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Purpose: The role of gap junctions in seizures is an area of intense research. Many groups have reported anticonvulsant effects of gap junction blockade, strengthening the case for a role for gap junctions in ictogenesis. The cerebral cortex is underrepresented in this body of research. We have investigated the effect of gap junction blockade on seizure-like activity in rat and mouse cerebral cortex slices.
Methods: Seizure-like activity was induced by perfusing with low-magnesium artificial cerebrospinal fluid. The effect of three gap junction blockers was investigated in rat cortical slices; quinine (200 and 400 μm), quinidine (100 and 200 μm), and carbenoxolone (100 and 200 μm). In addition, the effect of mefloquine was investigated in wild-type mice and connexin36 knockout mice. The data were analyzed for the effect on frequency and amplitude of seizure-like events.
Results: Paradoxical excitatory effects on seizure-like activity were observed for all three agents in rat cortical slices. Quinine (200 μm) and carbenoxolone (100 μm) increased both the frequency and amplitude of seizure-like events. Quinidine (100 μm) increased the frequency of events. Higher doses of quinine (400 μm) and carbenoxolone (200 μm) had biphasic excitatory–inhibitory effects. Similar excitatory effects were observed in adult wild-type mouse cortical slices perfused with mefloquine (5 μm or 10 μm), but were absent in slices from connexin36-deficient mice.
Discussion: In conclusion, we have shown a paradoxical proseizure effect of pharmacologic gap junction blockade in a cortical model of seizure-like activity. We suggest that this effect is probably due to a disruption of inhibitory interneuron coupling secondary to connexin36 blockade.
There has been increased interest in recent years in the role of gap junctions in seizure mechanisms. An important hallmark of seizure activity in the brain is believed to be synchronous neuronal population activity. Direct electrical coupling between neurons in the form of gap junctions is thought to contribute to ictogenesis by facilitating the rapid spread of electrical activity between neurons and by enhancing synchronous activity (Carlen et al., 2000; Perez Velazquez & Carlen, 2000). Traub et al. (2001) have suggested that gap junctions may also be important for seizure initiation.
Many groups have reported anticonvulsant effects of gap junction blockers, strengthening the case for a central role for gap junctions in seizure mechanisms (Carlen et al., 2000; Jahromi et al., 2002; Medina-Ceja et al., 2008). The majority of these studies have focused directly on the hippocampus, or are in vivo studies where the hippocampus may be implicated in cortically recorded phenomena (Gajda et al., 2005); Amabeoku & Chikuni, 1992; Amabeoku & Farmer, 2005). Less is known about the relationship between seizures and pharmacologic manipulation of gap junctions in the isolated cerebral cortex. One recent study has shown that carbenoxolone, a broad-spectrum gap junction blocker, causes paradoxical excitatory effects at the level of individual neurons within the cerebral cortex (Yang & Ling, 2007). An excitatory effect of carbenoxolone has also been observed at the cellular level in the hippocampus (Jahromi et al., 2002). These results seem to contradict previous findings showing inhibitory effects of gap junction blockade on population neural activity (Carlen et al., 2000; Jahromi et al., 2002; Medina-Ceja et al., 2008).
The aim of this study was to investigate the effect of gap junction blockade on seizure-like activity originating from the isolated cerebral cortex. We were particularly interested in whether the excitatory effects of gap junction blockade, previously observed by Jahromi et al. (2002) at the single cell level, could be observed as proseizure effects at the population level. We investigated in adolescent rats the effect of three gap junction blockers—carbenoxolone, quinine, and quinidine—on seizure-like activity induced by perfusion of cerebral cortical slices with low magnesium artificial cerebrospinal fluid (ACSF). A consistent finding was that gap junction blockade had the propensity to enhance low-magnesium seizure-like activity. In a second series of experiments we compared the effect mefloquine on seizure-like activity in wild-type mice and knockout mice lacking the gene for connexin36.
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In previous reports, pharmacologic blockade of gap junctions has been consistently associated with anticonvulsant effects, in both the cortex and the hippocampus (Carlen et al., 2000; Jahromi et al., 2002; Gajda et al., 2005; Nilsen et al., 2006; Bostanci & Bagirici, 2007; Medina-Ceja et al., 2008). Our study has shown that in the cerebral cortex, the anticonvulsant effects of quinine, quinidine, and carbenoxolone are preceded by excitatory phenomena, reflected in an increase in the frequency and amplitude of seizure-like events. To our knowledge, this is the first report directly linking gap junction blockade with excitatory effects on population activity within the cerebral cortex.
