Activation of Astroglial Connexin Is Involved in Concentration-Dependent Double-Edged Sword Clinical Action of Clozapine

Clozapine (CLZ) is a gold-standard antipsychotic against treatment-refractory schizophrenia, but is one of the most toxic antipsychotic agents. Pharmacological mechanisms of the double-edged sword clinical action of CLZ remain to be clarified. To explore the mechanisms of CLZ, the present study determined the astroglial transmission associated with connexin43 (Cx43), which is the most principal expression in astrocytes and myocardial cells, and expression of Cx43 in primary cultured astrocytes. Both acute and subchronic administrations of CLZ concentration-dependently increased Cx43-associated astroglial release of l-glutamate and d-serine, whereas therapeutic-relevant concentration of CLZ acutely did not affect but subchronically increased astroglial release. In contrast, after the subchronic administration of therapeutic-relevant concentration of valproate (VPA), acute administration of therapeutic-relevant concentration of CLZ drastically increased Cx43-associated astroglial releases. VPA increased Cx43 expression in cytosol fraction without affecting plasma membrane fraction, whereas CLZ increased Cx43 expression in both fractions. Acute administration of therapeutic-relevant concentration of CLZ drastically increased Cx43 expression in the plasma membrane fraction of astrocytes subchronically treated with VPA. The present findings suggest that CLZ-induced the activation of Cx43-associated channel activity and transported Cx43 to plasma membrane, probably contribute to the double-edged sword clinical action of CLZ, such as improvement of cognitive dysfunction and CLZ-induced myocarditis.


Introduction
It has been established that dysfunctions of both dopaminergic and glutamatergic transmission play important roles in the pathophysiology of schizophrenia, with various antipsychotics improving dysfunctions of mesolimbic and mesocortical dopaminergic transmissions with thalamocortical glutamatergic transmission [1][2][3][4][5][6][7]; however, schizophrenia is considered to be a heterogeneous disorder that is unlikely to be caused by a single etiological factor, but rather by a complex network of interacting pathogenic influences [8,9]. Impairment of the N-methyl-d-aspartate (NMDA)/glutamate receptor (NMDAR) contributes to the pathophysiology of schizophrenia [1][2][3]6,[9][10][11][12]. Several clinical reports have demonstrated that NMDAR antagonists (e.g., phencyclidine and ketamine) can generate schizophrenia-like positive and negative symptoms in healthy volunteers [1,13,14] and exacerbate psychosis in patients with schizophrenia [15]. Moreover, NMDAR antagonist-induced psychosis Our recent preclinical study demonstrated that CLZ acutely activated astroglial Cxs activity, in a concentration-dependent manner. Clinically, survival analysis has suggested that the lower limit of the therapeutic range of CLZ serum concentration is 0.6 µM [37], whereas exceeding 4 µM significantly increases the risk of seizures [38]. Lower than 3 µM CLZ did not affect the astroglial Cxs, whereas toxic concentrations of CLZ (higher than 10 µM) activated astroglial Cxs activity. These clinical and preclinical findings suggest that the mechanism of clinical action of CLZ is a candidate in the function of Cxs associated with Cx43; however, the effect of chronic administration of CLZ on tripartite synaptic transmission associated with Cxs remains to be clarified. Based on these backgrounds, to clarify the mechanisms of double-edged sword clinical action of CLZ, the present study determined the concentration-dependent effects of acute and subchronic administrations of CLZ and VPA on astroglial releases of l-glutamate and d-serine associated with Cxs and the astroglial expression of Cx43 using primary cultured astrocytes.

Preparation of Primary Astrocyte Culture
All animal care and experimental procedures described in this report complied with the Ethical Guidelines established by the Institutional Animal Care and Use Committee at Mie University, Japan (No. 2019-3-R1) and are reported in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines [40]. Astrocytes were prepared using a protocol adapted from previously described methods [5,6,12,24,[41][42][43][44].

