The Divergent Effects of CDPPB and Cannabidiol on Fear Extinction and Anxiety in a Predator Scent Stress Model of PTSD in Rats, John Shallcross et al., 2019

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

John Shallcross, Peter Hámor, Allison R. Bechard, Madison Romano, Lori Knackstedt and Marek Schwendt

Frontiers in Behavioral Neuroscience, May 2019 | Volume 13 | Article 91

published: 10 May 2019
doi: 10.3389/fnbeh.2019.00091

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-1Hpyrazol- 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.

Keywords : fear extinction, TMT, resilient, mGlu5, mPFC, BLA, Fos


Post-traumatic stress disorder (PTSD) develops in a subset of individuals following a traumatic event (Perkonigg et al., 2000). A characteristic feature of PTSD is impaired fear memory extinction (Orr et al., 2000; Guthrie and Bryant, 2006), which contributes to the persistent anxiety and hyperarousal experienced by affected individuals (Herman, 1992; Norrholm et al., 2011). Fear extinction is an active learning process where stimuli that previously elicited fear are repeatedly presented in the absence of threat to produce a gradual reduction in fear response (Bouton et al., 2006). While extinction-based exposure therapies are frequently used as a strategy for treating anxiety-like disorders, PTSD-associated extinction deficits reduce the efficacy of these treatments (Schottenbauer et al., 2008). Consequently, there is a need to improve currently available therapies for PTSD. One approach that directly addresses extinction deficits in PTSD would involve the co-administration of extinctionenhancing pharmacological agents with exposure therapy to improve treatment outcomes (e.g., Rothbaum et al., 2014).

Animal models are essential to interrogate the neurobiology underlying fear extinction and for the development of novel extinction-enhancing therapeutics. The most commonly used models are grounded in Pavlovian fear conditioning principles (Pavlov, 1927; Rescorla, 1988). Fear conditioning involves pairing an unconditioned aversive stimulus (US; e.g., mild electric shock) with neutral conditioned stimuli (CS; e.g., a discrete cue or context) until a conditioned fear response (CR; e.g., freezing, changes in heart rate) is produced following delivery of the CS alone. Like exposure therapy, fear extinction training involves prolonged, or repeated presentations of the CS alone, and ideally results in the gradual elimination of the CR (Rothbaum and Schwartz, 2002; Barad, 2005).

Footshock stress is commonly used to study fear learning, and although these models have contributed substantially to our understanding of neural circuits involved in conditioning and extinction of fear, several alternatives to footshock have been established, each offering unique and complementary contributions to the field. Notably, inescapable exposure to species-relevant predator odors (also termed as predator scent stress, PSS) can evoke persistent alterations in behavioral and physiological response in rats that mirror the symptom profile of fear and anxiety related disorders such as PTSD. Exposure of rodents to 2, 3, 5-Trimethyl-3-thiazoline (TMT), a synthetically derived component of fox feces (Vernet-Maury et al., 1984) induces hyperarousal (Hebb et al., 2003), anxiety (Rosen et al., 2015), social dysfunction (Stockman and McCarthy, 2017), vulnerability to substance use (Schwendt et al., 2018), and contextually cued defensive behaviors (Fendt and Endres, 2008; Homiack et al., 2017), indicating the incidence of both sensitized and conditioned fear and anxiety like behaviors.

A key advantage of using PSS models is the ability to examine physiological features associated with the individual differences in vulnerability to such stress. As previously established for PSS using cat odor (Cohen et al., 2003, 2014; Nalloor et al., 2011), rats can be separated into Susceptible, Resilient and Intermediate phenotypes based on scores in both the elevated plus maze (EPM) and habituation in the acoustic startle response (ASR) 7 days after PSS exposure. Control rats are placed into the PSS context without predator odor and are later assessed in the EPM and ASR. Most humans exposed to trauma initially display symptoms of distress and anxiety which dissipate within 1–4 weeks following the trauma (Foa et al., 2006). A similar pattern is observed in the PSS model: 1 day following PSS exposure, approximately 90% of PSS exposed rats are classified as Susceptible; by the 7th day postexposure, this rate drops to 25%, nicely paralleling the human condition (Cohen et al., 2003). We and others have demonstrated that in unstressed Control rats, the percent of rats classified as Susceptible is much lower, at 1.33–4% (Cohen et al., 2003; Schwendt et al., 2018). Thus, the anxiety phenotype in Susceptible rats is induced by PSS exposure, and is not present in the absent of such exposure.

Likewise, we have recently reported that a single 10- min exposure to TMT gives rise to distinct stress-Susceptible and Resilient phenotypes in Sprague-Dawley rats, with each group presenting distinct behavioral, hormonal, and molecular signatures (Schwendt et al., 2018). Notably, we found that while all TMT-exposed rats and Control rats displayed similar freezing during the PSS exposure, only Susceptible rats displayed increased freezing upon re-exposure to the PSS context whereas Resilient and Control rats did not. Furthermore, Susceptible rats do not decrease freezing over the course of 5 days of extinction
exposures to the PSS context (Schwendt et al., 2018). Taken together, these findings indicate phenotypic heterogeneity among populations of stressed animals which may have an unseen influence on the conclusions gained measuring fear extinction within the entire population of stressed animals. Thus, studies addressing differential vulnerabilities may reveal novel fearassociated adaptations.

