Upregulated Connexin 43 Induced by Loss-Of-Functional S284L-Mutant 4

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Article Upregulated Connexin 43 Induced by Loss-of-Functional S284L-Mutant α4 Subunit of Nicotinic ACh Receptor Contributes to Pathomechanisms of Autosomal Dominant Sleep-Related Hypermotor Epilepsy

Kouji Fukuyama 1, Masashi Fukuzawa 2, Ruri Okubo 1 and Motohiro Okada 1,* 1 Department of Neuropsychiatry, Division of Neuroscience, Graduate School of Medicine, Mie University, Tsu, Mie 514-8507, Japan; [email protected] (K.F.); [email protected] (R.O.) 2 Department of Biology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8560, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-59-231-5018

Received: 8 March 2020; Accepted: 27 March 2020; Published: 29 March 2020

Abstract: To study the pathomechanism and pathophysiology of autosomal dominant sleep-related hypermotor epilepsy (ADSHE), this study determined functional abnormalities of glutamatergic transmission in the thalamocortical motor pathway, from the reticular thalamic nucleus (RTN), motor thalamic nuclei (MoTN) tosecondary motor cortex (M2C) associated with the S286L-mutant α4β2-nicotinic acetylcholine receptor (nAChR) and the connexin43 (Cx43) hemichannel of transgenic rats bearing the rat S286L-mutant Chrna4 gene (S286L-TG), which corresponds to the human S284L-mutant CHRNA4 gene using multiprobe microdialysis, primary cultured astrocytes and a Simple Western system. Expression of Cx43 in the M2C plasma membrane fraction of S286L-TG was upregulated compared with wild-type rats. Subchronic nicotine administration decreased Cx43 expression of wild-type, but did not aﬀect that of S286L-TG; however, zonisamide (ZNS) decreased Cx43 in both wild-type and S286L-TG. Primary cultured astrocytes of wild-type were not aﬀected by subchronic administration of nicotine but was decreased by ZNS. Upregulated Cx43 enhanced glutamatergic transmission during both resting and hyperexcitable stages in S286L-TG. Furthermore, activation of glutamatergic transmission associated with upregulated Cx43 reinforced the prolonged Cx43 hemichannel activation. Subchronic administration of therapeutic-relevant doses of ZNS compensated the upregulation of Cx43 and prolonged reinforced activation of Cx43 hemichannel induced by physiological hyperexcitability during the non-rapid eye movement phase of sleep. The present results support the primary pathomechanisms and secondary pathophysiology of ADSHE seizures of patients with S284L-mutation.

Keywords: idiopathic epilepsy; zonisamide; microdialysis; connexin; hemichannel

1. Introduction Until recently, numerous gene mutations of ion channels, which regulate neuronal excitabilities in the central nervous system, have been identiﬁed in the various pedigrees of idiopathic epilepsy syndromes. In vitro expression studies using Xenopus oocytes or human embryonic kidney cells have demonstrated the functional abnormalities of mutant ion channels, whereas these abnormalities cannot fully resemble the situation in vivo [1]. In contrast, animal models bearing the mutant gene corresponding to the human mutation have the advantage that the functional consequences of a given mutation can be studied in the complex context of an intact living organism [1].

Pharmaceuticals 2020, 13, 58; doi:10.3390/ph13040058 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2020, 13, 58 2 of 23

