR β3 subunits from the vmPFC tissues of rats prepared at different time points after extinction training (A: 2, 4, 8, and 18 h) or (B: 4 h). Bottom, Quantification of the S/I ratio of GABA_AR β3 subunit levels from Western blot data. Error bars indicate mean ± SEM (n = 6–8). *p < 0.05, compared with the no-extinction control or vehicle control groups (one-way or two-way ANOVA followed by Bonferroni's post hoc test). **p < 0.01, compared with the no-extinction control or vehicle control groups (one-way or two-way ANOVA followed by Bonferroni's post hoc test). The magnitude of LTP was the average of the last 5 min recordings expressed as the mean ± SEM percentage of the baseline fEPSP. Data were analyzed with two-tailed Student's t tests.

Figure 2.

Extinction training increases synaptic localization of Arc to regulate GABA_AR endocytosis and CPA extinction. A, CPA rats at 1 h after extinction training showed elevation of Arc protein expression in the vmPFC, detected by using immunohistochemical analysis. B, Increase of synaptic Arc protein levels in the vmPFC was detected at 2 h after extinction training by immunobloting. C, Lentivirus-infected regions in the vmPFC were visualized by fluorescence microscope. D, Infection of the vmPFC with lentivirus expressing Arc/Arg3.1-shRNA attenuated synaptic Arc protein expression within the vmPFC. E, Infection of the vmPFC with lentivirus expressing Arc/Arg3.1-shRNA inhibited GABA_AR β3 subunit endocytosis. F, Infection of the vmPFC with lentivirus expressing Arc/Arg3.1-shRNA impaired CPA extinction. Error bars indicate mean ± SEM (n = 6–8). **p < 0.01, compared with no-extinction or control shRNA groups. ***p < 0.001 compared with no-extinction or control shRNA groups. ^##p < 0.01, compared with control shRNA groups (two-tailed Student's t test, or one-way or two-way ANOVA followed by Bonferroni's post hoc test).

Figure 3.

Synaptic localization of Arc depends on actin polymerization induced by Rac1-mediated phosphorylation of Pak1 and cofilin. A, B, Extinction training resulted in activation of Rac1, but not Cdc42, in the vmPFC. C, D, Extinction training led to phosphorylation of Pak1 and cofilin, and both were blocked by bilateral intra-vmPFC infusions of Rac1 inhibitor NSC23766 (Fig. 3-1). E, Extinction training-induced actin polymerization could be inhibited by NSC23766 (0.5 μg/ 0.5 μl/side) and actin polymerization inhibitor latrunculin A (250 ng/0.5 μl/side), whereas they have no effects on total actin (left). F, Blockade of actin polymerization by NSC23766 and latrunculin A disrupted extinction training-induced enhancement of Arc protein levels at synaptic membrane preparations (left) but had no effects on extinction training-induced Arc protein levels in the vmPFC homogenates (right). Error bars indicate mean ± SEM (n = 8). *p < 0.05, compared with no-extinction control groups (one-way ANOVA followed by Bonferroni's post hoc test). **p < 0.01, compared with no-extinction control groups (one-way ANOVA followed by Bonferroni's post hoc test).

Figure 4.

Suppression of actin polymerization by NSC23766 and latrunculin A inhibits GABA_AR endocytosis, LTP facilitation, and CPA extinction. A, B, Bilateral intra-vmPFC infusion of NSC23766 (0.5 μg/0.5 μl/side) 30 min before extinction training inhibited the endocytosis of GABA_AR β3 subunits and impaired CPA extinction. C, HFS at 50 Hz failed to elicit LTP induction in slices from rats that were pretreated with Rac1 inhibitor NSC23766 before extinction training. D, E, Bilateral intra-vmPFC infusion of latrunculin A (250 ng/0.5 μl/side) 10 min before extinction training inhibited the endocytosis of GABA_AR β3 subunits and impaired CPA extinction. Error bars indicate mean ± SEM (n = 6–8). **p < 0.01, compared with no-extinction control groups. ^#p < 0.05, compared with vehicle control groups (one-way ANOVA followed by Bonferroni's post hoc test). ^##p < 0.01, compared with vehicle control groups (one-way ANOVA followed by Bonferroni's post hoc test). The magnitude of LTP was the average of the last 5 min recordings expressed as the mean ± SEM percentage of the baseline fEPSP. Data were analyzed with two-tailed Student's t tests.

