Cannabidiol inhibits synaptic transmission in rat hippocampal cultures and slices via multiple receptor pathways.

BACKGROUND AND PURPOSE: Cannabidiol (CBD) has emerged as an interesting compound with therapeutic potential in several CNS disorders. However, whether it can modulate synaptic activity in the CNS remains unclear. Here, we have investigated whether CBD modulates synaptic transmission in rat hippocampal cultures and acute slices. EXPERIMENTAL APPROACH: The effect of CBD on synaptic transmission was examined in rat hippocampal cultures and acute slices using whole cell patch clamp and standard extracellular recordings respectively. KEY RESULTS: Cannabidiol decreased synaptic activity in hippocampal cultures in a concentration-dependent and Pertussis toxin-sensitive manner. The effects of CBD in culture were significantly reduced in the presence of the cannabinoid receptor (CB(1) ) inverse agonist, LY320135 but were unaffected by the 5-HT(1A) receptor antagonist, WAY100135. In hippocampal slices, CBD inhibited basal synaptic transmission, an effect that was abolished by the proposed CB(1) receptor antagonist, AM251, in addition to LY320135 and WAY100135. CONCLUSIONS AND IMPLICATIONS: Cannabidiol reduces synaptic transmission in hippocampal in vitro preparations and we propose a role for both 5-HT(1A) and CB(1) receptors in these CBD-mediated effects. These data offer some mechanistic insights into the effects of CBD and emphasize that further investigations into the actions of CBD in the CNS are required in order to elucidate the full therapeutic potential of CBD.

Interaction between anandamide and sphingosine-1-phosphate in mediating vasorelaxation in rat coronary artery.

BACKGROUND AND PURPOSE: Anandamide and sphingosine-1-phosphate (S1P) both regulate vascular tone in a variety of vessels. This study aimed to examine the mechanisms involved in the regulation of coronary vascular tone by anandamide and S1P, and to determine whether any functional interaction occurs between these receptor systems. EXPERIMENTAL APPROACH: Mechanisms used by anandamide and S1P to regulate rat coronary artery (CA) reactivity were investigated using wire myography. Interactions between S1P and the cannabinoid (CB)(2) receptor were determined using human embryonic kidney 293 (HEK293) cells that stably over-express recombinant CB(2) receptor. KEY RESULTS: Anandamide and S1P induced relaxation of the rat CA. CB(2) receptor antagonists attenuated anandamide-induced relaxation, while S1P-mediated relaxation was dependent on the vascular endothelium and S1P(3). Anandamide treatment resulted in an increase in the phosphorylation of sphingosine kinase-1 within the CA. Conversely, anandamide-mediated relaxation was attenuated by inhibition of sphingosine kinase. Moreover, S1P(3), specifically within the vascular endothelium, was required for anandamide-mediated vasorelaxation. In addition to this, S1P-mediated relaxation was also reduced by CB(2) receptor antagonists and sphingosine kinase inhibition. Further evidence that S1P functionally interacts with the CB(2) receptor was also observed in HEK293 cells over-expressing the CB(2) receptor. CONCLUSIONS AND IMPLICATIONS: In the vascular endothelium of rat CA, anandamide induces relaxation via a mechanism requiring sphingosine kinase-1 and S1P/S1P(3). In addition, we report that S1P may exert some of its effects via a CB(2) receptor- and sphingosine kinase-dependent mechanism, where subsequently formed S1P may have privileged access to S1P(3) to induce vascular relaxation.

Benzodiazepine dependence: from neural circuits to gene expression.

The neural mechanisms underlying benzodiazepine dependence remain equivocal. The present studies tested the hypothesis that similar neural systems are recruited during diazepam tolerance and withdrawal, and that these are associated with changes in GABA(A) receptor properties. 2-Deoxyglucose quantitative autoradiography was employed to map the brain structures affected during chronic treatment and withdrawal from diazepam (5 mg/kg i.p. daily) in rats. Acute administration of diazepam evoked widespread reductions in local rates of cerebral glucose (LCGU) utilization throughout the brain. Brain structures associated with sensory processing developed tolerance to these depressant effects of diazepam after 3 days of treatment, whereas tolerance occurred in the Papez circuit of emotion after 28 days of treatment. These data suggest that adaptive changes in different neuroanatomical circuits may underlie tolerance to the various effects of diazepam. During flumazenil-precipitated withdrawal from diazepam there were marked increases in glucose use in structures of the Papez circuit, the nucleus accumbens, and the basolateral amygdala. These data suggest that the Papez circuit features strongly in diazepam tolerance and withdrawal and supports a common adaptive process being involved in these phenomena. While GABA enhancement of benzodiazepine binding was reduced in the nucleus accumbens after repeated diazepam treatment, there was little evidence to support adaptive changes in GABA(A) receptors or GABA(A) subunit gene expression (gamma2, alpha1, or alpha4) as underlying the functional changes in the identified circuits. Alternative neurochemical mechanisms, such as changes in glutamatergic function should be considered.

Changes in benzodiazepine-GABA receptor coupling in an accumbens-habenula circuit after chronic diazepam treatment.

