Cannabidiol and the Remainder of the Plant Extract Modulate the Effects of Δ9-Tetrahydrocannabinol on Fear Memory Reconsolidation.

Background: Δ9-Tetrahydrocannabinol (THC, a CB1 receptor agonist) and Cannabidiol (CBD, a non-competitive antagonist of endogenous CB1 and CB2 ligands) are two primary components of Cannabis species, and may modulate fear learning in mammals. The CB1 receptor is widely distributed throughout the cortex and some limbic regions typically associated with fear learning. Humans with posttraumatic disorder (PTSD) have widespread upregulation of CB1 receptor density and reduced availability of endogenous cannabinoid anandamide, suggesting a role for the endocannabinoid system in PTSD. Pharmacological blockade of memory reconsolidation following recall of a conditioned response modulates the expression of learned fear and may represent a viable target for the development of new treatments for PTSD. In this study, we focused on assessing the impact of the key compounds of the marijuana plant both singly and, more importantly, in concert on attenuation of learned fear. Specifically, we assessed the impact of THC, CBD, and/or the remaining plant materials (post-extraction; background material), on reconsolidation of learned fear. Method: Male Sprague-Dawley rats received six 1.0 mA continuous foot shocks (contextual training). Twenty-four hours later, rats were re-exposed to the context. Immediately following memory retrieval (recall) rats received oral administration of low dose THC, high dose THC, CBD, CBD + low THC, CBD + high THC [as isolated phytochemicals and, in separate experiments, in combination with plant background material (BM)]. Rodents were tested for freezing response context re-exposure at 24 h and 7 days following training. Results: CBD alone, but not THC alone, significantly attenuated fear memory reconsolidation when administered immediately after recall. The effect persisted for at least 7 days. A combination of CBD and THC also attenuated the fear response. Plant BM also significantly attenuated reconsolidation of learned fear both on its own and in combination with THC and CBD. Finally, THC attenuated reconsolidation of learned fear only when co-administered with CBD or plant BM. Conclusion: CBD may provide a novel treatment strategy for targeting fear-memories. Furthermore, plant BM also significantly attenuated the fear response. However, whereas THC alone had no significant effects, its effects were modulated by the addition of other compounds. Future research should investigate some of the other components present in the plant BM (such as terpenes) for their effects alone, or in combination with isolated pure cannabinoids, on fear learning.

Extract and Active Principal of the Neotropical Vine Souroubea sympetala Gilg. Block Fear Memory Reconsolidation.

Background: Souroubea sympetala Gilg. is a neotropical vine native to Central America, investigated as part of a targeted study of the plant family Marcgraviaceae. Our previous research showed that extract of S. sympetala leaf and small branch extract had anxiolytic effects in animals and acts as an agonist for the GABAA receptor at the benzodiazepine binding site. To date, the potential effects of S. sympetala and its constituents on reconsolidation have not been assessed. Reconsolidation, the process by which formed memories are rendered labile and susceptible to change, may offer a window of opportunity for pharmacological manipulation of learned fear. Here, we assessed the effects of S. sympetala crude extract and isolated phytochemicals (orally administered) on the reconsolidation of conditioned fear. In addition, we explored whether betulin (BE), a closely related molecule to betulinic acid (BA, an active principal component of S. sympetala), has effects on reconsolidation of learned fear and whether BE may synergize with BA to enhance attenuation of learned fear. Method: Male Sprague-Dawley rats received six 1.0-mA continuous foot shocks (contextual training). Twenty-four hours later, rats were re-exposed to the context (but in the absence of foot shocks). Immediately following memory retrieval (recall), rats received oral administration of S. sympetala extract at various doses (8-75 mg/kg) or diazepam (1 mg/kg). In separate experiments, we compared the effects of BA (2 mg/kg), BE (2 mg/kg), and BA + BE (2 mg/kg BA + 2 mg/kg BE). The freezing response was assessed either 24 h later (day 3) or 5 days later (day 7). Effects of phytochemicals on fear expression were also explored using the elevated plus maze paradigm. Results: S. sympetala leaf extract significantly attenuated the reconsolidation of contextual fear at the 25- and 75-mg/kg doses, but not at the 8-mg/kg dose. Furthermore, BA + BE, but not BA or BE alone, attenuated the reconsolidation of learned fear and exerted an anxiolytic-like effect on fear expression.

