The divergent effects of cdppb and cannabidiol on fear extinction and anxiety in a predator scent stress model of ptsd in rats

Post-traumatic stress disorder (PTSD) currently has no FDA-approved treatments that reduce symptoms in the majority of patients. The ability to extinguish fear memory associations is impaired in PTSD individuals. As such, the development of extinction-enhancing pharmacological agents to be used in combination with exposure therapies may benefit the treatment of PTSD. Both mGlu5 and CB1 receptors have been implicated in contextual fear extinction. Thus, here we tested the ability of the mGlu5 positive allosteric modulator 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) and cannabidiol (CBD) to reduce both conditioned and unconditioned fear. We used a predator-threat animal model of PTSD which we and others have previously shown to capture the heterogeneity of anxiety responses observed in humans exposed to trauma. Here, 1 week following a 10-min exposure to predator scent stress, rats were classified into stress-Susceptible and stress-Resilient phenotypes using behavioral criteria for elevated plus maze and acoustic startle response performance. Two weeks after classification, rats underwent 3 days of contextual fear extinction and were treated with vehicle, CDPPB or CBD prior to each session. Finally, the light-dark box test was employed to assess phenotypic differences and the effects of CDPPB and CBD on unconditioned anxiety. CDPBB but not CBD, reduced freezing in Susceptible rats relative to vehicle. In the light-dark box test for unconditioned anxiety, CBD, but not CDPPB, reduced anxiety in Susceptible rats. Resilient rats displayed reduced anxiety in the light-dark box relative to Susceptible rats. Taken together, the present data indicate that enhancement of mGlu5 receptor signaling in populations vulnerable to stress may serve to offset a resistance to fear memory extinction without producing anxiogenic effects. Furthermore, in a susceptible population, CBD attenuates unconditioned but not conditioned fear. Taken together, these findings support the use of predator-threat stress exposure in combination with stress-susceptibility phenotype classification as a model for examining the unique drug response profiles and altered neuronal function that emerge as a consequence of the heterogeneity of psychophysiological response to stress. © 2019 Shallcross, Hámor, Bechard, Romano, Knackstedt and Schwendt

The Divergent Effects of CDPPB and Cannabidiol on Fear Extinction and Anxiety in a Predator Scent Stress Model of PTSD in Rats

Post-traumatic stress disorder (PTSD) currently has no FDA-approved treatments that reduce symptoms in the majority of patients. The ability to extinguish fear memory associations is impaired in PTSD individuals. As such, the development of extinction-enhancing pharmacological agents to be used in combination with exposure therapies may benefit the treatment of PTSD. Both mGlu5 and CB1 receptors have been implicated in contextual fear extinction. Thus, here we tested the ability of the mGlu5 positive allosteric modulator 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) and cannabidiol (CBD) to reduce both conditioned and unconditioned fear. We used a predator-threat animal model of PTSD which we and others have previously shown to capture the heterogeneity of anxiety responses observed in humans exposed to trauma. Here, 1 week following a 10-min exposure to predator scent stress, rats were classified into stress-Susceptible and stress-Resilient phenotypes using behavioral criteria for elevated plus maze and acoustic startle response performance. Two weeks after classification, rats underwent 3 days of contextual fear extinction and were treated with vehicle, CDPPB or CBD prior to each session. Finally, the light-dark box test was employed to assess phenotypic differences and the effects of CDPPB and CBD on unconditioned anxiety. CDPBB but not CBD, reduced freezing in Susceptible rats relative to vehicle. In the light-dark box test for unconditioned anxiety, CBD, but not CDPPB, reduced anxiety in Susceptible rats. Resilient rats displayed reduced anxiety in the light-dark box relative to Susceptible rats. Taken together, the present data indicate that enhancement of mGlu5 receptor signaling in populations vulnerable to stress may serve to offset a resistance to fear memory extinction without producing anxiogenic effects. Furthermore, in a susceptible population, CBD attenuates unconditioned but not conditioned fear. Taken together, these findings support the use of predator-threat stress exposure in combination with stress-susceptibility phenotype classification as a model for examining the unique drug response profiles and altered neuronal function that emerge as a consequence of the heterogeneity of psychophysiological response to stress

