Introduction

Background 

Despite the need, there are currently only 2 FDA-approved medications for treating PTSD, the selective serotonin reuptake inhibitors (SSRIs) sertraline and paroxetine [8-10]. If these are ineffective or have undesirable side effects, additional off label medications include other SSRIs (e.g., fluoxetine), selective norepinephrine reuptake inhibitors (e.g., venlafaxine) and/ or antiepileptic drugs (e.g., topiramate [9]. Additionally, beta adrenergic antagonists can treat hyperarousal and have been shown to enhance fear extinction in patients with PTSD [11]. Unfortunately, SSRIs only help 50% of PTSD patients and less than 30% of patients experience full remission even after years of treatment [12-14]. While antidepressants may help with mood and anxiety in some patients there is no approved treatment for the cognitive symptomology of PTSD [14]. Psychotherapy for PTSD includes cognitive behavioral therapy, and eye movement desensitization and reprocessing [9]. However, deficits in extinction learning in patients with PTSD reduce the effectiveness of psychotherapy; therefore, to improve PTSD treatment, future research targeting novel extinction-enhancing pharmacotherapies that can be used in conjunction with psychotherapy is desperately needed [1,15].

Cannabis and the endocannabinoid system

Cannabis is a plant containing over 100 cannabinoids that are non-psychoactive and non-addictive including the most wellknown, cannabidiol [16,17]. The infamous and main psychoactive compound in cannabis is ∆9 tetrahydrocannabinol [16]. Two cannabinoid receptors, CB₁ and CB₂, are believed to be primarily responsible for the pharmacological actions of cannabis [1,18]. Most of cannabis’ known effects are produced via the CB₁ receptor, which is densely expressed in the brain [1,18], particularly in areas that regulate stress, emotions, learning, anxiety-like behaviors and fear learning, including the prefrontal cortex, hippocampus and amygdala [1,19,20]. Blocking or deleting CB₁ receptors impairs extinction of fear learning and induces an anxious-like phenotype in rodents [1,21]. CB₁ receptor agonists decrease the reconsolidation of fear memories while enhancing fear extinction in rodents [11].

Additionally, CBD has been shown to produce anxiolytic-like effects by binding to both CB₁ and CB₂ receptors [1,22]. These data suggest that CB₁ and CB₂ regulate stress, anxiety, cognition and learning processes, highlighting these receptors as potential targets for treating PTSD [1,19,20,22].

Indeed, several recent clinical studies recording real-time cannabis use reported reductions in feelings of irritability/agitation and stress, along with increases in mood nearly immediately following cannabis consumption [23-25]. These data suggest that cannabis may be used to treat several symptoms associated with PTSD, however, one remaining unanswered question is does cannabis facilitate the hallmark feature of PTSD: extinction of memories associated with the traumatic event. To address this knowledge gap, the present study utilized the stress-enhanced fear learning (SEFL) model to examine the effects of full spectrum hemp oil on extinction of fear memory in adult male rats. We hypothesized that administering full spectrum hemp oil prior to extinction sessions would facilitate extinction of fear memory.

Materials and methods

Animals

Subjects were experimentally naïve male Long-Evans hooded rats (N=35, PND 110-111 at the time of trauma exposure) born and reared in the Logan Hall Animal Research Facility at the University of New Mexico. Following weaning (~PND 21), rats were pair housed in standard home cages (21.6 x 45.7 x 17.8 cm) in a temperature-controlled colony room (~22°C) with a reverse 12:12-h light-dark cycle (lights off at 10 am); 3 days prior to the start of the experiment rats were single housed for the remainder of the experiment. Food and water were available ad libitum in the rat’s home cages. Experimental and husbandry procedures followed the Guide for the Care and Use of Laboratory Animals Committee [26] and were approved by the University of New Mexico’s Institutional Animal Care and Use Committee.