In healthy humans, neuroimaging studies have revealed an important role for neural activity in the circuitry encompassing medial prefrontal cortex (mPFC) and amygdala during fear extinction. Increased activity is observed in the ventral medial prefrontal cortex (vmPFC) and decreased activity observed
in the dorsal lateral prefrontal cortex (dlPFC; Milad et al., 2007) and amygdala (LaBar et al., 1998). Opposite patterns are demonstrated in humans with PTSD, with low vmPFC activity and high activity in both dlPFC and amygdala (Milad et al., 2009). As noted above, the neural correlates of fear extinction in rodents have been extensively studied using footshock models, and suggest a conserved mechanism also involving functional interactions between the mPFC and amygdala. In the rodent mPFC, the prelimbic (PL) and infralimbic (IL) cortices (analogous to the dlPFC and vmPFC in humans, respectively) are strongly interconnected with the basolateral amygdala (BLA; Hoover and Vertes, 2007). The BLA is required for extinction of conditioned footshock (Falls et al., 1992), and serves to regulate fear response through output to the central amygdala (CeA) and brainstem regions (Royer et al., 1999; Haubensak
et al., 2010). Chemogenetic, or electrical stimulation of IL or PL pathways targeting the BLA reveal opposing influences (Herry et al., 2008; Senn et al., 2014), with IL enhancing, and PL impairing extinction (Sierra-Mercado et al., 2011). Additionally, inhibitory and excitatory IL and PL projections (respectively) regulate BLA excitability, stabilizing fear response inhibition (Cho et al., 2013). This evidence suggests that extinction of footshock conditioned fear requires a switch from PL- to ILmediated
reciprocal signaling through the BLA. Indeed, the assessment of neuronal activity using c-Fos immunoreactivity reveals high Fos expression in the IL, but not PL following extinction, and PL and BLA Fos expression correlating with extinction resistance (Knapska and Maren, 2009). While TMT exposure is also associated with changes in amygdala, IL, and PL activity (Sevelinges et al., 2004; Hwa et al., 2019), and recent studies implicate these regions in the extinction of conditioned fear with alternative predator odors, how the coordinated activity across these regions may contribute to the suppression of TMT conditioned fear remains undetermined.

Glutamate receptor signaling has been the focus of many efforts in the development of extinction-enhancing agents. Metabotropic glutamate receptor 5 (mGlu5) subtype regulates bidirectional synaptic plasticity in fear-associated brain regions including the mPFC and BLA (Niswender and Conn, 2010). Pharmacological and genetic inhibition of mGlu5 impairs extinction of both cues and contexts paired with footshock (Xu et al., 2009; Fontanez-Nuin et al., 2011; Sepulveda-Orengo et al., 2013; Sethna and Wang, 2016), and administration of mGlu5 positive allosteric modulators (PAMs) enhances extinction of a footshock-paired context (e.g., Sethna and Wang, 2014). Although antagonism of mGlu5 receptors impairs consolidation of extinction memory, these drugs have also been found to produce anxiolytic effects (Porter et al., 2005; Rahman et al., 2017). The consequences of glutamate receptor modulation on the extinction of predator odor conditioned fear has been assesses in only one study that demonstrated partial agonism of NMDA receptors with D-cycloserine enhanced extinction of a cat odorpaired context (Sarıdo˘gan et al., 2015). We have previously found increased mGlu5 gene expression in the amygdala and mPFC of Resilient rats following re-exposure to the TMTassociated context (Schwendt et al., 2018). In the same study, daily systemic treatment with the mGlu5 PAM CDPPB during extinction of the TMT paired context increased freezing in a cohort of Susceptible rats that previously underwent cocaine self-administration (Schwendt et al., 2018). However, as chronic cocaine can alter both function and mGlu5 receptors numbers in brain regions associated with fear signaling (Hao et al., 2010; Ghasemzadeh et al., 2011; Schmidt et al., 2011), a primary goal here was to examine the effects of CDPPB on fear extinction in cocaine-naïve rats using this model.

Like mGlu5, CB1 receptors are abundantly expressed in the BLA and mPFC and are important modulators of fear and anxiety signaling (Chhatwal and Ressler, 2007). Previous studies have revealed dysregulated expression of CB1 receptors and abnormal levels endocannabinoids in subjects with PTSD (Neumeister et al., 2015), as well as in rodent PTSD models (Schwendt et al., 2018). In rodents, genetic or pharmacological inhibition of CB1 receptors impairs extinction (Marsicano et al., 2002), while CB1 agonists have extinction-enhancing effects (Chhatwal et al., 2005; Campolongo et al., 2009). However, CB1 agonists can also produce biphasic anxiogenic and anxiolytic effects (Haller et al., 2004; Sink et al., 2010), which may compromise their clinical usefulness. Several recent studies have demonstrated that cannabidiol (CBD), a component of cannabis which lacks THC-like psychoactive effects (Campos et al., 2012b), may serve to mitigate symptoms of PTSD by increasing extinction and reducing post-trauma anxiety in both humans and rodents (Bitencourt et al., 2008; Das et al., 2013).

Here we evaluated the effects of CDPPB and CBD on the extinction of contextual fear in a PSS model. We focused our investigation on rats with stress-Susceptible phenotype, as Resilient and Control rats do not demonstrate freezing upon reexposure to the conditioning context (Schwendt et al., 2018). Further, this study explored possible changes in neuronal activity (via Fos expression) produced by fear extinction training within the PL, IL, and BLA regions. Given the involvement of PL, IL, and BLA neuronal activity in extinction to conditioned footshock (Cho et al., 2013), and evidence indicating an important role for mGlu5 receptor function (Sethna and Wang, 2016), we predicted that treatment with CDPPB would (a) enhance extinction of contextual fear, and (b) increase Fos expression in all three regions. Finally, this study also considered the effects of CDPPB and CBD treatment on unconditioned anxiety, as anxiogenic effects may compromise the utility of these drugs for fearextinction therapies.