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Δ9-Tetrahydrocannabinol (THC), the primary psychoactive component of Cannabis (sativa, indica, and ruderalis), has been reported to affect fear memory, expression, consolidation, and extinction (Phan et al., 2008; Lemos et al., 2010; Klumpers F. et al., 2012; Klumpers L. E. et al., 2012; Klumpers et al., 2013; Rabinak et al., 2013, 2014). In addition, Cannabidiol (or CBD), another component of the plant, has also been reported to impact fear memory. Medical cannabis (or marijuana for medical purposes, MMP) is widely used to self-medicate for a variety of medical conditions, including disorders rooted in fear learning such as post-traumatic stress disorder, PTSD (Lucas and Walsh, 2017). However, the effects of marijuana on fear memory reconsolidation have been only sparsely explored. Additionally, MMP is often utilized as a whole plant material. However, the effects of combined doses of THC and CBD in varying concentrations, as well as the role of the remaining (non-THC, non-CBD) plant material and its interactions with THC and CBD, remain largely unexplored. Here, we aimed to identify whether combined THC and CBD could affect fear memory reconsolidation both in isolation and when combined at varying concentrations. In addition, since it is highly relevant for the use of whole plant material as MMP, we also sought to determine whether the effects of THC and CBD on reconsolidation are modulated by the inclusion of the remaining plant material.

Behaviorally, in many respects CBD has been shown to produce effects which are opposite those of THC. One functional magnetic resonance imaging (fMRI) study found CBD and THC had opposite effects on regional activation in the hippocampus, amygdala, superior temporal cortex, and occipital cortex (Bhattacharyya et al., 2010). The same study found that pretreatment with 5 mg CBD intravenously (IV) attenuated the severity of psychotic symptoms induced by THC.

There is some data from central studies to suggest that targeting the endocannabinoid (ECB) system may be a viable strategy for pharmacologically attenuating established fear memories. While THC acts as an agonist for the CB1 and CB2 receptors, both of which have been implicated in fear-learning (Lafenêtre et al., 2007; Ruehle et al., 2012), the actions of CBD are complex. CBD exerts some of its effects indirectly by inhibiting the actions of endogenous CB1 and CB2 agonists. CBD has been shown to act as a potent antagonist for CB1 and CB2 ligands, while displaying low binding affinity for the CB1 receptor (Bisogno et al., 2001; Thomas et al., 2004, 2007). CBD has also been shown to act as an indirect agonist of 5-HT_1A receptors, and may exert some of its effects via this mechanism (Rock et al., 2012; McPartland et al., 2015). CB1 receptors are expressed in the hippocampus, basolateral and lateral amygdala, and medial prefrontal cortex (mPFC; Tsou et al., 1998)—key regions implicated in fear learning—but are absent in the central and medial nuclei of the amygdala (Katona et al., 2001), regions involved in fear expression. Thus, CB1 likely affects fear expression via an indirect neuromodulatory mechanism. CB1 receptors are found on GABAergic neurons of the basolateral amygdala (BLA), and their activation dampens BLA inhibitory interneuron activity. This disinhibition increases output from BLA projections (Marsicano et al., 2002; Pistis et al., 2004). BLA stimulation induces long-term potentiation (LTP) along the BLA-prelimbic (PLC) pathway, and blockade of CB1 transmission prevents this (Tan et al., 2010). The same study also demonstrated that pharmacological blockade of BLA-PLC CB1 signaling blocks encoding of fear learning. Thus there is a strong theoretical framework for the notion that pharmacologically modulating the ECB system may allow for the attenuation of traumatic memories. It is possible that the blockade of CB1 agonists by CBD impedes BLA-PLC signaling, thereby exerting its effects on fear learning.

The distribution of central receptors in the ECB system has already been implicated in human PTSD. Evidence from positron emission tomography (PET) imaging suggests that among those with PTSD there is a widespread upregulation of CB1 receptor density particularly in regions implicated in learned fear (the amygdala, hippocampus, orbitofrontal cortex, and anterior cingulate; Neumeister et al., 2013). Combined with behavioral evidence of cannabinoid involvement in fear-learning, this suggests the endocannabinoid system may be involved in the mediation of fear memories and may represent a viable target for the mitigation of some PTSD symptoms. Indeed, some research points to positive effects of oral THC in the reduction of hyper-arousal and frequency of nightmares among those affected by PTSD (Roitman et al., 2014).

