Cannabidiol displays antiepileptiform and antiseizure properties in vitro and in vivo

Plant-derived cannabinoids (phytocannabinoids) are compounds with emerging therapeutic potential. Early studies suggested that cannabidiol (CBD) has anticonvulsant properties in animal models and reduced seizure frequency in limited human trials. Here, we examine the anti-epileptiform and anti-seizure potential of CBD using in vitro electrophysiology and an in vivo animal seizure model, respectively. CBD (0.01-100 muM) effects were assessed in vitro using the Mg(2+)-free and 4-aminopyridine (4-AP) models of status epilepticus-like epileptiform activity in hippocampal brain slices via multi-electrode array (MEA) recordings. In the Mg(2+)-free model, CBD decreased epileptiform local field potential (LFP) burst amplitude (in CA1 and dentate gyrus (DG) regions) and burst duration (in all regions) and increased burst frequency (in all regions). In the 4-AP model, CBD decreased LFP burst amplitude (in CA1, only at 100 muM CBD), burst duration (in CA3 and DG), and burst frequency (in all regions). CBD (1, 10 and 100 mg/kg) effects were also examined in vivo using the pentylenetetrazole (PTZ) model of generalised seizures. CBD (100 mg/kg) exerted clear anticonvulsant effects with significant decreases in incidence of severe seizures and mortality in comparison to vehicle-treated animals. Finally, CBD acted with only low affinity at cannabinoid CB(1) receptors and displayed no agonist activity in [(35)S]GTPgammaS assays in cortical membranes. These findings suggest that CBD acts to inhibit epileptiform activity in vitro and seizure severity in vivo. Thus, we demonstrate the potential of CBD as a novel anti-epileptic drug (AED) in the unmet clinical need associated with generalised seizures.

Cholinergic modulation of intrinsic fibre-evoked excitatory transmission contains a nicotinic component in immature but not adult rat piriform cortex, in vitro

The piriform cortex (PC) is highly prone to epileptogenesis, particularly in immature animals, where decreased muscarinic modulation of PC intrinsic fibre excitatory neurotransmission is implicated as a likely cause. However, whether higher levels of acetylcholine (ACh) release occur in immature vs. adult PC remains unclear. We investigated this using in vitro extracellular electrophysiological recording techniques. Intrinsic fibre-evoked extracellular field potentials (EFPs) were recorded from layers II to III in PC brain slices prepared from immature (P14-18) and adult (P>40) rats. Adult and immature PC EFPs were suppressed by eserine (1muM) or neostigmine (1muM) application, with a greater suppression in immature ( approximately 40%) than adult ( approximately 30%) slices. Subsequent application of atropine (1muM) reversed EFP suppression, producing supranormal ( approximately 12%) recovery in adult slices, suggesting that suppression was solely muscarinic ACh receptor-mediated and that some 'basal' cholinergic 'tone' was present. Conversely, atropine only partially reversed anticholinesterase effects in immature slices, suggesting the presence of additional non-muscarinic modulation. Accordingly, nicotine (50muM) caused immature field suppression ( approximately 30%) that was further enhanced by neostigmine, whereas it had no effect on adult EFPs. Unlike atropine, nicotinic antagonists, mecamylamine and methyllycaconitine, induced immature supranormal field recovery ( approximately 20%) following anticholinesterase-induced suppression (with no effect on adult slices), confirming that basal cholinergic 'tone' was also present. We suggest that nicotinic inhibitory cholinergic modulation occurs in the immature rat PC intrinsic excitatory fibre system, possibly to complement the existing, weak muscarinic modulation, and could be another important developmentally regulated system governing immature PC susceptibility towards epileptogenesis.

Developmental changes in presynaptic muscarinic modulation of excitatory and inhibitory neurotransmission in rat piriform cortex in vitro: relevance to epileptiform bursting susceptibility

Suppression of depolarizing postsynaptic potentials and isolated GABA-A receptor-mediated fast inhibitory postsynaptic potentials by the muscarinic acetylcholine receptor agonist, oxotremorine-M (10 microM), was investigated in adult and immature (P14-P30) rat piriform cortical (PC) slices using intracellular recording. Depolarizing postsynaptic potentials evoked by layers II-III stimulation underwent concentration-dependent inhibition in oxotremorine-M that was most likely presynaptic and M2 muscarinic acetylcholine receptor-mediated in immature, but M1-mediated in adult (P40-P80) slices; percentage inhibition was smaller in immature than in adult piriform cortex. In contrast, compared with adults, layer Ia-evoked depolarizing postsynaptic potentials in immature piriform cortex slices in oxotremorine-M, showed a prolonged multiphasic depolarization with superimposed fast transients and spikes, and an increased 'all-or-nothing' character. Isolated N-methyl-d-aspartate receptor-mediated layer Ia depolarizing postsynaptic potentials (although significantly larger in immature slices) were however, unaffected by oxotremorine-M, but blocked by dl-2-amino-5-phosphonovaleric acid. Fast inhibitory postsynaptic potentials evoked by layer Ib or layers II-III-fiber stimulation in immature slices were significantly smaller than in adults, despite similar estimated mean reversal potentials ( approximately -69 and -70 mV respectively). In oxotremorine-M, only layer Ib-fast inhibitory postsynaptic potentials were suppressed; suppression was again most likely presynaptic M2-mediated in immature slices, but M1-mediated in adults. The degree of fast inhibitory postsynaptic potential suppression was however, greater in immature than in adult piriform cortex. Our results demonstrate some important physiological and pharmacological differences between excitatory and inhibitory synaptic systems in adult and immature piriform cortex that could contribute toward the increased susceptibility of this region to muscarinic agonist-induced epileptiform activity in immature brain slices.