Prior to the start of behavioral testing, rats were handled for 10 days (60-90 seconds each day) to adapt to experimenter handling stress (see Figure 1 for the experimental timeline). Next, rats were habituated to eating peanut butter for 15 days, which was used as the vehicle for cannabis administration. Finally, rats were randomly assigned to one of four experimental groups: no-trauma/ vehicle, no-trauma/hemp oil, trauma/vehicle and trauma/hemp oil.

Figure 1: Experimental timeline. The experimental design consisted of 10 days of handling, 15 days of habituation to the peanut butter vehicle, 1 day of trauma exposure (context A), 1 day of traumatic memory testing (context A), 1 day of new fear learning (context B), 1 day of new fear memory testing (context B) and 5 extinction trials (context B), each separated by 1 week.

Drugs

Full-spectrum hemp oil was cultivated by Organic-Energetic Solutions LLC (Albuquerque, NM, USA) and is commercially available under the “LyFeBaak” label. Briefly, the cannabis goes through an extraction procedure where the cannabinoids, terpenes, lipids, chlorophyll and wax compounds are stripped from mature hemp flower (“buds”) using an ethanol bath. Once all of the compounds from the plant are extracted, the alcohol solution is poured through a filter to remove any plant material. Next, the alcohol is evaporated, leaving only the cannabis compounds in an ultra-concentrated form. This ultra-concentrate is then combined with medium-chain triglyceride (MCT) oil to increase its bioavailability, for its final retail form.

Rats assigned to the two hemp oil groups received 1 mL or approximately .002mg/kg, of full-spectrum hemp oil [27] dissolved in a peanut butter (.152 g) vehicle; rats in the two control groups received the peanut vehicle. Peanut butter was chosen for the vehicle as it is high in fat and cannabis is fat soluble, thus increasing the bioavailability of the hemp oil.

Stress-Enhanced Fear Learning

The Stress-Enhanced Fear Learning (SEFL) procedures were adapted from an animal model of PTSD [28,29]. While no animal models currently capture all of the human complexities associated with PTSD, the SEFL model captures 3 critical components. First, in this model, the trauma consists of an uncued, single traumatic event, realistic to the development of PTSD in humans [28-30]. Second, the SEFL model affects new fear learning, shown by the exaggerated fear response to a new context, similar to symptomology seen in PTSD patients [28-30]. Third, this model leads to lasting behavioral changes including resistance to extinction [29]. To our knowledge, no other research has used this model to study the effects of cannabis on extinction of SEFL.

Stress-enhanced fear learning took place in two distinct contexts (A & B) that differed in lighting, flooring, sound and odor [29]. Each context consisted of a larger sound attenuating chamber that housed a smaller fear-conditioning shock chamber (25.4 X 29.21 X 29.21 cm; Coulbourn Instruments LLC; Whitehall, PA). Context A consisted of a white visible light and a fan set at 60 dB for background noise [28,30]. The shock chamber inside context A consisted of smooth steel rod flooring with 27 steel bars (4mm diameter) arranged vertically every .635 cm. 1% acetic acid was used to clean the chamber and underneath pan after each use and 11% coconut extract was used to scent the pan [28-30]. Context B consisted of red lighting and a white noise generator set at 60 dB for background noise [28-30]. The shock chamber inside context B consisted of 2 black plexiglass side walls at a 60° angle to create an A-frame and had heavily textured steel rod flooring with 18 steel bars (6mm diameter) arranged vertically every 1.27 cm [30]. 5% ammonium hydroxide was used to clean the chamber and underneath pan after each use and was also used to scent the pan [28]. In order to further minimize fear generalization, the two contexts were further differentiated by modes of transportation. Rats were either transported on a cart, in their uncovered home cage to context A, or were transported by hand in a clean, empty covered cage to context B [29]. 