Autosomal dominant sleep-related hypermotor epilepsy (ADSHE) [2] was first identified as distinct familial idiopathic epilepsy (previously ADNFLE: autosomal dominant nocturnal frontal lobe epilepsy) in 1994 [3]. Typically, ADSHE seizures are symptomatically comparable to those seen in frontal lobe epilepsy and occur predominantly during the non-rapid eye movement sleep phase [2,4–6]. These seizures are complex, stereotyped hyperkinetic seizures that consist of three types of motor seizures, ‘nocturnal paroxysmal arousals’, ‘nocturnal paroxysmal dystonia’ and ‘episodic nocturnal wandering’ [2,4–6]. Onset ages of ADSHE are usually prior to age 20 (mean age of onset is 14 years) [2,4–6]. Although the majority of ADSHE patients are not progressive but lifelong, frequencies of ADSHE seizures are usually reduced age-dependently. Interestingly, a sporadic form of sleep-related hypermotor epilepsy (SHE) [2,7] (previously NFLE: nocturnal frontal lobe epilepsy), is clinically indistinguishable from those of ADSHE [4,5]. Therefore, any observed clinical phenotypes have been considered to belong uniformly to ADSHE/SHE syndrome [2]. The seizure features of ADSHE/SHE syndrome are mostly uniform; however, ADSHE/SHE syndrome is classified based on the characteristics in two major clinical features, antiepileptic drug sensitivity and comorbidity with cognitive deficit. In spite of the uniformity of ADSHE/SHE syndrome, various missense mutations in genes encoding α2 subunit (CHRNA2) of the nicotinic acetylcholine receptor (nAChR), α4-nAChR (CHRNA4), β2-nAChR (CHRNB2), corticotropin-releasing hormone (CHR), sodium-activated potassium channel subunit 1 (KCNT1) and DEP domain containing protein 5 (DEPDC5) have been identified in many ADSHE pedigrees [2,5,6]. Several clinical studies reported that gene mutations provide the variances in ADSHE/SHE syndrome, since the classical three ADSHE mutations of CHRNA4, S280F, S284L and insL, differ significantly with respect to their neuropsychiatric comorbidities and antiepileptic drug sensitivities. It has been well established that carbamazepine (CBZ) is the first-choice anticonvulsant for ADSHE/SHE and leads to remission in approximately 60% of ADSHE/SHE patients, including ADSHE patients with S280F and insL mutations [4,8,9]. However, ADSHE patients with S284L mutation are resistant to CBZ but responsive to other anticonvulsants such as zonisamide (ZNS) [5,10–13]. Usually ADSHE/SHE seizures are the sole major symptom of ADSHE/SHE syndrome, and additional neuropsychiatric features have been reported in just lower than 3% of ADSHE patients with S280F mutation [6,14–18]. Contrary, ADSHE patients with insL and S284L mutation have been known to be comorbid with cognitive disturbances such as schizophrenia-like psychosis, autism and intellectual disability [7,11–13,16,19,20]. Therefore, ADSHE is a syndrome of heterogeneous aetiologies characterized by the occurrence of sleep-related seizures with various motor manifestations, depending on the involved functional abnormalities of neuronal networks within the frontal cortex and its associated neural circuits. Indeed, there are intra- and inter-familial variations in both onset age and prognosis in the same ADSHE pedigree [2,4–6]. Recently, we demonstrated the pathophysiology of CBZ-resistant/ZNS-sensitive ADSHE seizures and pathomechanisms of comorbidity of cognitive deficits using a genetic ADSHE model rat, namely the S286L transgenic rat (S286L-TG), bearing the missense S286L mutation in the rat Chrna4 gene which corresponds to the S284L mutation in the human CHRNA4 gene [21,22]. These studies using S286L-TG demonstrated several pathomechanisms of ADSHE seizures associated with the S284L mutation [21,22]. The first was that the basal extracellular L-glutamate level in various brain regions of S286L-TG were larger compared with wild-type rats [21,22], similar to S284L-TG [23]. The second was that activation of S286L-mutant α4β2-nAChR in the reticular thalamic nucleus (RTN) of S286L-TG generated the relatively GABAergic disinhibition in the motor thalamic nuclei (MoTN) resulting in enhancement of glutamatergic transmission in the thalamocortical motor pathway, from the MoTN to the secondary motor cortex (M2C) [21], and in the thalamic hyperdirect pathway, from the MoTN to the subthalamic nucleus (STN) [22]. The third was that the M2C itself cannot independently generate epileptic discharge, whereas the M2C can integrate external excitatory inputs from the thalamocortical motor pathway (MoTN-M2C pathway), resulting in the generation of epileptic discharges in the M2C [21]. These findings suggest the pathomechanisms of three major ADSHE seizures, ‘nocturnal paroxysmal arousals’, ‘nocturnal paroxysmal dystonia’ and ‘episodic nocturnal wandering’ [21,22]. Pharmaceuticals 2020, 13, 58 3 of 23

In spite of these efforts, additional studies are needed to clarify the pathomechanisms and pathophysiology of ADSHE with the S284L mutation. Exploring the detailed mechanisms of enhanced basal extracellular L-glutamate levels in the MoTN, M2C and STN of S286L-TG [21–23], as well as the mechanisms of focus generation due to the integration of hyperglutamatergic transmission of the thalamocortical motor pathway in the M2C [21,22], will contribute to breakthroughs in the elucidation of epileptogenesis and/or ictogenesis of ADSHE with the S284L mutation. Furthermore, even if ADSHE seizures are controlled, once a patient experiences an ADSHE seizure, they will experience many ADSHE seizures during that same night [4]. These intra-individual variations of ADSHE seizure frequency suggest the possibilities that the event-related factors leading to epileptogenesis and/or ictogenesis of ADSHE probably play important roles in the secondary pathogenesis of ADSHE seizures. In vitro experiments revealed that α4β2-nAChR activation can decrease endothelial intercellular communication via the downregulation of connexin 43 (Cx43) [24]. Connexin is a family of 21 protein isoforms, and 11 connexin isoforms are expressed in the central nervous system [25–27]. Six connexin proteins assemble to form a homomeric/heteromeric connexon. Two connexon in two neighboring cells (including neuron, astrocyte, oligodendrocyte and microglia) form a gap-junction channel with an aqueous pore and charged surface walls [25–27]. A single connexon contributes to chemical connection between intra- and extra-cellular spaces as hemichannels [25–27]. Cx43 is the most widely expressed and predominantly expressed isoform in the central nervous system, including in astrocytes [28,29]. Accumulating evidence indicates that connexin is crucial to the coordination and maintenance of physiologic activity including neuronal excitability, synaptic plasticity, tripartite synaptic transmission and homeostasis maintenance in the central nervous system [26,27]. Furthermore, the expression of Cx43 is increased in glia but not in neurone in animal models and patients with epilepsy [30]. In particular, inhibitors of connexin channels can prevent the onset of epileptic seizures [26,31,32]. Recently, we demonstrated that an atypical antipsychotic, clozapine, was able to increase hemichannel activity as well as Cx43 expression in a concentration dependent manner in the plasma membrane of astrocytes [33,34]. The stimulatory effects of clozapine on Cx43 expression however plays a double-edged sword where it has demonstrated improvement in cognitive dysfunction in antipsychotic-refractory schizophrenia but has also resulted in clozapine-induced toxic myocarditis and convulsion via enhanced astroglial L-glutamate release [33,34]. Based on these clinical and preclinical findings, to explore the pathomechanisms and pathophysiology of ADSHE seizure associated with Cx43 induced by S284L-mutant α4β2-nAChR, the present study determined the effects of the subchronic administration of a therapeutic-relevant dose of ZNS on functional abnormality of transmission in the focus region (M2C) and the thalamocortical motor pathway associated with Cx43 and α4β2-nAChR of S286L-TG. This study used multiprobe microdialysis and the effects of nicotine and zonisamide on Cx43 expression in plasma membrane of subchronically administrated S286L-TG and primary cultured astrocytes using a Simple Western system.