Extinction vs. Abstinence: A Review of the Molecular and Circuit Consequences of Different Post-Cocaine Experiences

The intravenous cocaine self-administration model is widely used to characterize the neurobiology of cocaine seeking. When studies are aimed at understanding relapse to cocaine-seeking, a post-cocaine abstinence period is imposed, followed by “relapse” tests to assess the ability of drug-related stimuli (“primes”) to evoke the resumption of the instrumental response previously made to obtain cocaine. Here, we review the literature on the impact of post-cocaine abstinence procedures on neurobiology, finding that the prelimbic and infralimbic regions of the prefrontal cortex are recruited by extinction training, and are not part of the relapse circuitry when extinction training does not occur. Pairing cocaine infusions with discrete cues recruits the involvement of the NA, which together with the dorsal striatum, is a key part of the relapse circuit regardless of abstinence procedures. Differences in molecular adaptations in the NA core include increased expression of GluN1 and glutamate receptor signaling partners after extinction training. AMPA receptors and glutamate transporters are similarly affected by abstinence and extinction. Glutamate receptor antagonists show efficacy at reducing relapse following extinction and abstinence, with a modest increase in efficacy of compounds that restore glutamate homeostasis after extinction training. Imaging studies in humans reveal cocaine-induced adaptations that are similar to those produced after extinction training. Thus, while instrumental extinction training does not have face validity, its use does not produce adaptations distinct from human cocaine users

mGlu5 Receptors and Relapse to Cocaine-Seeking: The Role of Receptor Trafficking in Postrelapse Extinction Learning Deficits

We have previously demonstrated that MTEP, an allosteric antagonist of mGlu5, infused into the nucleus accumbens attenuates relapse after abstinence from cocaine self-administration. MTEP infused into the dorsolateral striatum (dlSTR) does not alter relapse but has long-lasting effects on subsequent extinction learning. Here we tested whether systemic MTEP would prevent relapse after abstinence or alter extinction learning. We also investigated the mechanism of action by which intra-dlSTR MTEP on test day alters extinction on subsequent days. Animals self-administered cocaine for 12 days followed by abstinence for 20-21 days. MTEP (0.5–5 mg/kg IP) was administered prior to placement into the operant chamber for a context-primed relapse test. A separate group of animals received intra-dlSTR MTEP prior to the relapse test and were sacrificed day later. Systemic administration of MTEP attenuated abstinent-relapse without significantly affecting extinction learning. Surface biotinylation analysis of protein expression in the dlSTR revealed that, in cocaine animals, intra-dlSTR MTEP administration decreased mGlu5 surface expression and prevented changes in Arc and GluA1/GluA2 observed in their vehicle counterparts. Thus, blockade of mGlu5 receptors may be utilized in future treatment strategies for relapse prevention in humans, although the effects of chronic blockade on extinction learning should be further evaluated

Extinction-Dependent Alterations in Corticostriatal mGluR2/3 and mGluR7 Receptors following Chronic Methamphetamine Self-Administration in Rats

Methamphetamine (meth) is a highly addictive and widely abused psychostimulant. Repeated use of meth can quickly lead to dependence, and may be accompanied by a variety of persistent psychiatric symptoms and cognitive impairments. The neuroadaptations underlying motivational and cognitive deficits produced by chronic meth intake remain poorly understood. Altered glutamate neurotransmission within the prefrontal cortex (PFC) and striatum has been linked to both persistent drug-seeking and cognitive dysfunction. Therefore, the current study investigated changes in presynaptic mGluR receptors within corticostriatal circuitry after extended meth self-administration. Rats self-administered meth (or received yoked-saline) in 1 hr/day sessions for 7 days (short-access) followed by 14 days of 6 hrs/day (long-access). Rats displayed a progressive escalation of daily meth intake up to 6 mg/kg per day. After cessation of meth self-administration, rats underwent daily extinction or abstinence without extinction training for 14 days before being euthanized. Synaptosomes from the medial PFC, nucleus accumbens (NAc), and the dorsal striatum (dSTR) were isolated and labeled with membraneimpermeable biotin in order to measure surface mGluR2/3 and mGluR7 receptors. Extended access to meth selfadministration followed by abstinence decreased surface and total levels of mGluR2/3 receptors in the NAc and dSTR, while in the PFC, only a loss of surface mGluR2/3 and mGluR7 receptors was detected. Daily extinction trials reversed the downregulation of mGluR2/3 receptors in the NAc and dSTR and mGluR7 in the PFC, but downregulation of surfac