The SEFL procedure occurred over 4 consecutive days followed by 5 extinction trials; each extinction trial was separated by 1 week (see Figure 1). Day one consisted of the trauma or notrauma exposure in context A. Trauma rats were placed into the shock box in context A and received 240 seconds of pre-exposure to the environment. After this pre-exposure period, rats received 15 1-second footshocks (1milliamp) randomly over a 90-minute period [28-30]. Rats in the no-trauma group were placed in the shock box in context A for the same amount of time but did not receive any footshocks [28-30]. On day 2, rats were placed back into context A for 8 minutes without receiving any footshocks. Rat behavior was recorded to quantify freezing as the primary index of fear (Blanchard & Blanchard, 1969; Fanselow, 1980). Freezing in context A on day two reflects the fear memory associated with the trauma. On day 3, all rats were placed into the novel context B for a 180-second baseline period; freezing during this baseline period reflects fear generalization [29]. Following the baseline period, all rats received a single 1-second footshock (1milliamp) and remained in the box for 30 seconds to observe the fear response (freezing) to the single shock [29]. On day 4, rats were placed back into context B for 8 minutes; freezing in context B reflects SEFL from context A [29].

Extinction commenced one week after day 4 and occurred once a week for 5 weeks. Prior to each extinction trial, rats were pretreated with their assigned drug (vehicle or hemp oil) 2 hours prior to being placed in context B for an 8-minute extinction session to examine the effects of full spectrum hemp oil on the extinction of stress-enhanced fear-learning behavior [31].

Data Analysis

Freezing was operationally defined as the lack of movement except for respiration (Blanchard & Blanchard, 1969; Fanselow, 1980). Percent freezing was analyzed on days 2, 3 and 4, as well as all 5 extinction trials by an experimenter blind to group conditionings. Percent freezing was calculated using the formula [(A1-A2)/A1] *100 where A1 represents the total time spent in the context and A2 represents the time spent moving in the context. In order to verify that the conditioning procedures produced SEFL, separate independent samples t-tests were run comparing trauma and no-trauma groups on percent freezing across days. First, to confirm that the traumatic memory was intact following the repeated footshocks, percent freezing was analyzed on day two during re-exposure to context A. In order to see if fear generalization occurred between contexts, we analyzed freezing in the novel context (B) during the baseline period on day 3. Next, following the single shock in context B, freezing was analyzed to examine whether an exaggerated fear response occurred after the single shock on day 3. In order to examine SEFL, we analyzed freezing in context B on day 4. Lastly, to examine the effects of full spectrum hemp oil on fear extinction, we used a repeated measures ANOVA with trauma and hemp oil groups as between subject factors and the 5 extinction trials as within subject factors. Independent sample t-tests were used to further probe significant interactions where appropriate. Levene’s Test for equality of variance was performed on each dependent variable, and Cohen’s d or Hedge’s g (correction) were computed to provide effect sizes for significant results. SPSS 29 was used to perform all statistical analyses with α = 0.05 for all comparisons. All data are reported as the mean ± standard error of the mean using Prism 9.0.

Results

Figure 2A indicates that rats in the trauma group froze significantly more in context A on day 2 compared to their notrauma counterparts indicating that the memory of the traumatic event was intact [t(1, 23.64) = 15.86, p<.001, g=4.92] Importantly, there was no significant difference between groups during the baseline period before the single shock on day 3, demonstrating that context A and B were sufficiently different and no fear generalization between the two contexts occurred [see Figure 2B; t(1, 22.79) = 2.02, p>.05, g=.63]. Figure 2Cindicates that rats from the trauma group froze significantly more than their no-trauma counterparts [t(1,33) = 6.82, p<.001, d=2.32] following the single 1-second 1-milliamp footshock, suggesting that previous exposure to the traumatic stressor created an exaggerated fear response to the mild stressor. Lastly, the trauma group froze significantly more than their no-trauma counterparts during re-exposure to context B (see Figure 2D), indicating that the exposure to the traumatic experience on day 1 enhanced fear learning to the mild stressor on day 3 [t(1,33) = 7.88, p<.001, d=2.68].