Palatable Food Dampens the Long-Term Behavioral and Endocrine Effects of Juvenile Stressor Exposure but May Also Provoke Metabolic Syndrome in Rats.

The juvenile period is marked by a reorganization and growth of important brain regions including structures associating with reward seeking behaviors such as the nucleus accumbens (NA) and prefrontal cortex (PFC). These changes are impacted by stressors during the juvenile period and may lead to a predisposition to stress induced psychopathology and abnormal development of brain reward systems. Like in humans, adult rodents engage certain coping mechanisms such as increases in the consumption of calorie-rich palatable foods to reduce stress, but this behavior can lead to obesity and metabolic disorders. In this study, we examined whether stressors during the juvenile period led to increased caloric intake when a palatable diet was accessible, and whether this diet attenuated adult stress responses. In addition, we examined if the stress buffering effects produced by the palatable diet were also accompanied by an offset propensity towards obesity, and by alterations in mRNA expression of dopamine (DA) receptors in the NA and PFC in adulthood. To this end, juvenile male Wistar rats underwent episodic stressor exposure (forced swim, elevated platform stress and restraint) on postnatal days (PD) 27-29 and received access to regular chow or daily limited access to a palatable diet until adulthood. At the age of 2 months, rats were tested on a social interaction test that screens for anxiety-like behaviors and their endocrine responses to an acute stressor. Animals were sacrificed, and their brains processed to detect differences in DA receptor subtype expression in the PFC and NA using qPCR. Results showed that rats that were stressed during the juvenile period displayed higher social anxiety and a sensitized corticosterone response as adults and these effects were attenuated by access to the palatable diet. Nevertheless, rats that experienced juvenile stress and consumed a palatable diet showed greater adiposity in adulthood. Interestingly, the same group displayed greater mRNA expression of DA receptors at the NA. This suggests that access to a palatable diet mitigates the behavioral and endocrine effects of juvenile stressor exposure in adulthood, but at the cost of metabolic imbalances and a sensitized dopaminergic system.

Sacred Maya incense, copal (Protium copal - Burseraceae), has antianxiety effects in animal models.

ETHNOPHARMACOLOGICAL RELEVANCE: The Maya have traditionally used copal, Protium copal, as incense during ceremonies since pre-Columbian times. Anecdotally, copal (when burned as incense), is thought to elicit mentally uplifting and calming effects. The main objective of this study was to determine whether the incense elicits anxiolytic-like behavior in animal models using rats. A second objective was to characterize active constituents and discern potential mechanism(s) of action, specifically the involvement of the GABAergic and endocannabinoid (eCB) systems. Despite the extensive Central American use of this resin, there are currently no known scientific behavioral or pharmacological studies done with the incense. MATERIALS AND METHODS: Quantification of the triterpenes in the copal resin and cold trapped incense was achieved by HPLC MS. Behavioral effects in rats were assessed using the elevated plus maze (EPM), social interaction (SI) test, conditioned emotion response (CER) and Novel object recognition (NOR) paradigms. Rats were exposed to burning copal (200 mg) over 5 min in a smoking chamber apparatus and then immediately tested in each behavioral paradigm. Follow-up SI tests were done using two antagonists flumazenil (1 mg/kg) and AM251 (1 mg/kg) administered systemically. Inhibition of MAGL (monoacylglycerol lipase) was measured by microplate assay with recombinant human enzyme and probe substrate. RESULTS: Phytochemical analysis revealed that copal resin and incense had high α- and ß-amyrins and low lupeol triterpene content. Exposure to Protium copal incense significantly reduced anxiety-like behavior in the SI and CER tests. In contrast, no anxiolytic effects were observed in the EPM. The CER effect was time dependent. Both flumazenil and AM251 blocked the anxiolytic activity of copal revealing the involvement of GABAergic and endocannabinoid systems. Copal, as well as the identified triterpenes, potently inhibited monoacylglycerol lipase (MAGL) activity in vitro (IC50 ≤ 811 ng/mL). CONCLUSIONS: This is the first study to show that copal incense from Protium copal elicits anxiolytic-like effects in fear and social interaction models as evidenced by a reduced learned fear behavior and an increase in active social interaction. It's high α and ß-amyrin content suggests behavioral effects may be mediated, in part, by the known action of these terpenes at the benzodiazepine receptor. Furthermore, P. copal's observed activity through the eCB system via MAGL offers a new potential mechanism underlying the anxiolytic activity.

