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 antiepileptiform and antiseizure potential of CBD using in vitro electrophysiology and an in vivo animal seizure model, respectively. CBD (0.01-100 M) effects were assessed in vitro using the Mg 2ϩ -free and 4-aminopyridine (4-AP) models of epileptiform activity in hippocampal brain slices via multielectrode array recordings. In the Mg 2ϩ -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 M 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 model of generalized seizures. CBD (100 mg/ kg) exerted clear anticonvulsant effects with significant decreases in incidence of severe seizures and mortality compared with vehicle-treated animals. Finally, CBD acted with only low affinity at cannabinoid CB 1 receptors and displayed no agonist activity in [35 S]guanosine 5Ј-O-(3-thio)-triphosphate assays in cortical membranes. These findings suggest that CBD acts, potentially in a CB 1 receptor-independent manner, to inhibit epileptiform activity in vitro and seizure severity in vivo. Thus, we demonstrate the potential of CBD as a novel antiepileptic drug in the unmet clinical need associated with generalized seizures.

-THCV (0.025-2.5 mg/kg) on pentylenetetrazole (PTZ)-induced seizures in adult rats were also assessed. Results: After induction of stable spontaneous epileptiform activity, acute D 9 -THCV application ( ‡20 lM) significantly reduced burst complex incidence and the amplitude and frequency of paroxysmal depolarizing shifts (PDSs). Furthermore, slices pretreated with 10 lM D 9 -THCV prior to induction of epileptiform activity exhibited significantly reduced burst complex incidence and PDS peak amplitude. In radioligand-binding experiments, -THCV exerts antiepileptiform and anticonvulsant properties, actions that are consistent with a CB1 receptor-mediated mechanism and suggest possible therapeutic application in the treatment of pathophysiologic hyperexcitability states.

The neuropeptide substance P evokes a long-term protein synthesis-dependent increase in the cycle frequency of locomotor network activity in the lamprey. Although cellular and synaptic mechanisms that could induce this effect have been identified, nothing is known of the underlying maintenance mechanisms. These mechanisms have been examined here.Substance P potentiates low-frequency-evoked EPSPs from excitatory network interneurons. It also converts the depression of the EPSP during spike trains into facilitation, an example of metaplasticity. The metaplasticity was associated with a reduction of the transmitter release probability but an increase in the number of release sites. Although the potentiation of low-frequency-evoked EPSPs recovered within 1 hr, the metaplastic facilitation had not recovered 3-4 hr after substance P application. The metaplasticity thus extended into the protein synthesis-dependent maintenance phase of the network modulation, making it the only identified cellular or synaptic effect of substance P to last this long. It also had the same induction and maintenance features as the network burst frequency modulation, further suggesting that the two effects were related.Long-term changes in synaptic properties are often associated with changes in synaptic organization. We have thus also examined the effects of substance P on synaptic ultrastructure up to 5 hr after substance P application. Substance P had several significant effects. These included an increase in the number of docked vesicles and a reduction of the synaptic gap.Substance P thus has long-term effects on synaptic organization and function. The relevance of these effects to the long-term locomotor network modulation is discussed.

. Variability is increasingly recognized as a characteristic feature of cellular, synaptic, and network properties. While studies have traditionally focused on mean values, significant effects can result from changes in variance. This study has examined cellular and synaptic variability in the lamprey spinal cord and its modulation by the neuropeptide substance P. Cellular and synaptic variability differed in different types of cell and synapse. Substance P reduced the variability of subthreshold locomotor-related depolarizations and spiking in motor neurons during network activity. These effects were associated with a reduction in the variability of spiking in glutamatergic excitatory network interneurons and with a reduction in the variance of excitatory interneuronevoked excitatory postsynaptic potentials (EPSPs). Substance P also reduced the variance of postsynpatic potentials (PSPs) from crossing inhibitory and excitatory interneurons, but it increased the variance of inhibitory postsynpatic potentials (IPSPs) from ipsilateral inhibitory interneurons. The effects on the variance of different PSPs could occur with or without changes in the PSP amplitude. The reduction in the variance of excitatory interneuron-evoked EPSPs was protein kinase A, calcium, and N-methyl-D-aspartate (NMDA) dependent. The NMDA dependence suggested that substance P was acting postsynaptically. This was supported by the reduced variability of postsynaptic responses to glutamate by substance P. However, ultrastructural analyses suggested that there may also be a presynaptic component to the modulation, because substance P reduced the variability of synaptic vesicle diameters in putative glutamatergic terminals. These results suggest that cellular and synaptic variability can be targeted for modulation, making it an additional source of spinal cord plasticity.

Chromogranin A (CgA), a member of the granin/secretogranin family of acidic glycoproteins that play multiple roles in the process of regulated secretion of peptide hormones and neurotransmitters, is specifically expressed in endocrine and neuroendocrine cells. We previously cloned and characterized the human (h) CgA gene and showed that nucleotides -55 to +32 relative to the transcriptional start site that contain a consensus cAMP element (CRE) and TATA-box motif were sufficient for neuroendocrine cell-specific expression. Here, we examined the role of the well conserved CRE in basal and cAMP-stimulated transcription in neuroendocrine cells. Transient transfection studies with hCgA gene promoter/chloroamphenical acetyl transferase (CAT) reporter constructs were conducted in a panel of neuroendocrine cell lines as well as in nonendocrine cell lines. Deletion or mutation of the CRE resulted in loss of neuroendocrine cell specific transcriptional activity. Mutation of a well conserved region (the TG-box) located between the CRE and the TATA box had no effect or resulted in only a modest decrease in activity. Mutation of the CRE in 5'-extended (-2300 to +32 and -700 to +32) constructs resulted in a 50-75% decrease in basal activity in neuroendocrine cells. This emphasized the importance of the CRE in basal transcription and also suggested that other elements between -700 and -55 may act independently of the CRE to contribute to full basal activity in some neuroendocrine cells. Dibutyryl cAMP stimulated transcriptional activity in neuroendocrine cells, and this was abolished by mutation of the CRE. In the presence of a PKA inhibitor, dibutyryl cAMP-induced activity was completely abolished and basal activity was decreased by up to 85%. Similar protein-DNA complexes were formed in gel retardation assays with a CgA-CRE oligonucleotide and nuclear extracts from both neuroendocrine and nonendocrine cells. A predominant complex that was supershifted by addition of a CREB antibody was identical in all cell types. By immunoblot analysis, levels of total CREB protein and phosphorylated (Ser 133) CREB did not differ between neuroendocrine and nonendocrine cells. Phosphorylated CREB was increased by forskolin treatment, an effect that was blocked by a PKA-inhibitor. Expression of the transcriptional cointegrator, CREB-binding protein (CBP), assessed by both RT-PCR and Western blot analysis, did not differ between neuroendocrine and nonendocrine cells. In summary, the CRE in the hCgA gene proximal promoter is critical for both basal and cAMP-induced expression in neuroendocrine cells via a PKA-mediated pathway. However, the neuroendocrine specificity of hCgA gene transcription mediated by the CRE is not a function of levels of total CREB or phosphorylated CREB or its cointegrator CBP. Specificity may be achieved by a PKA-responsive CRE-binding protein other than CREB expressed specifically in neuroendocrine cells, expression of a repressor molecule that binds CREB in nonendocrine cells, or may lie downstream of a CRE-binding pro...