Gajda and colleagues (2005) anecdotally reported “pro-convulsive” effects of “high” dose (>80 μm) quinine in penicillin-induced neocortical seizures in vivo; however, the data relating to this effect were not presented. They attributed these excitatory effects to unspecified nonspecific (i.e., extra-gap junctional) effects of the drug. Mefloquine and quinine have also been reported to have proconvulsant effects in vivo when administered systemically at high dose (approximately 250 and 600 μm, respectively) (Amabeoku & Chikuni, 1992; Amabeoku & Farmer, 2005). Interestingly these effects seem to be related to a disturbance in GABAergic mechanisms (Amabeoku & Chikuni, 1992; Amabeoku & Farmer, 2005).
We suggest that the proconvulsant activity of quinine and its analogs is due to blockade of connexin36 gap junctions, based on the following evidence. First and most important, the excitatory effects of mefloquine observed in wild-type mice were not observed in mice lacking the gene for connexin36. Mefloquine potently and specifically (between 1 and 5 μm) blocks connexin36 gap junctions (Cruikshank et al., 2004), strongly suggesting that the excitatory effects observed in the wild-type animals were a specific connexin36 effect. Second, if nonspecific actions were responsible for the excitatory effects observed in this study, it is unlikely that completely different classes of drug (carbenoxolone and quinine/quinidine) would show similar effects. Third, connexin36 gap junctions are maximally blocked by quinine at a dose of approximately 100 μm (Srinivas et al., 2001) and twice this dose has no direct effects on neuronal excitability (Bikson et al., 2002). The dose of quinine used in the present study was 200 μm. Therefore, the proconvulsive effects of quinine were observed at a dose associated with strong gap junction blockade, but that has no direct effects on neuronal excitability.
The results of this study are consistent with recent findings showing that carbenoxolone (a broad-spectrum gap junction blocker (Gajda et al., 2005; Nilsen et al., 2006) causes paradoxical excitatory effects at the level of individual neurons within the cerebral cortex (Yang & Ling, 2007). In this study by Yang and Ling, an increase in excitatory postsynaptic potential amplitude was shown to be indirectly caused by uncoupling of (GABAergic) inhibitory interneurons. Connexin36 gap junctions are restricted primarily to a subclass of interneurons, parvalbumin-containing basket cells (Deans et al., 2001; Liu & Jones, 2003; Markram et al., 2004; Baude et al., 2007), which synapse onto pyramidal cells in the region of the soma or proximal dendrites (Deans et al., 2001; Liu & Jones, 2003; Markram et al., 2004). Blocking direct electrical communication within this network of cells by closing gap junctions is likely to have a disinhibitory effect on pyramidal cell activity, hence the excitatory effects observed at both the cellular level (Yang & Ling, 2007) and at the population level in the present study.
In this study we did not observe any difference in baseline seizure activity between wild-type and connexin36 knockout animals. Given the excitatory effects observed with pharmacologic blockade of connexin36 gap junctions, we might have expected the connexin36 knockout animals to show enhanced baseline seizure activity. The most likely explanation is that connexin36 knockouts develop compensatory responses to chronic gap junction blockade. Wide-reaching compensatory changes in neuronal gene regulation have been documented for connexin knockouts (Iacobas et al., 2005; Spray & Iacobas, 2007), although the functional correlates of these changes remains a matter for further investigation. One report has shown morphologic and functional neuronal effects at the subcortical level in connexin36 knockouts (De Zeeuw et al., 2003). Preliminary results from our laboratory have failed to identify any compensatory changes in the mRNA levels for a range of glial and neuronal connexins and pannexins (results not shown). An alternative, and less likely explanation in our view, is that the excitatory effects of mefloquine in wild-type animals are due to off-target effects (see subsequent text for further discussion). If this was the case, however, one would expect similar excitatory effects in both wild-type and knockout animals.
