The effects of two adamantane derivatives, 1‐trimethylammonio‐5‐(l‐adamantane‐methylammoniopentane dibromide) (IEM‐1460) and 1‐ammonio‐5‐(l‐adamantane‐methylammoniopentane dibromide) (IEM‐1754) on kainate‐induced currents were studied in Xenopus oocytes expressing recombinant ionotropic glutamate receptors and in freshly isolated neurones from rat hippocampal slices. The adamantane derivatives caused use‐and voltage‐dependent block of open channels of recombinant AMPA receptors. This antagonism was dependent on receptor subunit composition; channels gated by recombinant, homomeric GluRl and GluR3 receptors exhibited a higher sensitivity to block than those gated by receptors containing edited GluR2 subunits. In the former cases, IEM‐1460 had an IC50 of 1.6 μm at a holding potential (Vh) of −80 mV and IEM‐1754 was 3.8 times less potent than IEM‐1460. In contrast, 100 μm IEM‐1460 inhibited responses to 100 μm kainate of receptors containing edited GluR2 subunits by only 7.8 ± 2.4% (n= 5 oocytes) at a Vh of −80 mV. Native AMPA/kainate receptors in isolated hippocampal cells were inhibited by adamantane derivatives in a use‐ and voltage‐dependent manner. This antagonism was dependent on cell type: pyramidal neurones were less sensitive to IEM‐1460 (IC50= 1617 μm at Vh=−80mV) than interneurones (IC50= 1.6 μm at Vh=−80 mV). IEM‐1460 and IEM‐1754 were equipotent when applied to pyramidal neurones, but IEM‐1754 was less potent (∼3 times) than IEM‐1460 when applied to interneurones. It is concluded that the presence of the edited GluR2 subunit in recombinant AMPA receptors and native AMPA/kainate receptors inhibits channel block by organic cations and that adamantane derivatives are potentially valuable tools for identifying classes of AMPA/kainate receptors and their roles in synaptic transmission.
Some forms of seizure activity can be stopped by gap junctional (GJ) blockade. Here, we found that GJ blockers attenuate hippocampal seizure activity induced by a novel seizuregenic protocol using Co 2+ . We hypothesized that this activity may occur because of the altered expression of connexin (Cx) and/or pannexin (Panx) mRNAs and protein.We found a 1.5-, 1.4-, and 2-fold increase in Panx1, Panx2, and Cx43 mRNAs, respectively. Significant post-translational modifications of the proteins Cx43 and Panx1 were also observed after the Co 2+ treatment. No changes were observed in the presence of tetrodotoxin, indicating that seizure activity is required for these alterations in expression, rather than the Co 2+ treatment itself. Further analysis of the QPCR data showed that the Cx and Panx transcriptome becomes remarkably re-organized. Pannexin (Panxs 1 and 2) and glial connexin mRNA became highly correlated to one another; suggesting that these genes formed a transcriptomic network of coordinated gene expression, perhaps facilitating seizure induction. These data show that seizure activity up-regulates the expression of both glial and neuronal GJ mRNAs and protein while inducing a high degree of coordinate expression of the GJ transcriptome. We have shown previously that mechanisms involving activity-dependent facilitation of GJ communication may play a major role in Co 2+ -induced epileptiform discharges (He et al. 2009). To further understand the underlying cellular mechanism of the Co 2+ -induced seizure activity, the expression levels of Cx and Panx mRNA and protein were analyzed using quantitative RT-PCR (QRT-PCR) and western blotting in an in vitro mouse hippocampal model. Materials and methodsAll experimentation conducted in this study has been reviewed and approved by the animal care committee of our institution.Preparation of mouse hippocampal tissues C57BL/6 male mice (postnatal 15 days; Charles River Laboratories, Montreal, QC, Canada) were decapitated under isoflurane anesthesia, and their brains were quickly dissected out and maintained in an ice-cold, oxygenated artificial cerebrospinal fluid (ACSF in mM: 125 NaCl, 3.5 KCl, 1.25 NaH 2 PO 4 , 25 NaHCO 3 , 2 CaCl 2 , 1.3 MgSO 4 , and 10 glucose) at pH 7.4 when aerated with 95% O 2 -5% CO 2 for a few minutes before further dissection. For preparing whole hippocampal isolates (Wu et al. 2002), we used a fine glass probe and gently separated the dentate gyrus from the CA1 area, and then removed the dentate gyrus area while preserving the CA3-CA1 tissues. The whole hippocampal isolates were maintained in warmed ACSF (31-32°C) for 1 h before further experimental manipulations. Co 2+ treatmentWhole hippocampal isolates (left and right) from C57BL/6 male mice were prepared and pre-incubated separately in warmed ACSF (31-32°C) for 1 h before further experimental manipulations. Then, 100 lM CoCl 2 (Sigma-Aldrich, Oakville, ON, Canada) was added to the ACSF of one of the isolates. Both control (without cobalt) and treated hippocampal isolates were continu...