Concentration-Dependent Effects of Acute and Subchronic Administration of VPA on Astroglial
Releases of l-glutamate and d-serine (Study_3) To determine the effects of acute administration of VPA on astroglial releases of l-glutamate and d-serine, during DIV21 to DIV28, astrocytes were incubated in fDMEM, not containing any target agents. On DIV28 during pretreatment incubation, astrocytes were incubated in ACSF containing VPA (0, 300, 1000, or 3000 µM) for 60 min. After pretreatment incubation, astrocytes were incubated in ACSF, MK-ACSF, or HK-ACSF containing the same agent of pretreatment incubation for 20 min.
To determine the effects of subchronic administration of VPA on astroglial releases of l-glutamate and d-serine, during DIV21 to DIV28, astrocytes were incubated in fDMEM containing VPA (0, 300, 1000, or 3000 µM). On DIV28 during pretreatment incubation, astrocytes were incubated in ACSF containing the same agent for 60 min. After pretreatment incubation, astrocytes were incubated in ACSF, MK-ACSF, or HK-ACSF containing the same agent of pretreatment incubation for 20 min.

Simple Western Analysis
Simple Western analyses were performed using Wes (ProteinSimple, Santa Clara, CA) according to the ProteinSimple user manual [27]. Lysate of primary cultured astrocytes was mixed with a master mix (ProteinSimple) to a final concentration of 1×sample buffer, 1×fluorescent molecular weight markers, and 40 mM dithiothreitol, then heated at 95 • C for 5 min. The samples, blocking reagent, primary antibodies, horseradish peroxidase (HRP)-conjugated second antibodies, chemiluminescent substrate, and separation and stacking matrices were also dispensed to designated wells in a 25-well plate. After plate loading, the separation electrophoresis and immunodetection steps took place in the capillary system and were fully automated. Simple Western analysis was carried out at room temperature, and instrument default settings were used. Capillaries were first filled with separation matrix, followed by stacking matrix and about 40 nL sample loading. During electrophoresis, proteins were separated on the basis of molecular weight through the stacking and separation matrices at 250 volts for 40-50 min and then immobilized on the capillary wall using proprietary photo-activated capture chemistry. The matrices were then washed out. Capillaries were next incubated with a blocking reagent for 15 min, and target proteins were immunoprobed with primary antibodies, followed by HRP-conjugated secondary antibodies. Antibodies of GAPDH (NB300-322SS, Novus Biologicals, Littleton, CO) and Cx43 (C6219, Sigma-Aldrich, St. Louis, MO, USA) were diluted in antibody diluent (ProteinSimple) with 1:100 dilution. The antibody incubation time was 0-120 min with antibody diluents. Luminol and peroxide (ProteinSimple) were then added to generate chemiluminescence, which was captured by a charge coupled device (CCD) camera. The digital image was analyzed with Compass software (ProteinSimple), and the quantified data of the detected protein were reported as molecular weight, signal/peak intensity.

Statistical Analysis
All experiments were designed with equal-sized groups (N = 6) that were predetermined based on our previous studies [5,12,[41][42][43][44]. All values are expressed as mean ± SD, and a p value less than 0.05 was considered statistically significant. Statistical analyses were performed in BellCurve for Excel Version 3.2. (Social Survey Research Information Co., Ltd., Tokyo, Japan). Astroglial transmitter concentrations were analyzed by Mauchly's sphericity test, followed by multivariate analysis of variance (MANOVA) using BellCurve for Excel. When the data did not violate the assumption of sphericity (p > 0.05), the F-value of MANOVA was analyzed using sphericity-assumed degrees of freedom. However, if the assumption of sphericity was violated (p < 0.05), the F-value was analyzed using Chi-Muller's-corrected degrees of freedom. When the F-value for the agent/concentration factors of MANOVA was significant, the data was analyzed by Tukey's post hoc test. Protein expression of Cx43 in cytosol and plasma membrane fractions was analyzed by two-way analysis of variance (ANOVA) with Tukey's post hoc test using BellCurve for Excel.

Concentration-Dependent Effects of Acute Administration of CLZ on Astroglial Releases of L-glutamate and D-serine (Study_1)
Acute administration of CLZ (0, 1, 3, 10, 30, and 100 µM) concentration-dependently increased basal and K + -evoked astroglial releases of L-glutamate (F stimulation (2,15) Figure 1A,B). The K + -evoked stimulation (50 and 100 mM) concentration-dependently increased astroglial releases of L-glutamate and D-seine ( Figure 1A,B). The threshold concentration of acute administration of CLZ on basal releases of L-glutamate and D-serine was 100 µM. The threshold concentration of acute administration of CLZ on 50 mM K + -evoked releases of L-glutamate and D-serine was 100 µM and 30 µM, respectively. The threshold concentration of acute administration of CLZ on 100 mM K + -evoked releases of L-glutamate and D-serine was 30 µM.