1. The effects of subacute and of chronic diazepam treatment upon binding to the GABAA receptor have been examined by use of receptor autoradiography for determining flunitrazepam (FNZP) binding, GABA enhancement of FNZP binding. SR 95531 2-(3'-carboxy-2',propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide) binding and GABA binding in parallel sections from rat brain. Prior to the autoradiographic procedures, a behavioural assessment of the rats was made in the elevated plus-maze test of anxiety. 2. Rats receiving diazepam either subacutely (3 days) or chronically (28 days) by both continuous release, from previously implanted subcutaneous silastic capsules, or by daily injection (5 mg kg-1) did not display changes in FNZP or GABA binding in any of the 47 brain structures analysed. Similarly, there were no significant effects of treatment upon mean total entries or on the open:total ratio for entries in the elevated plus-maze. 3. There were reductions in the GABA enhancement of FNZP binding in the nucleus accumbens and central grey after subacute diazepam treatment. This effect persisted in the nucleus accumbens after chronic treatment. Less marked effects occurred in the lateral habenula, dorsal raphe and substantia nigra pars compacta. In the dorsal tegmental nucleus, GABA enhancement of FNZP binding was enhanced after chronic treatment and this was accompanied by reductions in SR 95531 binding. Treatment did not otherwise affect SR 95531 binding, with the exception of the dorsal raphe where binding was decreased after subacute treatment. 4. In general, the patterns of binding produced by the two different treatment routes were very similar. However, SR 95531 binding was lower in certain hippocampal fields in the i.p. treated animals compared to the rats implanted with silastic capsules. 5. It is concluded that repeated administration of diazepam evokes changes in benzodiazepine and GABA receptor coupling, and to a lesser extent changes in low affinity GABA binding, in certain interrelated brain structures of which an accumbens-habenula circuit is a central feature. These changes occur soon after the initiation of diazepam treatment, suggesting that they are unlikely to account for tolerance to the anxiolytic effects of diazepam but may trigger and/or accompany other critical neurochemical events.

The benzodiazepine receptor inverse agonist FG 7142 induces cholecystokinin gene expression in rat brain.

The effects of acute administration of the anxiogenic benzodiazepine receptor ligand, N-methyl-beta-carboline-3-carboxamide (FG 7142) and of a single exposure to the elevated plus-maze test of anxiety on preprocholecystokinin mRNA levels in rat brain were examined using the technique of in situ hybridisation. Administration of FG 7142 (10 mg/kg i.p.), but not elevated plus-maze exposure, increased cholecystokinin (CCK) mRNA levels in the basolateral amygdala and the CA3 pyramidal cell layer of the hippocampus. Neither stimulus produced changes in thalamic structures. These data suggest that drug-induced anxiety can induce CCK gene expression in brain structures previously implicated in anxiety.

Continuous exposure to FG 7142: behavioural sensitisation is not accompanied by changes in benzodiazepine/GABA receptor coupling.

Chronic intermittent high-dose treatment with N-methyl-ß-carboline-3-carboxamide (FG 7142) leads to kindling accompanied by reduction in γ-aminobutyric acid (GABA) receptor function, whereas chronic continuous administration may result in behavioural effects in the opposite direction from those of acute FG 7142. In the present study, we have investigated the effects of continuous administration of low doses of FG 7142 on the response to an acute challenge dose of FG 7142 in an ethologically based model of anxiety. Rats treated continuously for 14 days with FG 7142 delivered by osmotic minipump at a rate of 1.2-1.5 mg/kg/day showed sensitisation to the anxiogenic effects of a challenge dose of FG 7142 (6 mg/kg), as measured in the elevated plus-maze. This was not accompanied by any change in benzodiazepine/GABA receptor coupling, as assessed by the 'GABA shift'. These results indicate that continuous low-dose treatment with FG 7142 can elicit sensitisation to the behavioural effects of FG 7142, but that this is unlikely to be mediated by changes in benzodiazepine/GABA receptor coupling.

Muscimol-associated changes in local cerebral glucose use following chronic diazepam administration.

Local cerebral glucose use (LCGU) was determined in parallel groups of conscious rats receiving muscimol (1.5 mg/kg i.v.) after either saline pretreatment (28 days i.p.), saline pretreatment (27 days i.p.) followed by a single dose of diazepam (5 mg/kg i.p.) 24 h prior to muscimol administration, or chronic diazepam pretreatment (5 mg/kg i.p. daily for 28 days). Acute administration of muscimol produced a significant reduction in LCGU in 25 out of 66 structures examined compared with vehicle-treated controls. The pattern of reductions was heterogeneous. Thalamic and most cortical areas showed reductions of the order of 30-45%, whereas more modest depressions of 15-20% were observed in some limbic structures (e.g. basolateral amygdala, anterior thalamic nuclei, nucleus accumbens, subiculum). This contrasts with the more extensive and homogeneous pattern of LGCU reductions (around 20%) produced by diazepam. Neither acute diazepam treatment the previous day nor chronic diazepam pretreatment altered the LGCU response to muscimol. These data suggest that high-affinity GABA receptor-mediated responses are unchanged by both acute and chronic benzodiazepine pretreatment. It would appear unlikely that alterations in these responses contribute to the mechanism of benzodiazepine tolerance.

Chronic handling modifies the anxiolytic effect of diazepam in the elevated plus-maze.

Rats treated for 3 days with diazepam demonstrated a clear anxiolytic action of the drug as assessed in the elevated plus-maze. However, in a chronic experiment, in which all rats were handled for 28 days, no anxiolytic effect of diazepam could be shown either in rats treated for 3 days or in those treated for 28 days. These results suggest that there is an interaction between handling and the anxiolytic effect of diazepam.