We cannot completely rule out pharmacologic effects on gap junctions other than connexin36 in this study. Connexin45, for example, is expressed in cortical neurons of adult mice (Maxeiner et al., 2003) and rats (Condorelli et al., 2003) and quinine partially blocks this gap junction at a dose of 300 μm (Srinivas et al., 2001). Effects on astrocytic gap junctions may also be relevant. Astrocytes are important regulators of synaptic excitability (Fellin & Haydon, 2005; Tian et al., 2005; Fellin et al., 2006) and changes in astrocytic signaling can alter neuronal population activity (Tian et al., 2005). Mefloquine at a dose of 10 μm causes a 40% blockade of connexin43 (Cruikshank et al., 2004), an astrocytic gap junction protein in the central nervous system. Furthermore, the effect of quinine and its derivatives on connexin30, another astrocytic gap junction protein (Rash et al., 2001), has not been documented. “Off-site” effects, however, do not provide a complete explanation for the observed results, for two reasons. First, the effect of mefloquine was abolished in the connexin36 knockout animals, strongly implying a specific connexin36 effect. Second, the doses of quinine (200 μm) and mefloquine (5–10 μm) used are such that effects on connexin45 and connexin43, respectively, are likely to have been minimal.
A major drawback of blocking gap junctions pharmacologically is the possibility of non–gap junctional drug effects. Mefloquine has been shown to disrupt calcium homeostasis in neurons, although at a concentration four times that used in the present study (Caridha et al., 2008). Carbenoxolone is known to have extra–gap junctional effects. For example, 100 μm carbenoxolone has direct inhibitory effects on neural network activity independent of its effects on gap junctions (Rouach et al., 2003; Chepkova et al., 2008). In addition, carbenoxolone has mineralocorticoid actions, which may reduce seizure propensity (Nilsen et al., 2006). Quinidine and its analogs are also known to have a multitude of actions, many unrelated to gap junction effects (Gattass & De Meis, 1978; Garlid et al., 1986; Pederson et al., 1986). The potential importance of these off-target effects in studies that have documented a reduction in seizure activity with these agents cannot be underestimated, and call into question the significance of gap junctions in ictogenesis. In the present study, a reduction in seizure-like activity was observed at the higher drug doses only, where nonspecific pharmacologic effects may predominate. At the very least, the present results strongly suggest that connexin36 gap junction coupling is not critical for ictogenesis in the low-magnesium cortical model.
In hippocampal (Bikson et al., 2002) and cortical seizure models (Gajda et al., 2005), quinine has been shown to increase seizure frequency concurrently with a reduction in seizure duration (and amplitude). An increase in event frequency may result from a reduction in refractory period due to the shorter duration of each seizure event (Bikson et al., 2002). This explanation does not account for the proseizure effects observed in our study. The baseline duration of each seizure-like event in the present study was already much shorter than in these other models (from 1 to 3 s) and was not significantly reduced during the period of heightened activity.
A potential confounder in this study is the effect of DMSO, a versatile solvent that is widely used to aid delivery of drug compounds that are poorly soluble in aqueous solution. A DMSO concentration of 1% is reported to be a “safe” experimental concentration (Kahler, 2000); however, a close look at the literature reveals that much lower concentrations (as low as 0.02%) might have significant effects on central nervous system function (Tsvyetlynska et al., 2005; Nasrallah et al., 2008). At the maximum dose used (0.2%), no consistent or significant effect on seizure-like event characteristics was observed, supporting earlier reports that this dose does not modulate neuronal excitability in the mammalian cortical slice preparation (Rosen & Andrew, 1991; Nasrallah et al., 2008). However, a marked reduction in seizure-like activity was noted in a subset of slices from a single animal, which was reversible with a return to low-magnesium ACSF. This may explain the small, but significant reduction in seizure-like event amplitude during mefloquine infusion in connexin36 knockout animals. The possibility of an antiseizurogenic effect of DMSO, although an important issue that deserves further attention, does not compromise the present results; if anything, this would have minimized the excitatory effects that we observed.
In conclusion, we have shown a paradoxical proseizure effect of pharmacologic gap junction blockade in a cortical model of seizure-like activity. We suggest that this effect is due to a disruption of inhibitory interneuron coupling resulting from connexin36 blockade. This finding also suggests that in the low-magnesium model, connexin36 gap junctions are not critical for ictogenesis.