Acute Effects of CLZ on Astroglial Releases of L-glutamate and D-serine from Astrocytes Subchronically
Administrated with Therapeutic-Relevant Concentration of VPA (Study_5) Acute administration of CLZ concentration-dependently increased K + -evoked astroglial releases of L-glutamate and D-serine ( Figure 5A   After wash-out, astrocytes were incubated in ACSF containing the same concentration of CLZ without (control: opened circles) or with therapeutic-relevant concentration of VPA (1000 µM: closed circles) for 60 min (pretreatment incubation). After pretreatment, to determine the K + -evoked astroglial releases of L-glutamate and D-serine, astrocytes were incubated in MK-ACSF (50.0 mM K + : black circles) or HK-ACSF (100.0 mM K + : blue circles) containing the same concentrations of CLZ and VPA during pretreatment incubation for 20 min, and then incubate medium (MK-ACSF or HK-ACSF) was collected for analysis. Ordinate: mean ± SD (n = 6) of extracellular levels of l-glutamate and d-serine (µM). Abscissa: concentration of CLZ (µM). * p < 0.05 and ** p < 0.01 vs. CLZ free by MANOVA with Tukey's post hoc test.

Acute Effects of CLZ on Astroglial Releases of L-glutamate and D-serine from Astrocytes Subchronically Administrated with Therapeutic-Relevant Concentration of VPA (Study_5)
Acute administration of CLZ concentration-dependently increased K + -evoked astroglial releases of L-glutamate and D-serine ( Figure 5A Figure 5A,B). Subchronic VPA administration also reduced the threshold concentration of acute administration of CLZ on 100 mM K + -evoked releases of L-glutamate (from 30 µM to 3 µM) and D-serine (from 10 µM to 3 µM). Astrocytes were incubated in fDMEM without (control: opened circles) or with VPA (1000 μM: closed circles). After wash-out, astrocytes were incubated in ACSF containing the same concentration of VPA with CLZ (0, 1, 3, 10, 30, 100 μM) for 60 min (pretreatment incubation). After pretreatment incubation, to determine the K + -evoked astroglial releases of L-glutamate and D-serine, astrocytes were incubated in MK-ACSF (50.0 mM K + : black circles) or HK-ACSF (100.0 mM K + : blue circles) containing the same concentration of CLZ and VPA during pretreatment incubation for 20 min, and then incubate medium (MK-ACSF or HK-ACSF) was collected for analysis. Ordinate: mean ± SD (n = 6) of extracellular levels of L-glutamate and D-serine (μM). Abscissa: concentration of CLZ (μM). * p < 0.05 and ** p < 0.01 vs. CLZ free, and @@ p < 0.01 vs. VPA free (control) by MANOVA with Tukey's post hoc test.

Effects of VPA and CLZ on Astroglial Transmission Associated with Cxs.
The present study demonstrated that CLZ enhanced astroglial releases of L-glutamate and Dserine, in a concentration-dependent manner. Survival analysis has suggested that effective relapse prevention requires the maintenance of patients at CLZ serum concentrations above 0.6 μM [37]; however, exceeding 4 μM can significantly increase the risk of adverse effects such as seizures [38]. Therefore, acute and subchronic administrations of 1 and 3 μM CLZ are considered therapeuticrelevant range, whereas higher than 10 μM CLZ is considered supratherapeutic range. The threshold concentrations of acute administration of CLZ on basal, 50 mM and 100 mM K + -evoked astroglial releases of L-glutamate and D-serine were 100 μM, 30-100 μM and 10-30 μM, respectively. The K +evoked astroglial release was composed of astroglial exocytosis [12,24,44], output through glutamate transporter [5,41,42], and Cxs [6]. Cxs activities were regulated by Ca 2+ , K + , and plasma membrane voltage [6,31]. During resting stage, Cxs exhibits a low open probability; however, the elevation of plasma membrane voltage activates Cxs [33]. In the present study, both nonselective Cxs inhibitor, CBX, and selective Cx43 inhibitor, GAP19, reduced the K + -evoked astroglial releases of L-glutamate and D-serine. These results suggest that the K + -evoked astroglial releases of L-glutamate and D-serine, at least partially, comprise the output through Cxs. Therefore, acute administration of CLZ concentration-dependently enhances astroglial releases of L-glutamate and D-serine associated with Cxs, but the stimulatory effects of CLZ on astroglial releases were observed in the supratherapeutic range. The therapeutic concentration of VPA was considered, ranged 350-700 μM [49]. Neither acute administration of therapeutic-relevant nor supratherapeutic concentrations of VPA (300-3000 μM) affected basal, 50 mM or 100 mM K + -evoked astroglial releases of L-glutamate and D-serine. Therefore, acute administration of VPA did not affect Cxs activities.

