The direct actions of cannabidiol and 2-arachidonoyl glycerol at GABAA receptors.

Cannabidiol (CBD) is a major non-intoxicating component of cannabis and possesses anti-epileptic, anxiolytic and anti-hyperalgesic properties. The mechanism of action of CBD in producing such effects remains unclear. Despite evidence that some endogenous and synthetic cannabinoids interact with GABAA receptors, no-one has yet investigated the effects of CBD. Here we used two-electrode voltage clamp electrophysiology to compare the actions of CBD with those of the major central endocannabinoid, 2-arachidonoyl glycerol (2-AG) on human recombinant GABAA receptors (synaptic α1-6ßγ2 and extrasynaptic α4ß2δ) expressed on Xenopus oocytes. CBD and 2-AG were positive allosteric modulators at α1-6ßγ2 receptors, with low micromolar potencies. The maximal level of enhancement seen with either CBD or 2-AG were on α2-containing GABAA receptor subtypes, with approximately a 4-fold enhancement of the GABA EC5 evoked current, more than twice the potentiation seen with other α-subunit receptor combinations. Further we observed ß-subunit selectivity, whereby modulatory activity was higher at ß2/ß3 over ß1 subunits. The ß1-subunit homologous mutant ß2(V436T) substantially diminished the efficacy of both drugs to a third of that obtained with wild-type ß2 subunit combinations, but without changing potency. The potency of CBD increased and efficacy preserved in binary α1/α2ß2 receptors indicating that their effects do not involve the classic benzodiazepine site. Exploration of extrasynaptic α4ß2δ receptors revealed that both compounds enhanced GABA EC5 evoked currents at concentrations ranging from 0.01-1µM. Taken together these results reveal a mode of action of CBD on specifically configured GABAA receptors that may be relevant to the anticonvulsant and anxiolytic effects of the compound.

A pharmacological characterization of GABA, THIP and DS2 at binary α4ß3 and ß3δ receptors: GABA activates ß3δ receptors via the ß3(+)δ(-) interface.

There is growing evidence that GABA (γ-aminobutyric acid) can activate GABAA receptors (GABAARs) in the absence of an α subunit. In this study, we compared the pharmacology of homomeric and binary α4, ß3 or δ subunits with ternary α4ß3δ to identify subunit interfaces that contribute to the pharmacology of GABA, THIP, and DS2, and the antagonists, Zn(2+), gabazine and bicuculline. ß3δ receptors form functional GABA-gated channels when expressed in Xenopus oocytes with a pharmacology that differs to homomeric ß3, binary α4ß3 and ternary α4ß3δ receptors. GABA had similar potency at α4ß3 and ß3δ receptors (25µM and 26µM, respectively) but differed at α4ß3δ receptors where GABA exhibited a biphasic concentration-response (EC50 (1)=12.6nM; EC50 (2)=6.3µM). THIP activated ß3δ receptors (EC50=456µM) but was a more potent activator of α4ß3 (EC50=27µM) and α4ß3δ receptors (EC50 (1)=27.5nM; EC50 (2)=29.5µΜ), indicating that the α4 subunit significantly contribute to its potency. The δ-preferring modulator, DS2 had marginal or no effect at ß3δ and α4ß3 receptors, indicating a role for both the α4 and δ subunits for its potency. Gabazine inhibited GABA-elicited currents at ß3δ receptors whereas bicuculline activated these receptors. Mutational analysis verified that GABA binds to the ß3(+)δ(-) interface formed by the ß3 and δ subunits. In conclusion, evaluating agents against binary GABAARs such as ß3δ and α4ß3 receptors enables identification of interfaces that may contribute to the pharmacology of the more complex ternary α4ß3δ receptors.

Regional Fos-expression induced by γ-hydroxybutyrate (GHB): comparison with γ-butyrolactone (GBL) and effects of co-administration of the GABAB antagonist SCH 50911 and putative GHB antagonist NCS-382.

γ-Hydroxybutyrate (GHB) has a complex array of neural actions that include effects on its own high-affinity GHB receptor, the release of neuroactive steroids, and agonist actions at GABAA and GABAB receptors. We previously reported partial overlap in the c-Fos expression patterns produced by GHB and the GABAB agonist, baclofen in rats. The present study extends these earlier findings by examining the extent to which GHB Fos expression and behavioral sedation are prevented by (2S)-(+)-5,5-dimethyl-2-morpholineacetic acid (SCH 50911), a GABAB antagonist, and NCS-382, a putative antagonist at the high-affinity GHB receptor. We also compare Fos expression caused by GHB and its precursor γ-butyrolactone (GBL), which is a pro-drug for GHB but lacks the high sodium content of the parent GHB molecule. Both GHB (1,000 mg/kg) and GBL (600 mg/kg) induced rapid sedation in rats that lasted over 90 min and caused similar Fos expression patterns, albeit with GBL causing greater activation of the nucleus accumbens (core and shell) and dentate gyrus (granular layer). Pretreatment with SCH 50911 (100mg/kg) partly reversed the sedative effects of GHB and significantly reduced GHB-induced Fos expression in only four regions: the tenia tecta, lateral habenula, dorsal raphe and laterodorsal tegmental nucleus. NCS-382 (50mg/kg) had no effect on GHB-induced sedation or Fos expression. When given alone, both NCS-382 and SCH 50911 increased Fos expression in the bed nucleus of the stria terminalis, central amygdala, parasubthalamic nucleus and nucleus of the solitary tract. SCH 50911 alone affected the Islands of Calleja and the medial, central and paraventricular thalamic nuclei. Overall, this study shows a surprising lack of reversal of GHB-induced Fos expression by two relevant antagonists, both of which have marked intrinsic actions. This may reflect the limited doses tested but also suggests that GHB Fos expression reflects mechanisms independent of GHB and GABAB receptors.