The effects of full spectrum hemp oil on extinction over the course of the 5 trials are shown in Figure 3. The repeated measures ANOVA detected a significant main effect of extinction [F(4,124) = 20.87, p<.001)]and an extinction by trauma interaction [F(4,124) = 18.48, p<.001]. Subsequent t-tests (see Figure 3A) indicate that rats in the trauma group spent more time freezing than the notrauma controls on extinction days 1 [t(1,29.18) = 6.45, p<.001, g=2.04], 2 [t(1,33) = 5.04, p<.001, d=1.71], 3 [t(1,33) = 4.28, p<.001, d=1.45] and 4 [t(1,29.09) = 2.88, p<.005, g=.91], but day 5 was only a trend [t(1,33) = 1.58, p=.12, d=.54]. These data indicate that compared to no-trauma controls, rats in the trauma group showed delayed extinction. The ANOVA failed to detect a significant interaction between extinction and hemp oil [F(4,124) = .85, p>.05] and the three-way interaction between extinction, trauma and hemp oil was not significant [F(4, 124) = .28, p>.05]. These data suggest that full spectrum hemp oil did not facilitate rates of extinction in the trauma (see Figure 3B) or no-trauma (see Figure 3C) groups.

 

Figure 2: Freezing in context A and B on days 1 through 4. (A) Freezing in context A on day 2. The trauma group spent significantly more time freezing than the no-trauma group the day after the trauma (15 footshocks), indicating that the memory of the traumatic experience was intact. (B) Freezing baseline in context B on day 3. Prior to receiving the single footshock in context B, there was no significant difference in time spent freezing between the trauma and no-trauma groups (independent samples t-test, p>.05), indicating that there was no fear generalization between the two distinct contexts. (C) Freezing after the single shock in context B on day 3. The trauma group spent significantly more time freezing than the no-trauma group immediately following the single footshock on day 3, indicating an exaggerated fear response. (D) Freezing in context B on day 4. The trauma group spent significantly more time freezing than the no-trauma group the day after the single footshock, indicating enhanced fear learning in the trauma group. Asterisk (*) represents a significant difference compared to no-trauma controls (independent samples t-test, p<.001 in each case).

 

Figure 3: Freezing in context B across the 5 extinction trials.(A) Freezing in trauma and no trauma groups across the 5 extinction trials.The ANOVA indicated a main effect of extinction and a trauma by extinction interaction (p<.001 in each case). Subsequent independent sample t-tests indicated that rats in the trauma group froze significantly more than rats in the no-trauma group on extinction days 1 through 4 (p<.005 in each case), indicating that the trauma group showed resistance to extinction. (B – C) Effects of hemp oil on freezing across the 5 extinction trials in the trauma (B) and no-trauma (C) groups. In contrast, the ANOVA failed to detect a hemp oil by extinction interaction or a hemp oil by trauma by extinction interaction (p>.05 in each case), suggesting that full spectrum hemp oil did not facilitate fear extinction in the trauma or the no-trauma groups. Asterisk (*) and pound sign (#) represent significant differences compared to no-trauma controls (*p<.001; #p<.005).

Discussion

Our results replicate previous reports indicating that the SEFL model is a robust model of PTSD-like symptoms in adult male rats [28-30,32,33]. As expected, the trauma group (15 footshocks) showed higher levels of freezing compared to the no-trauma group (no footshocks) when they were returned to the traumatic context on day 2 (Figure 2A), indicating that the memory of the traumatic stressor was intact. Both groups showed minimal freezing in the novel context during the baseline period on day 3 (Figure 2B), suggesting that the enhanced freezing that occurred in this context following the single footshock (Figure 2C) was due to the mild stressor interacting with the prior trauma (context A) to produce an exaggerated fear response and was not simply the result of fear generalization from the trauma context. In addition to this heightened fear response following the single footshock, trauma rats re-exposed to context B on day 4 (Figure 2D), showed enhanced freezing compared to no-trauma rats indicating a stressenhanced fear response (i.e., SEFL). It should be noted that this enhanced fear response is not due to generalization (Figure 2B) or increased fear expression (freezing) as the traumatic stressor must come before the mild stressor to increase fear of the novel context [28]. Collectively, the present data along with results from previous studies [28-30,32,33] indicate that the SEFL model produces reliable and long-lasting enhanced fear learning that is consistent with the symptomology seen in PTSD patients, suggesting it is a valid rodent model to probe the neural mechanisms of PTSD.