Effects of VPA and CLZ on Astroglial Transmission Associated with Cxs
The present study demonstrated that CLZ enhanced astroglial releases of L-glutamate and D-serine, in a concentration-dependent manner. Survival analysis has suggested that effective relapse prevention requires the maintenance of patients at CLZ serum concentrations above 0.6 µM [37]; however, exceeding 4 µM can significantly increase the risk of adverse effects such as seizures [38]. Therefore, acute and subchronic administrations of 1 and 3 µM CLZ are considered therapeutic-relevant range, whereas higher than 10 µM CLZ is considered supratherapeutic range. The threshold concentrations of acute administration of CLZ on basal, 50 mM and 100 mM K + -evoked astroglial releases of L-glutamate and D-serine were 100 µM, 30-100 µM and 10-30 µM, respectively. The K + -evoked astroglial release was composed of astroglial exocytosis [12,24,44], output through glutamate transporter [5,41,42], and Cxs [6]. Cxs activities were regulated by Ca 2+ , K + , and plasma membrane voltage [6,31]. During resting stage, Cxs exhibits a low open probability; however, the elevation of plasma membrane voltage activates Cxs [33]. In the present study, both nonselective Cxs inhibitor, CBX, and selective Cx43 inhibitor, GAP19, reduced the K + -evoked astroglial releases of L-glutamate and D-serine. These results suggest that the K + -evoked astroglial releases of L-glutamate and D-serine, at least partially, comprise the output through Cxs. Therefore, acute administration of CLZ concentration-dependently enhances astroglial releases of L-glutamate and D-serine associated with Cxs, but the stimulatory effects of CLZ on astroglial releases were observed in the supratherapeutic range. The therapeutic concentration of VPA was considered, ranged 350-700 µM [49]. Neither acute administration of therapeutic-relevant nor supratherapeutic concentrations of VPA (300-3000 µM) affected basal, 50 mM or 100 mM K + -evoked astroglial releases of L-glutamate and D-serine. Therefore, acute administration of VPA did not affect Cxs activities.
Contrary to acute administrations, chronic administration of CLZ also increased basal and K + -evoked astroglial releases of L-glutamate and D-serine. The stimulatory effects of subchronic administration of CLZ on basal and K + -evoked astroglial releases were predominant compared with those of acute CLZ administration, since the threshold concentrations of subchronic administration of CLZ on basal, 50 mM and 100 mM K + -evoked astroglial releases of L-glutamate and D-serine were reduced to 30 µM (acute: 100 µM), 30 µM (acute: 30-100 µM), and 10 µM (acute: 10-30 µM), respectively. Similar to CLZ, subchronic administration of supratherapeutic concentration of VPA (3000 µM) increased K + -evoked astroglial releases of L-glutamate and D-serine without affecting basal release. These discrepancies between acute and subchronic administrations of CLZ and VPA on basal and K + -evoked astroglial releases of L-glutamate and D-serine suggest that subchronic administration of CLZ and VPA possibly increases expression of Cxs in plasma membrane.
Indeed, simple western analysis detected the subchronic administration of CLZ and VPA increased astroglial expression of Cx43, which is the principal Cx isoform in astrocytes [50]; however, the expression patterns of Cx43 induced by subchronic administrations of VPA and CLZ are not identical. Subchronic administration of VPA increased Cx43 content in cytosol fraction concentration-dependently without affecting that in plasma membrane fraction. Contrary to subchronic administration of CLZ increased Cx43 content in both cytosol and plasma membrane fractions concentration-dependently, whereas an increase in expression of Cx43 in plasma membrane fraction was predominant, rather than those in cytosol fraction. Cx isoforms which are structural proteins in Cxs have an average half-life about 2-3 h [51]. Therefore, the increased Cx43 content induced by subchronic administration of VPA and CLZ is probably mediated by activation of Cx43 turnover.
The Cx gene regulation system, transcriptional factors (Sp1, activator protein 1 complex, cyclic adenosine monophosphate, Wnt signaling pathway), and epigenetic processes (histone modifications, DNA methylation and microRNA species) have been identified [31,52]. Histone deacetylase (HDAC) inhibition is considered to be one of the most principal pharmacological features of VPA, which inhibits Class I and IIa HDAC isoforms [53]. HDAC inhibitors are a class of drugs that increase the acetylation of histone and non-histone proteins to activate transcription, enhance gene expression, and modify the function of target proteins [53]. Both HDAC inhibitors, suberoylanilide hydroxamic acid and trichostatin A, increase expression of Cx43 mRNA and protein [54,55]. Additionally, subchronic administration of Class I and IIa HDAC inhibitor, 4-phenylbutyrate, for 5 days also increases Cx43 expression in NGC-407 cell [56]; however, two previous studies demonstrated VPA did not affect the astroglial expression of Cx43 [57,58]. Subacute (for 24 h) administration of VPA (300-1500 µM) did not affect total lysate fraction of Cx43 expression in astrocytes co-cultured with 5% or 30 % microglia [57]. The Cx43 expression in prefrontal cortex total lysate fraction of rat was not affected by chronically intraperitoneal administration of VPA (300 mg/kg/day for 21 days) [58]; however, the estimated concentration of VPA during subchronic administration of 300 mg/kg/day was lower than therapeutic-relevant range (about 300 µM) [49]. Therefore, taken together with these previous demonstrations, in spite of short half-life of Cx43 (about 2-3 h) [51], the present results suggest that the elevation of Cx43 expression in cytosol fraction of astrocytes induced by VPA needs more than 5 days exposure with higher than therapeutic-relevant concentration of VPA (1000 µM).
Contrary to VPA, CLZ activates the inhibitory and stimulatory expression regulating system on Cx43 synthesis, such as HDAC2, Wnt pathway, transcription factor specificity protein 1, and activator protein 1 complex [59][60][61][62]. The effect of CLZ on Cx43 expression has more impact against post-translational modification than gene expression, since the CLZ-induced Cx43 expression in plasma membrane fraction was higher than that in the cytosol fraction. The post-translational modification of Cx43, including phosphorylation, acetylation, nitrosylation, sumoylation, and ubiquitylation, plays important roles in the Cxs function [31]. Especially, inhibition of ubiquitylation of Cx43 results in the accumulation of Cx43 in plasma membrane [63]. Unfortunately, the effects of CLZ on phosphorylation, acetylation, nitrosylation, sumoylation, and ubiquitylation systems have not been clarified. The detailed mechanism of an increase in the Cx43 expression in the astroglial plasma membrane shall be studied.
Cx43 is the principal Cxs in astrocytes, but Cx26 and Cx30 are also expressed in astrocytes [30]. In spite of lower quantities of Cx26 and Cx30, the hemichannel composed of Cx26 and Cx30 also contributes to astroglial transmission [30]. In the present study, the inhibitory effects of nonselective Cxs inhibitor, CBX, on astroglial releases of L-glutamate and D-serine were predominant compared with selective Cx43 inhibitor, GAP19. Therefore, to clarify the mechanisms of CLZ and VPA on astroglial transmission associated with Cxs, further study should determine the effects of them on astroglial transmission associated with Cx26 and Cx30.