Further characterization of muscarinic agonist-induced epileptiform bursting activity in immature rat piriform cortex, in vitro

The characteristics of muscarinic acetylcholine receptor agonist-induced epileptiform bursting seen in immature rat piriform cortex slices in vitro were further investigated using intracellular recording, with particular focus on its postnatal age-dependence (P+14-P+30), pharmacology, site(s) of origin and the likely contribution of the muscarinic acetylcholine receptor agonist-induced post-stimulus slow afterdepolarization and gap junction functionality toward its generation. The muscarinic agonist, oxotremorine-M (10 microM), induced rhythmic bursting only in immature piriform cortex slices; however, paroxysmal depolarizing shift amplitude, burst duration and burst incidence were inversely related to postnatal age. No significant age-dependent changes in neuronal membrane properties or postsynaptic muscarinic responsiveness accounted for this decline. Burst incidence was higher when recorded in anterior and posterior regions of the immature piriform cortex. In adult and immature neurones, oxotremorine-M effects were abolished by M1-, but not M2-muscarinic acetylcholine receptor-selective antagonists. Rostrocaudal lesions, between piriform cortex layers I and II, or layer III and endopiriform nucleus in adult or immature slices did not influence oxotremorine-M effects; however, the slow afterdepolarization in adult (but not immature) lesioned slices was abolished. Gap junction blockers (carbenoxolone or octanol) disrupted muscarinic bursting and diminished the slow afterdepolarization in immature slices, suggesting that gap junction connectivity was important for bursting. Our data show that neural networks within layers II-III function as primary oscillatory circuits for burst initiation in immature rat piriform cortex during persistent muscarinic receptor activation. Furthermore, we propose that muscarinic slow afterdepolarization induction and gap junction communication could contribute towards the increased epileptiform susceptibility of this brain area.

A novel component of cannabis extract potentiates excitatory synaptic transmission in rat olfactory cortex in vitro

Cannabis is a potential treatment for epilepsy, although the few human studies supporting this use have proved inconclusive. Previously, we showed that a standardized cannabis extract (SCE), isolated Delta(9)-tetrahydrocannabinol (Delta(9)-THC), and even Delta(9)-THC-free SCE inhibited muscarinic agonist-induced epileptiform bursting in rat olfactory cortical brain slices, acting via CB1 receptors. The present work demonstrates that although Delta(9)-THC (1microM) significantly depressed evoked depolarizing postsynaptic potentials (PSPs) in rat olfactory cortex neurones, both SCE and Delta(9)-THC-free SCE significantly potentiated evoked PSPs (all results were fully reversed by the CB1 receptor antagonist SR141716A, 1microM); interestingly, the potentiation by Delta(9)-THC-free SCE was greater than that produced by SCE. On comparing the effects of Delta(9)-THC-free SCE upon evoked PSPs and artificial PSPs (aPSPs; evoked electrotonically following brief intracellular current injection), PSPs were enhanced, whereas aPSPs were unaffected, suggesting that the effect was not due to changes in background input resistance. Similar recordings made using CB1 receptor-deficient knockout mice (CB1(-/-)) and wild-type littermate controls revealed cannabinoid or extract-induced changes in membrane resistance, cell excitability and synaptic transmission in wild-type mice that were similar to those seen in rat neurones, but no effect on these properties were seen in CB1(-/-) cells. It appears that the unknown extract constituent(s) effects over-rode the suppressive effects of Delta(9)-THC on excitatory neurotransmitter release, which may explain some patients' preference for herbal cannabis rather than isolated Delta(9)-THC (due to attenuation of some of the central Delta(9)-THC side effects) and possibly account for the rare incidence of seizures in some individuals taking cannabis recreationally

Investigation of the effects of the novel anticonvulsant compound carisbamate(RWJ-333369) on rat piriform cortical neurones in vitro

Background and purpose: Carisbamate is being developed for adjuvant treatment of partial onset epilepsy. Carisbamate produces anticonvulsant effects in primary generalized, complex partial and absence-type seizure models, and exhibits neuroprotective and antiepileptogenic properties in rodent epilepsy models. Phase IIb clinical trials of carisbamate demonstrated efficacy against partial onset seizures; however, its mechanisms of action remain unknown. Here, we report the effects of carisbamate on membrane properties, evoked and spontaneous synaptic transmission and induced epileptiform discharges in layer II-III neurones in piriform cortical brain slices. Experimental approach: Effects of carisbamate were investigated in rat piriform cortical neurones by using intracellular electrophysiological recordings. Key results: Carisbamate (50–400 mmol·L-1) reversibly decreased amplitude, duration and rise-time of evoked action potentials and inhibited repetitive firing, consistent with use-dependent Na+ channel block; 150–400 mmol·L-1 carisbamate reduced neuronal input resistance, without altering membrane potential. After microelectrode intracellular Cl- loading, carisbamate depolarized cells, an effect reversed by picrotoxin. Carisbamate (100–400 mmol·L-1) also selectively depressed lateral olfactory tract-afferent evoked excitatory synaptic transmission (opposed by picrotoxin), consistent with activation of a presynaptic Cl conductance. Lidocaine (40–320 mmol·L-1) mimicked carisbamate, implying similar modes of action. Carisbamate (300–600 mmol·L-1) had no effect on spontaneous GABAA miniature inhibitory postsynaptic currents and at lower concentrations (50–200 mmol·L-1) inhibited Mg2+-free or 4-aminopyridine-induced seizure-like discharges. Conclusions and implications: Carisbamate blocked evoked action potentials use-dependently, consistent with a primary action on Na+ channels and increased Cl- conductances presynaptically and, under certain conditions, postsynaptically to selectively depress excitatory neurotransmission in piriform cortical layer Ia-afferent terminals.