Contrary to our prediction, results from the present study indicate that full spectrum hemp oil did not facilitate extinction of SEFL across the 5 extinction trials (Figure 3). One possible reason that we failed to detect a significant reduction in freezing is that the hemp oil dose (.002 mg/kg) was insufficient. A study by Franzen and colleagues found that CBD (3.0 and 10 mg/kg) administered before conditioned context exposure reduced freezing in adult female rats. Further, they found that these doses of CBD reduced anxiety-like behavior in the elevated plus-maze (EPM). Interestingly, in an unpublished pilot study conducted in our lab the lower dose of hemp oil (.002 mg/kg) produced similar results to Franzen et al.’s study on anxiety-like behavior in the EPM. However, because the EPM and fear conditioning models reflect different underlying constructs (i.e., anxiety vs. fear), as well as the different procedures between the fear conditioning procedures in the present study and Franzen study (i.e., SEFL vs. standard fear conditioning), a higher hemp oil dose may be required to reduce conditioned freezing in the SEFL model.

A second possible reason we did not detect effects of full spectrum hemp oil on extinction of SEFL might be that hemp oil alters locomotor activity. CBD is thought to have little to no effects on locomotion, but when locomotor activity is altered, CBD is thought to balance out these deficits. For example, one study suggests that CBD increases locomotor activity after a spinal injury while other studies suggest that CBD decreases locomotor activity in cases of Tardive Dyskinesis as well as from cocaineinduced hyperactivity [34-36]. CBD is also thought to decrease catalepsy and oral movements in patients with Parkinson’s Disease [37]. Research on the effects of CBD on spontaneous locomotor activity is mixed. One study concluded that in rodents, CBD does not affect locomotor activity in the rotarod test, however, it decreased locomotor activity in the open field test [38]. This directly contradicts another study that reported CBD did not alter locomotor activity in the open field test [39]. Other studies suggest that CBD does not affect locomotor activity in tests like the light/ dark test or the EPM, however, CBD did increase time freezing in both the conditioned emotional response test as well as in fear conditioning models [37,40,41]. The latter result contradicts Franzen et al.’s study that found CBD decreased freezing in a standard contextual fear conditioning model [42]. In our study, the full spectrum hemp oil treated rats did not show altered freezing when compared to their non-hemp oil counterparts, however, the possible effect of CBD on freezing in fear conditioning models may explain why we found no effect of full spectrum hemp oil on conditioned freezing following SEFL.

A third possible reason we did not detect effects of full spectrum hemp oil on extinction of SEFL may be due to what is known as the “entourage effect”. The entourage effect occurs when all of the 100 or more compounds in the cannabis plant are combined, including cannabinoids, flavonoids and terpenes, and are hypothesized to work together to produce the desired effects of cannabis [43]. However, Federal laws limit the legal amount of THC in any type of Cannabis plant to be equal to or less than 0.3% weight/dried flower [44]. In the present study, we choose to examine the effects full spectrum hemp oil containing less than 0.3% THC as there is a large population of people who likely want the health effects of cannabis without the psychoactive effects of THC and follow the federal legality of hemp oil. In either case, a higher percentage of THC might be needed for the entourage effect to take effect and facilitate fear extinction following SEFL. Indeed, a recent study looking at THC to CBD ratios and their effects on memory found that cannabis use with higher THC content had the greatest effect on memory; therefore, it is possible that a higher THC content would better facilitate the extinction of SEFL [45].