New Botanical Anxiolytics for Use in Companion Animals and Humans.

As part of our ongoing research into botanical therapies for anxiety disorders, the neotropical vine Souroubea sympetala was chosen for study as a phytochemical discovery strategy focusing on rare Central American plant families. When orally administered to male Sprague-Dawley rats, the crude plant extract, its ethyl acetate fraction, supercritical carbon dioxide fraction, or its isolated triterpenes reduced anxiety and/or fear-related behavior in standardized behavioral models. Pharmacological studies showed that the extracts acted at the benzodiazepine GABAA receptor and reduced corticosterone levels. A preparation containing Souroubea fortified with a second triterpene containing plant, Platanus occidentalis, was shown to be safe in a 28-day feeding trial with beagles at 5 times the intended dose. Subsequent trials with beagles in a thunderstorm model of noise aversion showed that the material reduced anxiety behaviors and cortisol levels in dogs. The formulation has been released for the companion animal market in Canada and the USA under the Trademark "Zentrol." Ongoing research is exploring the use of the material in treatment of anxiety and post-traumatic stress in humans.

Effects of intranasal and peripheral oxytocin or gastrin-releasing peptide administration on social interaction and corticosterone levels in rats.

The intranasal route of drug administration has gained increased popularity as it is thought to allow large molecules, such as peptide hormones, more direct access to the brain, while limiting systemic exposure. Several studies have investigated the effects of intranasal oxytocin administration in humans as this peptide is associated with prosocial behavior. There are, however, few preclinical studies investigating the effects of intranasal oxytocin administration in rodents. Oxytocin modulates hypothalamic-pituitary-adrenal (HPA) axis functioning and it has been suggested that oxytocin's ability to increase sociability may occur through a reduction in stress reactivity. Another peptide that appears to influence both social behavior and HPA axis activity is gastrin-releasing peptide (GRP), but it is not known if these GRP-induced effects are related. With this in mind, in the present study, we assessed the effects of intranasal and intraperitoneal oxytocin and GRP administration on social interaction and release of corticosterone in rats. Intranasal and intraperitoneal administration of 20, but not 5 µg, of oxytocin significantly increased social interaction, whereas intranasal and peripheral administration of GRP (20 but not 5 µg) significantly decreased levels of social interaction. In addition, while intranasal oxytocin (20 µg) had no effect on blood corticosterone levels, a marked increase in blood corticosterone levels was observed following intraperitoneal oxytocin administration. With GRP, intranasal (20 µg) but not peripheral administration increased corticosterone levels. These findings provide further evidence that intranasal peptide delivery can induce behavioral alterations in rodents which is consistent with findings from human studies. In addition, the peptide-induced changes in social interaction were not linked to fluctuations in corticosterone levels.

Ethnopharmacology of Souroubea sympetala and Souroubea gilgii (Marcgraviaceae) and identification of betulinic acid as an anxiolytic principle.