Weekly gamma-hydroxybutyrate exposure sensitizes locomotor hyperactivity to low-dose 3,4-methylenedioxymethamphetamine in rats.

Users of the popular party drug 3,4-methylenedioxymethamphetamine (MDMA) sometimes report combining MDMA with gamma-hydroxybutyrate (GHB) to enhance the pleasurable effects of both drugs. However, very few studies have examined the influences of this drug combination. The present study investigated the development of locomotor sensitization in laboratory rats given 7 once-weekly exposures to either MDMA, GHB or their combination (MDMA/GHB). The drugs were administered at a high ambient temperature (28 degrees C) to mimic nightclub conditions. MDMA (5 mg/kg), given once weekly, produced a progressively greater locomotor and hyperthermic response over time. In contrast, GHB (500 mg/kg) administered weekly produced consistent low levels of locomotor activity and few changes in body temperature. Rats receiving the mixture of MDMA (5 mg/kg) and GHB (500 mg/kg) showed asymptotic levels of sensitized locomotor activity similar to those seen in rats given MDMA alone, but the development of locomotor sensitization was delayed by coadministered GHB. GHB also delayed the development of MDMA-induced hyperthermia. After a washout period of 5 weeks, rats pre-exposed to MDMA, GHB and MDMA/GHB showed no hyperactivity when tested drug-free in the context in which they had previously received drugs, but displayed a sensitized locomotor response to a low challenge dose of MDMA (2.5 mg/kg). The response to a low dose of methamphetamine (0.5 mg/kg) did not differ among groups. Neurochemical analysis using high-performance liquid chromatography revealed few lasting changes in serotonin, dopamine or their metabolites in the striatum or prefrontal cortex of MDMA- or GHB-pre-exposed rats. These results indicate that GHB modulates the locomotor and hyperthermic response to acute MDMA and that pre-exposure to GHB can sensitize the locomotor response to low doses of MDMA.

The distribution of gamma-hydroxybutyrate-induced Fos expression in rat brain: comparison with baclofen.

gamma-Hydroxybutyrate (GHB) is a euphoric, prosocial and sleep inducing drug that binds with high affinity to its own GHB receptor site and also more weakly to GABA(B) receptors. GHB is efficacious in the treatment of narcolepsy and alcoholism, but heavy use can lead to dependence and withdrawal. Many effects of GHB (sedation, hypothermia, catalepsy) are mimicked by GABA(B) receptor agonists (e.g. baclofen). However other effects (euphoric and prosocial effects and a therapeutic effect in narcolepsy) are not. The present study used Fos immunohistochemistry to assess the neural activation produced in rat brain by medium to high doses of GHB (250, 500 and 1000 mg/kg) and a high dose of baclofen (10 mg/kg) that produced similar sedation to 500 mg/kg GHB. Results showed many common regions of activation with these two drugs including the supraoptic, paraventricular, median preoptic and ventral premammillary nuclei of the hypothalamus, the central nucleus of the amygdala, Edinger-Westphal nucleus, lateral parabrachial nucleus, locus coeruleus, and nucleus of the solitary tract. GHB (500 mg/kg), but not baclofen (10 mg/kg), induced significant Fos expression in the median raphe nucleus and lateral habenula, while a higher dose of GHB (1000 mg/kg) induced additional Fos expression in the islands of Calleja, dentate gyrus (polymorphic layer) and arcuate nucleus, and in various regions implicated in rapid and non-rapid eye movement sleep (laterodorsal tegmental nucleus, tuberomammillary nucleus and the ventrolateral and anterodorsal preoptic nuclei). Surprisingly, Fos immunoreactivity was not observed with either GHB or baclofen in reward-relevant regions such as the nucleus accumbens, striatum and ventral tegmental area. Overall these results indicate a distinctive signature of brain activation with GHB that may be only partly due to GABA(B) receptor effects. This confirms a unique neuropharmacological profile for GHB and indicates key neural substrates that may underlie its characteristic influence on sleep, body temperature, sociability and endocrine function.