Finally, we may have failed to detect an effect of full spectrum hemp oil on the facilitation of extinction as a result of the extinction protocol that was utilized. For example, hemp oil may have facilitated fear extinction if the trials were run consecutively across days instead of weekly. Additionally, it is possible that more than 5 extinction sessions, either run consecutively or weekly, may be needed for hemp oil to accelerate fear extinction. Future studies are needed to address these possible explanations.

Conclusions

Collectively, the present results indicate that full spectrum hemp oil did not facilitate extinction of SEFL. Given that THC may have greater effects on memory, a broader THC:CBD ratio dose range should be investigated using this experimental design. Previous studies have reported conflicting results with CBD impacting locomotion while other studies report no change [3741]. Further research needs to be conducted to understand the relationship between CBD and locomotion, as well as full spectrum hemp oil, as this could impact the present results. While we found no effect of full spectrum hemp oil on extinction in males, Franzen et al., [42] found an effect in females suggesting sex may play a significant role in on how cannabis affects the extinction of fear memories. Thus, future research examining the effects of hemp oil on extinction of SEFL in female rats is needed. Lastly, a promising direction for future PTSD research could be to combine cannabis with other pharmacotherapies and/or psychotherapies. This approach might yield a more effective PTSD treatment since monotherapy is rarely successful in the treatment and remission of PTSD in humans. Future research should continue to investigate the link between the endocannabinoid system and its potential treatment for PTSD. Replication and extension of the present results using additional doses and/or cannabis combinations are needed to fully clarify if cannabis represents a viable treatment for PTSD.