Interaction between VPA and CLZ
The incidence of seizures with CLZ is ranged from 2% to 7.5%, and the risk of CLZ-induced seizures increased with higher doses [18]. The risk of CLZ-induced seizure was increased dose-dependently: lower than 300 mg/day (3%), between 325 and 500 mg/day (8%), and higher than 500 mg/day (38%) [18]. In 50% cases of CLZ-induced seizures, reduced CLZ dose was sufficient to prevent recurrence of seizures [18]. VPA is recommended as the first-line drug in prevention of CLZ-induced seizure and augmentation therapy [20,21]. In the present study, acute administration of therapeutic-relevant concentration of VPA did not affect astroglial releases of L-glutamate and D-serine from astrocytes subchronically administrated with CLZ. Contrary to our expectations, the present study failed to detect the specific novel mechanism of VPA on astroglial transmission of chronically CLZ-exposed astrocytes. Most of the cognitive and behavioral functions associated with astroglial Cx43 have been implicated in the ability to regulate astroglial transmitter release through Cxs during resting stage [64]. Therefore, the present demonstration, the lack of acute VPA administration on astroglial transmission of chronically CLZ-exposed astrocytes, supports the mechanisms of efficacious and safe augmentation agents of CLZ due to less cognitive impairment induced by Cxs [20].
In the present study, neither acute nor subchronic administrations of therapeutic-relevant concentration of CLZ (1-3 µM) affected astroglial transmission; however, acute administration of therapeutic-relevant concentration of CLZ drastically increased astroglial releases of L-glutamate and D-serine from astrocytes which were chronically administrated with therapeutic-relevant concentration of VPA (1000 µM). Therapeutic-relevant concentration of CLZ (3 µM) also drastically increased Cx43 expression in the plasma membrane fraction without affecting that in cytosol fraction of therapeutic-relevant concentration of VPA-administrated astrocytes. These results suggest that increased Cx43 in the cytosol fraction induced by subchronic administration of therapeutic-relevant concentration of VPA (1000 µM) is transported to plasma membrane by CLZ. The rapid titration of CLZ dose and VPA administration at the commencing CLZ are significantly associated with increasing risks of CLZ-induced myocarditis/cardiomyopathies [22]. It has been well established that chronic downregulation of Cx43 is observed in several models of myocarditis and cardiomyopathies [65]. In contrast with the above observation of Cx43 downregulation, expression of Cx43 was increased in the early stage of hypertrophic and dilated cardiomyopathies, but with their progression into heart failure, the levels of Cx43 decreased [65]. Therefore, upregulation of Cx43 in acute stages of hypertrophic and dilated cardiomyopathies may act as the trigger of pathological or pathophysiological onset. Moreover, prolongation of QRS complex duration is one of the risk factors for morbidity and mortality in myocarditis. Recent preclinical study demonstrated that upregulation of Cx43 contributed to the prolongation of QRS complex duration [33]. Taken together with previous clinical and preclinical findings, the present demonstration suggests that the upregulation of cardiac Cx43 in cytosol by chronic administration of therapeutic-relevant concentration of VPA with enhancement of transported cytosol Cx43 to plasma membrane induced by CLZ plays an important role in the pathomechanisms of CLZ-induced myocarditis/cardiomyopathies. To clarify our hypothesis, further studies are needed.

