Summary:Purpose: The possible role of gap junctions in the manifestation and control of the duration of seizures was tested on the 4-aminopyridine-induced epilepsy model in rats in vivo, by using electrophysiologic, pharmacologic, and molecular biologic techniques.Methods: In electrophysiologic experiments, the functional states of the gap junctions were manipulated with a specific blocker (carbenoxolone) or opener (trimethylamine) at the already active focus of adult, anesthetized rats, 60 min after the induction of the first seizure, which was repeated spontaneously thereafter. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) amplification was used to measure the levels of connexin (Cx) 32, 43, and 36 messenger RNAs (mRNAs) prepared from the areas of the already active primary and mirror foci.Results: After repeated seizures, the expression levels of Cx32, Cx43, and Cx36 mRNAs at the epileptic foci were increased significantly. Blockade of the gap junctions with carbenoxolone shortened the duration of seizures and decreased the amplitude of the seizure discharges, whereas their opening with trimethylamine lengthened the duration and increased the amplitude. Secondary epileptogenesis was facilitated when the gap junctions were opened.Conclusions: Our findings support the idea that, in epileptic foci, the gap junctions are involved in the expression of rhythmic ictal discharges and in the control of the duration and propagation of the individual seizures in vivo. Key Words: Connexins-Gap junctions-4-AP-induced seizureCarbenoxolone-Trimethylamine.Gap junctions are dynamic structures that can be modulated by a number of intracellular and extracellular factors (1-6). The extent of coupling in the in vitro seizure models (7-10) is periodic: it is increased by alkalinization at the start of a seizure, and decreased as acidification occurs toward the end of an ictal period.Our previous work (11) revealed noteworthy upregulations of connexin (Cx)32 and Cx43 mRNAs after repeated seizures both at the primary focus (Pf) and at the mirror focus (Mf, homotopic area contralateral to the Pf). Accordingly, we examined whether manipulation of the functional state of the gap junctions with a specific blocker (carbenoxolone) or opener (trimethylamine, TMA) influences the manifestation, duration, and propagation of seizures. We also were interested in whether repeated seizures influence the expression of the Cx36 gene, coding for a gap-junction protein existing predominantly in neuronal cells of the mature brain (13).

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Heat shock proteins (HSPs) and metallothioneins (MTs) play important roles in protection against environmental stressors. The present study analyzes and compares the regulation of heat shock ( hsp70, hsc70-1 and hsp90alpha ) and metallothionein (MT-1 and MT-2) genes in the heart of common carp, in response to elevated temperature, cold shock and exposure to several heavy metal ions (As 3+ , Cd 2+ and Cu 2+ ), in whole-animal experiments. Among these metals, arsenate proved to be the most potent inducer of the examined stress genes; the hsp90alpha and MT-1 mRNA levels were elevated 11- and 10-fold, respectively, after a 24-h exposure. In contrast, Cd 2+ at 10 mg/L had no impact on the expression of hsp90alpha , and the MT genes also proved to be rather insensitive to Cd 2+ treatment in the heart: only a 2-2.5-fold induction was observed in response to 10 mg/L Cd 2+ . Heat shock resulted in a transient induction of hsp70 (19-fold) and hsp90alpha (15-fold), while elevated temperature had no effect on the expression of the MTs. Direct cold shock induced hsp70 expression (14-fold), while the hsp90alpha (26-fold) and MT-2 (2-fold) expressions peaked after the recovery period following a direct cold shock. The five stress genes examined in this study exhibited a unique, tissue-specific basal expression pattern and a characteristic sensitivity to metal treatments and temperature shocks.

The expression pattern of two metallothionein (MT) genes in response to temperature shock and exposure to Cd(2+) was investigated in the brain of common carp ( Cyprinus carpio ), in whole-animal experiments. The changes in the levels of MT-1 and MT-2 mRNA in the olfactory lobe, midbrain and cerebellum were followed by semiquantitative RT-PCR. The inducibility of the two MT genes was brain region and stressor-specific. Cd(2+) affected mostly the expression of MT-2, while the level of the MT-1 transcript did not change significantly in any of the brain regions examined. Moreover, the MT-2 expression was regulated spatially; MT-2 was induced significantly more strongly in the olfactory lobe than in the cerebellum or midbrain. A sudden temperature drop mainly affected the expression of the MT-1 gene; after 5 h of cold shock, the MT-1 mRNA level was about 25% of the basal value in the cerebellum and the midbrain region. The MT-2 expression did not change significantly during this treatment.