Analysis of mGluR2/3 and mGluR7 surface vs. intracellular distribution in rat brain synaptosomes.

<p>(A) Rat brain atlas coordinates (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034299#pone.0034299-Paxinos1" target="_blank">[77]</a>) used for tissue dissection and synaptosome preparation. (B) Representative immunoblots showing surface vs. intracellular distribution of mGluR2/3 and mGluR7 receptors (as well as marker proteins: calnexin, ERK1/2 and RasD1/AGS1) in synaptosomal fraction prepared from the dSTR. P2* - synaptosomal fraction resolved under strongly reducing conditions (100 mM DTT); T – total synaptosomal fraction after biotinylation resolved under weakly reducing conditions (>10 mM DTT); NB – non-biotinylated (intracellular) proteins; B – biotinylated (surface) proteins isolated by precipitation with streptavidin-agarose beads.</p

Extended access to intravenous meth followed by extinction results in decreased surface expression of mGluR2/3 in the PFC.

<p><i>Top panels:</i> Representative immunoblots show levels of mGluR2/3 and mGluR7 protein in total (T) and surface (B, biotinylated) synaptosomal fractions isolated from the PFC (A), NAc (B), and dSTR (C) following meth self-administration and 14 days of extinction. <i>Lower panels:</i> Quantitative analyses of immunoblots revealed significant decreases in surface mGluR2/3 levels in the PFC (A). Integrated density of each band was analyzed by one-way ANOVA and expressed as the percentage of the saline-treated animals. Data shown as mean ± S.E.M.; n = 8 samples per group. *p<0.05 vs. Sal.</p

Extended access to intravenous meth results in robust self-administration and escalation of meth intake.

<p>(A) Timeline of the experiment as described in detail in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034299#s2" target="_blank">Materials and Methods</a> section. Animals underwent 21 days of meth self-administration (or received yoked-saline) followed by 14 days of home cage abstinence or 14 daily extinction sessions. Tissues were collected at the end of the experiment. (B) Daily lever responding for meth and yoked-sal animals during self-administration (left graph) and extinction (right graph). (C) Escalation of daily meth intake during short (1 hr) and long (6 hr) self-administration sessions as detected in abstinent and extinction animals. Mean daily meth intake over the course of the first three days vs. last three days of short- and long-access self-administration was analyzed by one-way ANOVA and expressed as mg/kg/day of meth. (D) Total meth intake (mg/kg) in abstinent vs. extinction animals. Data shown as mean ± S.E.M.; n = 8–11 samples per group. **p<0.01 L12–L14 vs. L1–L3.</p

Extended access to intravenous meth followed by abstinence results in decreased surface and/or total expression of mGluR2/3 in the PFC and striatal subregions and decreased surface expression of mGluR7 in the PFC.

<p><i>Top panels:</i> Representative immunoblots show levels of mGluR2/3 and mGluR7 protein in total (T) and surface (B, biotinylated) synaptosomal fractions isolated from the PFC (A), NAc (B), and dSTR (C), following meth self-administration and 14 days of abstinence. <i>Lower panels:</i> Quantitative analyses of immunoblots revealed significant decreases in total mGluR2/3 levels (B, C), surface mGluR2/3 levels (A, B, C) and surface mGluR7 levels (A) in meth vs. saline rats. Integrated density of each band was analyzed by one-way ANOVA and expressed as the percentage of the saline-treated animals. Data shown as mean ± S.E.M.; n = 11 samples per group. *p<0.05, **p<0.01 vs. Sal.</p