References

1. Locci A, Pinna G (2019) Social isolation as a promising animal model of PTSD comorbid suicide: Neurosteroids and cannabinoids as possible treatment options. Prog Neuropsychopharmacol Biol Psychiatry 92:243-259.
2. American Psychiatric Association. (2017). Section II: Diagnostic criteria and codes. In Diagnostic and statistical manual of mental disorders: DSM-5. essay, CBS Publishers & Distributors, Pvt. Ltd. Retrieved February 1, 2023.
3. Bae SM, Kang JM, Chang HY, Han W, Lee SH (2018) PTSD correlates with somatization in sexually abused children: Type of abuse moderates the effect of PTSD on somatization. PLoS One 13:e0199138.
4. Dardis CM, Dichter ME, Iverson KM (2018) Empowerment, PTSD and revictimization among women who have experienced intimate partner violence. Psychiatry Res 266:103-110.
5. Hoge CW, Riviere LA, Wilk JE, Herrell RK, Weathers FW (2014) The prevalence of post-traumatic stress disorder (PTSD) in US combat soldiers: A head-to-head comparison of DSM-5 versus DSM-IV-TR symptom criteria with the PTSD Checklist. The Lancet Psychiatry 1:269-277.
6. Kilpatrick DG, Resnick HS, Milanak ME, Miller MW, Keyes KM, et al., (2013) National estimates of exposure to traumatic events and PTSD prevalence using DSM-IV and DSM-5 criteria. Journal of Traumatic Stress, 26:537-547.
7. Pompili M, Sher L, Serafini G, Forte A, Innamorati M, et al., (2013) Posttraumatic stress disorder and suicide risk among veterans. J Nerv Ment Dis 201:802-812.
8. Alexander W (2012) Pharmacotherapy for Post-traumatic Stress Disorder In Combat Veterans: Focus on Antidepressants and Atypical Antipsychotic Agents. P T 37:32-38.
9. Jeffreys M (2009, July 1). Va.gov: Veterans Affairs. Clinician’s Guide to Medications for PTSD. Retrieved February 1, 2023.
10. Krystal JH, Davis LL, Neylan TC, Raskind M, Schnurr PP, et al., (2017) It is time to address the crisis in the pharmacotherapy of posttraumatic stress disorder: A consensus statement of the PTSD Psychopharmacology Working Group. Biological Psychiatry 82: E51-E59.
11. Steckler T, Risbrough V (2012) Pharmacological treatment of PTSD – established and new approaches. Neuropharmacology 62:617-627.
12. Berger W, Mendlowicz MV, Marques-Portella C, Kinrys G, Fontenelle LF, et al., (2009) Pharmacologic alternatives to antidepressants in posttraumatic stress disorder: A systematic review. Prog Neuropsychopharmacol Biol Psychiatry 33:169-180.
13. Darves-Bornoz J.-M, Alonso J, de Girolamo G, Graaf R. de, Haro J.-M, et al., (2008) Main traumatic events in Europe: PTSD in the European study of the epidemiology of Mental Disorders Survey. Journal of Traumatic Stress 21:455-462.
14. Trezza V, Campolongo P (2013) The endocannabinoid system as a possible target to treat both the cognitive and emotional features of post-traumatic stress disorder (PTSD). Front Behav Neurosci 7.
15. Shallcross J, Hámor P, Bechard AR, Romano M, Knackstedt L, 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. Front Behav Neurosci 13.
16. Rock EM, Limebeer CL, Petrie GN, Williams LA, Mechoulam R, et al., (2017) Effect of prior foot shock stress and Δ9-tetrahydrocannabinol, cannabidiolic acid, and cannabidiol on anxiety-like responding in the light-dark emergence test in rats. Psychopharmacology 234:22072217.
17. Russo EB (2007) History of cannabis and its preparations in saga, science, and sobriquet. ChemInform 38.
18. Pertwee RG (2008) The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: D9 -tetrahydrocannabinol, cannabidiol and D9 -tetrahydrocannabivarin. British Journal of Pharmacology 153: 199-215.
19. McPartland JM, Glass M, Pertwee RG (2007) Meta-analysis of cannabinoid ligand binding affinity and receptor distribution: Interspecies differences. British Journal of Pharmacology 152:583593.
20. Viveros M, Marco E, File S (2005) Endocannabinoid system and stress and anxiety responses. Pharmacol Biochem Behav 81:331-342.
21. Marsicano G, Wotjak CT, Azad SC, Bisogno T, Rammes G, et al., (2002) The endogenous cannabinoid system controls extinction of Aversive Memories. Nature 418:530-534.
22. Austrich-Olivares A, García-Gutiérrez MS, Illescas L, Gasparyan A, Manzanares J (2022) Cannabinoid CB1 receptor involvement in the actions of CBD on anxiety and coping behaviors in mice. Pharmaceuticals 15:473.