Figure 5.

Genetically manipulating Rac1 activity in the vmPFC bidirectionally regulates drug withdrawal memory extinction and endocytosis of GABA_ARs. A–D, Knockdown of the Rac1 expression by Rac1-shRNA impaired CPA extinction and suppressed endocytosis of GABA_AR β3 subunits. A, Infection of the vmPFC with lentivirus expressing Rac1-shRNA was visualized by fluorescence microscope. B, Infection of lentivirus expressing Rac1-shRNA attenuated Rac1 expression within the vmPFC. C, Knockdown of the Rac1 expression in the vmPFC with Rac1-shRNA impaired CPA extinction. D, Knockdown of the Rac1 expression in the vmPFC with Rac1-shRNA suppressed endocytosis of GABA_AR β3 subunits. *p < 0.05, compared with control shRNA or no-extinction control groups. ^#p < 0.05, compared with control shRNA groups. E–H, Facilitation of CPA extinction and endocytosis of GABA_AR β3 subunits were observed in floxed mice to express constitutively active form of Rac1 induced by adeno-associated virus carrying Cre recombinase (AAV-Cre). E, Infection of the vmPFC with AAV-Cre was visualized by fluorescence microscope. B, Elevation of Rac1 activity was induced by expressing constitutively active Rac1 in the vmPFC through AAV-Cre. G, H, The constitutively active Rac1 mice displayed acceleration of CPA extinction and augmentation of endocytosis of GABA_AR β3 subunits. *p < 0.05, compared with AAV-GFP control groups (two-tailed Student's t test or two-way ANOVA followed by Bonferroni's post hoc test). ^#p < 0.05, compared with no-extinction control groups (two-tailed Student's t test or two-way ANOVA followed by Bonferroni's post hoc test). Error bars indicate mean ± SEM (n = 6).

Figure 6.

BDNF scavenger TrkB-FC suppresses extinction training-induced activation of Rac1, endocytosis of GABA_ARs, and induction of LTP. A, Extinction training increased BDNF protein expression in the vmPFC. B, Extinction training induced Rac1 activation was blocked by intra-vmPFC infusion of BDNF scavenger TrkB-FC (0.65 μg/0.5 μl/side) and Rac1 inhibitor NSC23766 before extinction training. C, Endocytosis of GABA_AR β3 subunits was suppressed by intra-vmPFC infusion of BDNF scavenger TrkB-FC before extinction training. D, HFS at 50 Hz failed to elicit LTP induction in slices from rats that were pretreated with TrkB receptor antagonist k252a (35.7 μm/μl/side), given by intra-vmPFC infusion bilaterally before extinction training. *p < 0.05, compared with no-extinction control groups. **p < 0.01, compared with no-extinction control groups. ^#p < 0.05, compared with vehicle control groups (one-way ANOVA followed by Bonferroni's post hoc test). The magnitude of LTP was the average of the last 5 min recordings expressed as the mean ± SEM percentage of the baseline fEPSP. Data were analyzed with two-tailed Student's t tests.

Figure 7.

A synaptic model proposed for extinction of memory. Extinction training activates Rac1 in a BDNF-dependent manner in the vmPFC. Active Rac1 induces synaptic actin polymerization via activating Pak1-cofilin signal pathway. Meanwhile, extinction training also results in Arc mRNA transcription. Arc mRNA is then translocated at dendritic spines through actin polymerization-dependent machinery. Within the dendritic spines, Arc proteins are rapidly translated and then form a complex with dynamin (Dyn) to facilitate GABA_AR endocytosis, thereby leading to LTP induction and CPA extinction.