Some findings have also suggested that THC and CBD may disrupt the reconsolidation of recalled fear memories (Lin et al., 2006; Stern et al., 2012), a novel therapeutic strategy that may have relevance for attenuating established memories of trauma. Reconsolidation blockade is the process by which the expression of formed memories is reduced by drugs administered following recall (during the reconsolidation window; Nader et al., 2000; Nader and Hardt, 2009; Pitman et al., 2011). This procedure may offer new avenues for treatment of fear-based disorders that are resistant to extinction. Although several medicinal plants and isolated compounds have been reported to affect fear expression and reconsolidation in rodents (Nader et al., 2000; Lin et al., 2006; Da Silva et al., 2008; Bustos et al., 2009; Stern et al., 2012; Murkar et al., 2016; de Carvalho and Takahashi, 2017), reconsolidation paradigms have had mixed success when translated in human studies (Brunet et al., 2008; Pitman et al., 2011; Spring et al., 2015). Thus, new targets are needed for future translational studies with humans and Cannabis spp. extracts may offer one such target.

Although anecdotal findings regarding CBD and THC are compelling, there is a dearth of information around the effectiveness of cannabinoids at blocking fear memory reconsolidation. There is some evidence to suggest that both CBD and THC may block fear memory reconsolidation (Stern et al., 2012, 2015). This is curious, since THC and CBD often produce opposite effects. The effects of combined doses of CBD and THC on fear memory reconsolidation have also been sparsely explored (Stern et al., 2015), albeit at very low doses. It is important to assess this, as the consumption of marijuana would entail exposure to both the main cannabinoids simultaneously. In addition, there may be other active components present in the plant material that may be biologically active and may modulate the effects of the key cannabinoids. However, the effects of other components of the plant in combination with cannabinoids (such as the terpenes) remain largely unexplored. In terms of relevance for human use of MMP, it is important to explore the effects of all components of the plant (since MMP, which is typically administered as whole plant material, does not solely consist of THC and CBD). It is also clear that the concentrations of THC and CBD can vary based on the specific species of the plant; thus it is important to identify and standardize the concentrations of THC and CBD in administered extracts and to verify the effects of the remainder of the plant extracts containing varying concentrations of THC and CBD.

Herein, our experiments examined whether combined doses of THC and CBD would block fear memory reconsolidation, as well as whether the effects of the phytocannabinoids are modulated by the remaining plant background material (BM; all remaining plant components following CBD/THC extraction). In order to simulate MMP preparations consisting of whole plant material (which is much more relevant for human medical cannabis use, which may contain other active non-cannabinoids affecting fear memory), we tested doses of isolated phytochemicals THC and CBD singly, or in combination with each other, and in combination with plant BM. In addition, in order to simulate the effects of varying concentrations of THC in plant material, we tested the effects of co-administration of CBD with both a low- and high-dose of THC (both with and without BM).

Our findings suggest that CBD can modulate reconsolidation of learned fear, potentially opening up new treatment avenues for fear-based disorders. Our results also demonstrated that THC at the doses used (the primary psychoactive component of the plant) had no discernible effects on its own, but when co-administered with CBD and/or whole plant BM, was effective in modulating the response (suggesting the effects of THC on fear learning are influenced by other components of the plant). Our studies also revealed an inverted “u” dose response, such that at low- and high-dose THC had opposite effects when co-administered with plant BM. Low-dose THC, but not high-dose THC, attenuated reconsolidation of learned fear, when co-administered with BM. However, these effects were dependent on mediation by co-administration of either CBD or BM. In contrast to previous work (Stern et al., 2015), we found no significant effects of pure THC on reconsolidation of contextual learned fear. We also observed no attenuation of the learned fear expression, when drugs were administered without recall (re-exposure to the conditioned stimulus), suggesting the effect was dependent upon recall of the fearful memory.

These findings partially support prior work which suggests the effects of THC are synergized by the addition of other compounds (either in combination with CBD or whole plant material; Carlini et al., 1974; Fairbairn and Pickens, 1981; Mechoulam and Ben-Shabat, 1999). Research suggests that the behavioral effects of THC are modified by co-administration of other compounds, increasing some potentially therapeutic effects while diminishing sedative and anxiogenic effects (Russo, 2011), and our findings would seem to partially confirm this. However, since BM significantly attenuated the reconsolidation of learned fear on its own, it is unclear whether this is a synergistic effect of THC administered with whole plant material. Since the effects of 5 mg/kg THC were not augmented by co-administration (and since the effect of BM persisted when administered without the addition of THC), it is possible that therapeutic effects resulted primarily from the BM alone rather than THC-BM synergism.