The neotropical lianas Souroubea gilgii and Souroubea sympetala (Marcgraviaceae) were chosen for study as part of a phytochemical discovery strategy focusing on rare plant families in Central America. In participatory research, Q'eqchi' healers in Belize reported the use of these plants to reverse psychological symptoms inflicted by witchcraft. Extracts of two Souroubea species showed significant anti-anxiety activity in the elevated plus maze, a standardized test paradigm. Bioassay guided isolation led to the active principle, the pentacyclic triterpene, betulinic acid, which had activity in the elevated plus maze at 0.5mg/kg. Other phytochemicals isolated included α- and ß-amyrin, 2-hydroxyursolic acid, taraxenyl trans-4-hydroxy-cinnamate, naringenin, methyl ursolate, eriodytiol, methyl 2-α-hydroxyursolate, methyl 2-α-hydroxymaslinate, methyl betulinate, and condrilla sterol.

Souroubea sympetala (Marcgraviaceae): a medicinal plant that exerts anxiolysis through interaction with the GABAA benzodiazepine receptor.

The mode of action of the anxiolytic medicinal plant Souroubea sympetala was investigated to test the hypothesis that extracts and the active principle act at the pharmacologically important GABAA-benzodiazepine (GABAA-BZD) receptor. Leaf extracts prepared by ethyl acetate extraction or supercritical extraction, previously determined to have 5.54 mg/g and 6.78 mg/g of the active principle, betulinic acid, respectively, reduced behavioural parameters associated with anxiety in a rat model. When animals were pretreated with the GABAA-BZD receptor antagonist flumazenil, followed by the plant extracts, or a more soluble derivative of the active principle, the methyl ester of betulinic acid (MeBA), flumazenil eliminated the anxiety-reducing effect of plant extracts and MeBA, demonstrating that S. sympetala acts via an agonist action on the GABAA-BZD receptor. An in vitro GABAA-BZD competitive receptor binding assay also demonstrated that S. sympetala extracts have an affinity for the GABAA-BZD receptor, with an EC50 value of 123 µg/mL (EtOAc leaf extract) and 154 µg/mL (supercritical CO2 extract). These experiments indicate that S. sympetala acts at the GABAA-BZD receptor to elicit anxiolysis.

Protracted effects of juvenile stressor exposure are mitigated by access to palatable food.

Stressor experiences during the juvenile period may increase vulnerability to anxiety and depressive-like symptoms in adulthood. Stressors may also promote palatable feeding, possibly reflecting a form of self-medication. The current study investigated the short- and long-term consequences of a stressor applied during the juvenile period on anxiety- and depressive-like behavior measured by the elevated plus maze (EPM), social interaction and forced swim test (FST). Furthermore, the effects of stress on caloric intake, preference for a palatable food and indices of metabolic syndrome and obesity were assessed. Male Wistar rats exposed to 3 consecutive days of variable stressors on postnatal days (PD) 27-29, displayed elevated anxiety-like behaviors as adults, which could be attenuated by consumption of a palatable high-fat diet. However, consumption of a palatable food in response to a stressor appeared to contribute to increased adiposity.

Characterization of the anxiolytic activity of Nunavik Rhodiola rosea.

Rhodiola rosea is a medicinal plant used by the indigenous Inuit people of Nunavik and Nunatsiavut, Eastern Canada, as a mental and physical rejuvenating agent. This traditional use led to the present investigation of R. rosea in the context of anxiety disorders. An alcohol extract of R. rosea roots was characterized phytochemically and orally administered for three consecutive days to Sprague-Dawley rats at 8 mg/kg, 25 mg/kg, and 75 mg/kg body weight. The rats were subjected to three behavioral paradigms of anxiety, including the elevated plus maze, social interaction, and contextual conditioned emotional response tests. Rhodiola rosea showed dose-dependent anxiolytic activity in the elevated plus maze and conditioned emotional response tests, with moderate effects in the higher-anxiety SI test. The active dose varied according to the anxiety test. In order to elucidate a mechanism, the extract was further tested in an in vitro GABAA-benzodiazepine receptor-binding assay, where it demonstrated low activity. This study provides the first comparative assessment of the anxiolytic activity of Nunavik R. rosea in several behaviour models and suggests that anxiolytic effects may be primarily mediated via pathways other than the GABAA-benzodiazepine site of the GABAA receptor.