Chronic (18 h) exposure of cultured hippocampal slices to the type-A GABA receptor blocker, bicuculline methiodide (BMI) 10 lM increased the levels of connexin 43 (Cx43) and connexin 32 (Cx32) mRNAs, but not connexin 26 and connexin 36, as demonstrated by RNase protection assays. The levels of Cx43 and Cx32 proteins in membrane fractions detected by western blotting were also significantly increased. Immunoblotting indicated that BMI also promoted a significant expression of the transcription protein c-fos. The rate of fluorescence recovery after photobleaching, an index of gap junctional coupling, was also significantly increased, whereas it was blocked by the gap junctional blocker, carbenoxolone (100 lM). Extracellular recordings in CA1 stratum pyramidale, performed in BMI-free solution, demonstrated that BMIexposed cultures possessed synaptic responses characteristic of epileptiform discharges: (i) significantly greater frequency of spontaneous epileptiform discharges, (ii) post-synaptic potentials with multiple population spikes, and (iii) significantly longer duration of primary afterdischarges. Carbenoxolone (100 lM), but not its inactive analog, oleanolic acid (100 lM), reversibly inhibited spontaneous and evoked epileptiform discharges. The findings of BMI-induced parallel increases in levels of gap junction expression and function, and the increase in epileptiform discharges, which were sensitive to gap junctional blockers, are consistent with the hypothesis that increased gap junctional communication plays an intrinsic role in the epileptogenic process. Keywords: bicuculline, carbenoxolone, connexins, epilepsy, gap junction. Abbreviations used: ACSF, artificial cerebrospinal fluid; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionate; APV, 2-amino-5-phosphonovaleric acid; BMI, bicuculline methiodide; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; CREB, cAMP response element binding protein; Cx, connexin; FRAP, fluorescence recovery after photobleaching; IPSC, inhibitory post-synaptic current; MPP, myelin proteolipid protein; PAD, primary afterdischarge; PPD, pair-pulse depression; Syn, synapsin.
This study presents a model of chronic, recurrent, spontaneous seizures in the intact isolated hippocampal preparation from mice aged P8-P25. Field activity from the CA1 pyramidal cell layer was recorded and recurrent, spontaneous seizure-like events (SLEs) were observed in the presence of low Mg2+ (0.25 mM) artificial cerebrospinal fluid (ACSF). Hippocampi also showed interictal epileptiform discharges (IEDs) of 0.9-4.2 Hz occurring between seizures. No age-specific differences were found in SLE occurrence (2 SLEs per 10 min, on average), duration, and corresponding frequencies. After long exposure to low Mg2+ ACSF (>3 h), SLEs were completely reversible within minutes with the application of normal (2 mM Mg2+) ACSF. The AMPA antagonist, CNQX, blocked all epileptiform activity, whereas the NMDA antagonist, APV, did not. The gamma-aminobutyric acid (GABA)A antagonist, bicuculline, attenuated and fragmented SLEs, implicating interneurons in SLE generation. The L-type Ca2+ blocker, nifedipine, enhanced epileptiform activity. Analysis of dual site recordings along the septotemporal hippocampus demonstrated that epileptiform activity began first in the temporal pole of the hippocampus, as illustrated by disconnection experiments. Once an SLE had been established, however, the septal hippocampus was sometimes seen to lead the epileptiform activity. The whole hippocampus with intact local circuitry, treated with low Mg2+, provides a realistic model of recurrent spontaneous seizures, which may be used, in normal and genetically modified mice, to study the dynamics of seizures and seizure evolution, as well as the mechanisms of action of anti-epileptic drugs and other therapeutic modalities.
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