23. Stith SS, Li X, Diviant JP, Brockelman FC, Keeling KS, et al., (2020) The effectiveness of inhaled cannabis flower for the treatment of agitation/irritability, anxiety, and common stress. J Cannabis Res 2:47.
24. Stith SS, Vigil JM, Brockelman F, Keeling K, Hall B (2018) Patientreported symptom relief following medical cannabis consumption. Front. Pharmacol 9.
25. Vigil JM, Stith SS, Chanel T (2022) Cannabis consumption and prosociality. Scientific Reports 12: 8352.
26. National Research Council (2011) Guide for the care and use of Laboratory Animals. National Academies Press.
27. Fishbein-Kaminietsky M, Gafni M, Sarne Y (2014) Ultralow doses of cannabinoid drugs protect the mouse brain from inflammation-induced cognitive damage. J Neurosci Res 92:1669-1677.
28. Rau V, DeCola JP, Fanselow MS (2005) Stress-induced enhancement of fear learning: An animal model of posttraumatic stress disorder. Neurosci Biobehav Rev 29:1207-1223.
29. Rajbhandari AK, Gonzalez ST, Fanselow MS (2018) Stress-enhanced fear learning, a robust rodent model of post-traumatic stress disorder. J Vis Exp 58306.
30. Long VA, Fanselow MS (2011) Stress-enhanced fear learning in rats is resistant to the effects of immediate massed extinction. Stress 15:627636.
31. Deiana S, Watanabe A, Yamasaki Y, Amada N, Arthur M, et al., (2011) Plasma and brain pharmacokinetic profile of cannabidiol (CBD), Cannabidivarine (CBDV), Δ9-tetrahydrocannabivarin (THCV) and Cannabigerol (CBG) in rats and mice following oral and Intraperitoneal administration and CBD action on obsessive–compulsive behaviour. Psychopharmacology 219:859-873.
32. Asch RH, Fowles K, Pietrzak RH, Taylor JR, Esterlis I (2023) Examining mGlu5 receptor availability as a predictor of vulnerability to PTSD: An [¹⁸F]FPEB and PET study in male and female rats. Chronic Stress 7:1-10.
33. Jones ME, Lebonville CL, Barrus D, Lysle DT (2015) The role of brain interleukin-1 in stress-enhanced fear learning. Neuropsychopharmaoclogy 40:1289-1296.
34. Kajero JA, Seedat S, Ohaeri J, Akindele A, Aina O (2020) Investigation of the effects of cannabidiol on vacuous chewing movements, locomotion, oxidative stress and blood glucose in rats treated with oral haloperidol. World J Biol Psychiatry 21:612-626.
35. Kwiatkoski M, Guimarães FS, Del-Bel E (2011) Cannabidiol-treated rats exhibited higher motor score after Cryogenic Spinal Cord Injury. Neurotox Res 21:271-280.
36. Ledesma JC, Manzanedo C, Aguilar MA (2021) Cannabidiol prevents several of the behavioral alterations related to cocaine addiction in miceProg Neuropsychopharmacol Biol Psychiatry 111:110390.
37. Calapai F, Cardia L, Calapai G, Di Mauro D, Trimarchi F, et al., (2022) Effects of cannabidiol on locomotor activity. Life 12:652.
38. Schleicher EM, Ott FW, Müller M, Silcher B, Sichler ME, et al., (2019) Prolonged cannabidiol treatment lacks on detrimental effects on memory, motor performance and anxiety in C57BL/6J MICE. Front Behav Neurosci 13: 94.
39. Viudez-Martínez A, García-Gutiérrez MS, Medrano-Relinque J, Navarrón CM, Navarrete F, et al., (2018) Cannabidiol does not display drug abuse potential in mice behavior. Acta Pharmacol Sin 40:358364.
40. ElBatsh MM, Assareh N, Marsden CA, Kendall DA (2011) Anxiogenic-like effects of chronic cannabidiol administration in rats. Psychopharmacology 221:239-247.
41. Koo JW, Duman RS (2009) Interleukin-1 receptor null mutant mice show decreased anxiety-like behavior and Enhanced Fear Memory. Neurosci Lett 456:39-43.
42. Franzen JM, Werle I, Vanz F, de Oliveira BB, Martins Nascimento LM, et al., (2023) Cannabidiol attenuates fear memory expression in female rats via hippocampal 5-HT1A but not CB1 or CB2 receptors. Neuropharmacology 223:109316.
43. Koltai H, Namdar D (2020) Cannabis phytomolecule “entourage”: From domestication to medical use. Trends Plant Sci 25:976-984.
44. Wakshlag JJ, Cital S, Eaton SJ, Prussin R, Hudalla C (2020) Cannabinoid, terpene, and heavy metal analysis of 29 over-thecounter commercial veterinary hemp supplements. Vet Med (Auckl) 11:45-55.
45. Curran T, Devillez H, YorkWilliams SL, Bidwell LC (2020) Acute effects of naturalistic THC vs. CBD use on recognition memory: A preliminary study. J Cannabis Res 2:28.