The effects of cannabinoids on fear learning might also be mediated by other factors. CB1 receptors have been shown to play a role in modulating the release of other neurotransmitters such as acetylcholine and dopamine (Piomelli, 2003; Terzian et al., 2011; Micale et al., 2013). Knock-out mice lacking CB1 receptors on dopamine expressing neurons (type-1 receptors; D1Rs) have enhanced expression of cued fear (Terzian et al., 2011), and mice lacking CB1 receptors on neurons expressing D1Rs also exhibited deficits in safety learning in a step-down avoidance task in one study (Micale et al., 2017). Similarly, the effects of CB1 activation on anxiety-like behavior in rodents is partially dependent upon GABAergic and glutamatergic factors (Rey et al., 2012). It is therefore likely that the neuromodulatory effects of CB1 activation on a variety of neurochemical networks play a complex role in the effects of cannabinoids on fear learning as well.

Since BM was essentially devoid of THC and CBD but contained all remaining plant components, a number of molecules could potentially have had effects on reconsolidation of learned fear on their own. Furthermore, THC and CBD precursors cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA) can be decarboxylated to CBD and THC (Marks et al., 2009), and were present in the BM in small quantities, and could potentially be transformed to active cannabinoid molecules THC and CBD over time. The quantity of THC in our BM sample was 3 ± 0.5% and a minute quantity of CBD (less than 0.3%). The resulting dose of active THC may have been sub-anxiolytic on its own; however, it is likely that the effects of low-dose THC in the BM could be potentiated by some of the other molecules in the BM (e.g., terpenoids). In such a case, we could anticipate a potentiation of the behavioral effects of 5 mg/kg THC by the BM when co-administered. In our experiments this was not the case, but a floor effect due to very low freezing levels among both conditions (BM alone and 5 mg/kg THC plus BM) may have masked such an effect.

Another possibility is that other non-THC, non-CBD constituents of the plant modulated fear learning on their own. Cannabis spp. BM contains a number of terpenoid molecules, some of which have been shown to affect anxiety and fear learning. β-Caryophyllene (BCP), for example, is present in Cannabis spp. and has been shown to exert anxiolytic-like activity in rodents (Bahi et al., 2014). Anxiolytic effects of BCP are blocked by CB2 antagonist AM630 (Bahi et al., 2014), but not by 5-HT_1A antagonist NAN-190 or the GABA_A Benzodiazepine partial agonist Flumazenil (Galdino et al., 2012). This suggests BCP may act through CB2 receptors to produce anxiolytic-like effects in rodents. However, the exact mechanism by which BCP exerts its effects remains unknown. In addition, the terpenoids present may vary significantly among different plant strains (in our case, a detailed analysis of the terpenoids present in the BM is not available). Clearly, further research is needed in this regard. Our experiments are also not without limitations. Here, we did not conduct an in-depth dose-response of BM. Furthermore, our animals were pair-housed which could potentially result in the social transmission of fear, and be a confounding factor. In our experience, however, the stress of isolation through single housing is potentially a more severe confounding factor. Last but not least, one might highlight the potential confounding effects of locomotor effects of THC on subsequent freezing as a limiting factor. However, the elimination of THC in the rat is biphasic, with an initial rapid drop in THC levels in the first 120 min post-administration followed by a slower elimination half-life of THC afterward (Klausner and Dingell, 1971; Tseng et al., 2004). 11-hydroxy- Δ9-THC (the primary active metabolite of THC) is rapidly eliminated, and levels of 11-OH- Δ9-THC return to baseline only 240 min post-administration in both male and female rats (Tseng et al., 2004). As a result, the behavioral effects of THC in the rat are short-lived. This is reflected in our data, since our THC 50 mg/kg group exhibited freezing levels that were nearly identical to those of the vehicle-treated animals, 24 h later.

In order to simulate MMP, dosages of BM in our studies were maintained at a constant 30% of total compounds administered for each group. Future studies should aim to conduct dose-response experiments with BM alone and in combination with THC and CBD. Studies should also aim to explore the effects of isolated terpenes (Cannabis-derived) alone and in combination with THC and/or CBD. Finally, future studies could benefit from central microinjection studies aimed at sites known to play a role in fear learning (e.g., BLA, CA1, mPFC), and attempt to block the effects with co-administration of antagonists, to help identify the potential locus (loci) of action.