Conclusions
The present study determined the concentration-and time-dependent effects of CLZ and VPA on astroglial transmission of L-glutamate and D-serine associated with Cx43, to explore the mechanisms of the multimodal action of CLZ. Both acute and subchronic administrations of CLZ and VPA increased astroglial releases of L-glutamate and D-serine concentration-dependently, but the therapeutic-relevant concentration of neither CLZ nor VPA affected these releases. The stimulatory effects of subchronic administrations of CLZ and VPA on astroglial releases were more predominant compared with those of acute administrations. Subchronic administrations of both VPA and CLZ concentration-dependently increased Cx43 expression in astrocytes, but the therapeutic-relevant concentration of neither CLZ nor VPA affected Cx43 expression. Especially, VPA increased Cx43 expression in cytosol fraction of astrocytes, whereas CLZ increased Cx43 expression in both cytosol and plasma membrane fractions. After subchronic administration of CLZ, acute administration of therapeutic-relevant concentration of VPA did not affect CLZ-induced astroglial transmitter releases; however, after subchronic administration of therapeutic-relevant concentration of VPA, acute administration of CLZ drastically increased astroglial transmitter releases. Therapeutic-relevant concentration of CLZ alone could not affect astroglial release, whereas after the subchronic administration of therapeutic-relevant concentration of VPA, acute administration of therapeutic-relevant concentration of CLZ could increase astroglial transmitter releases. During subchronic administration of therapeutic-relevant concentration of VPA, acute administration of therapeutic-relevant concentration of CLZ enhanced the transport of cytosol Cx43 to plasma membrane. Therefore, CLZ enhances astroglial functional Cxs via activation of transport of cytosol Cx43 to plasma membrane. These results suggest that the hyperactivation of astroglial Cxs activities induced by supratherapeutic concentration or rapid titration of CLZ, at least partially, contributes to CLZ-induced seizure, but inhibitory effects of VPA on CLZ-induced seizure are not modulated with astroglial transmission associated with Cxs. Interestingly, VPA intake at the commencing CLZ increases risk of CLZ-induced myocarditis/cardiomyopathies, probably via reciprocal activation of Cxs between VPA and CLZ.