With regards to humans, behavioral studies suggest that the response to marijuana in individuals self-medicating for PTSD varies with symptom type and severity. Wilkinson et al. (2015) for example, found that symptom severity and violent behavior are significantly worse among veterans with PTSD who self-medicated with marijuana. It may be the case that those individuals with greater symptom severity were more likely to seek out alternate means to self-medicate. Indeed, veterans with PTSD are more likely to use marijuana and synthetic cannabis products than veterans without PTSD (Grant et al., 2016). Further evidence suggests that individuals with PTSD with marijuana dependence have blunted emotional reactivity, supporting the notion that cannabinoids may affect fear expression (Tull et al., 2016). Our results suggest that by using reconsolidation paradigms, prolonged treatment (or chronic use) may not be necessary in order to alleviate learned fear. Also, since CBD by itself was effective in our study, administration of CBD alone (i.e., a non-psychoactive component) may potentially be effective without exposing individuals to long-term treatment with psychoactive substances.

While these insights are valuable, the bulk of recent research on the effects of cannabinoids in humans with PTSD has observed the effects through the lens of self-medication (rather than clinical trial), and the lack of experimental guidance of drug administration in studies utilizing raw plant material leaves open the question of whether differences in plant composition may have led to variability in results (and whether different components of the plants—e.g., THC, CBD, etc.—are present in differing ratios and hence differentially affect PTSD symptomology). Since our findings revealed an inverted-u shaped dose response for THC in combination with BM and CBD, it is important that (as we have done here) studies using plant-derived cannabinoids characterize the specific THC and CBD content of extracts and raw plant material. Our studies also demonstrated that the plant BM is not necessarily inert, and also exerts effects on reconsolidation of fear memory. This suggests THC and CBD are not the only fear memory modulating molecules contained within the plant. Although CBD and THC (when co-administered with other compounds) may modulate fear learning, future research should be cautious to identify and quantify the THC, CBD, and other compounds that may be contributing to the measured effects on behavior.

Karniol et al. examined the effects of CBD alone as early as 1974 (Karniol et al., 1974), and found that oral CBD reduced THC-induced anxiety. The same group later demonstrated that CBD could block the effects of THC in normal, healthy participants (Zuardi et al., 1982). More recently, synthetic cannabinoid Nabilone has been demonstrated to effectively reduce the frequency of nightmares in sufferers of PTSD (Fraser, 2009; Cameron et al., 2014). Interestingly, oral THC has been shown to reduce amygdala activation in response to images of threat-related faces (Ballard et al., 2012); however, this contradicts earlier findings showing THC alone may be anxiogenic (Karniol et al., 1974). As a result, it is clear that our current understanding of the role of the endocannabinoid system in PTSD, anxiety, and learned fear is far from complete. Our findings seem to suggest that—at least at the doses used—THC by itself is not sufficient to modulate fear learning, but needs to be co-administered either with CBD, or with other compounds in the cannabis plant, to be effective. Since evidence suggests that PTSD is characterized by upregulation of CB1 receptors and reduced availability of anandamide, it may be the case that CB1 receptor activation by pharmacologic agents might serve to compensate for this receptor upregulation and restore the normal “tone” of the endocannabinoid system over time. However, effects of acute vs. chronic activation by pharmacological agents may not necessarily be the same. Future research should, therefore, aim to clarify the acute effects of CB1 agonists vs. chronic use, as well as examining differences in effects among normal, healthy subjects vs. those at risk of having altered endocannabinoid activity (e.g., sufferers of PTSD).

Both past and recent data cumulatively support the notion that CBD may impart anxiolytic action (Blessing et al., 2015). In addition, the action of THC in animal and human models of fear-learning warrants further clinical research to elucidate whether cannabinoids may serve as a novel intervention(s) for fear-related disorders such as PTSD. It will be important for these trials to identify the other potentially non-psychoactive components of Cannabis spp. to determine if and how they mediate fear learning. It goes without saying that ongoing and future studies aimed at unraveling the mechanism(s) of action of THC and CBD are critically important to fully exploit the therapeutic potential of these pharmacologic targets. Finally, it would be interesting to better understand if and how various pharmacologically active components of Cannabis spp. may interact to modulate the signaling of relevant brain circuits (e.g., pathways linking cortex and the amygdala) to affect encoding, consolidation, and reconsolidation of learned fear.

All experiments were conducted in accordance with the guidelines established by the Canadian Council on Animal Care and approved by the University of Ottawa Animal Care Committee.

AM wrote the article, analyzed data, performed experiments, and contributed to study design. JJ and CC performed experiments. ZM and PK contributed to study design and revised the article. TD provided the plant extracts and pure compounds used in the experiments.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.