2. Materials and Methods

2.1. Chemical Agents The selective α4β2-nAChR agonist, (E)-N-Methyl-4-(3-pyridinyl)-3-buten-1-amine oxalate (RJR2403) was obtained from Cosmo Bio (Tokyo, Japan) [35]. Amino-3-(3-hydroxy-5-methyl -isoxazol-4-yl)propanoic acid (AMPA) and nicotine ditartrate [35] were obtained from Wako Chemicals (Osaka, Japan). The selective Cx43 inhibitor, GAP19 [33,36] was obtained from Funakoshi (Tokyo, Japan). Zonisamide sodium salt (ZNS) was provided by Dainippon-Sumitomo Pharma (Osaka, Japan). All compounds were prepared on the day of the experiment. RJR2403, AMPA, GAP19 and ZNS were dissolved in modified Ringer’s solution (MRS) or 100 mM potassium containing MRS (HKMRS) directly for microdialysis study. ZNS and nicotine were also dissolved in artificial cerebrospinal fluid (ACSF) or Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (fDMEM) for the Pharmaceuticals 2020, 13, 58 4 of 23 primary cultured astrocyte study. The detailed compositions of ACSF, fDMEM, MRS and HKMRS are described in the following section. Previous studies have reported that the chronic administration of a therapeutic-relevant dose of ZNS ranged from 25 mg/kg/day to 50 mg/kg/day [37–39]. The therapeutic-relevant plasma concentration of ZNS against epilepsy ranged from 47 to 330 µM[38,40]. According to previous reports, in the present study, to study the subchronic effects of ZNS, each rat was subcutaneously administered ZNS (40 mg/kg/day for 7 days) [23] using a subcutaneous osmotic pump (2ML_1, Alzet, Cupertino, CA; the nominal pumping rate and duration was 10 µL/h for 7 days). Primary cultured astrocytes were subchronically administrated the lowest therapeutic-relevant concentration of ZNS (50 µM). To study the effects of nicotine on Cx43 expression in the plasma membrane, rats were subchronically administrated nicotine (10, 25 and 50 mg/kg/day) for 7 days using a subcutaneous osmotic pump (2ML_1, Alzet), according to a previous clinical study [41]. Primary cultured astrocytes were administered 1 and 3 µM nicotine for 7 days.

2.2. Preparation of the Microdialysis System Animal care, the experimental procedures, and protocols for animal experiments were approved by the Animal Research Ethics Committee of the Mie University School of Medicine (No. 24–37-R1). All studies involving animals have been reported in accordance with the ARRIVE guidelines for reporting experiments involving animals [42]. A total of 204 rats were used in the experiments described. Male S286L-TG [21,22] and wild-type littermates were anesthetized with 1.8% isoflurane and then placed in a stereotactic frame. A concentric direct-insertion type dialysis probe (0.22 mm diameter, 2 mm exposed membrane: Eicom, Kyoto, Japan) was implanted in the secondary motor cortex (M2C: A = +1.8 mm, L = +1.6 mm, V = 2.4 mm, relative to bregma), motor thalamic nuclei − comprising ventroanterior and ventrolateral thalamic nuclei (MoTN: A = 2.0 mm, L = +1.4 mm, − V = 7.2 mm, relative to bregma), and reticular thalamic nucleus (RTN: A = 1.4 mm, L = +1.2 mm, − − V = 7.2 mm, relative to bregma) [43]. − Perfusion experiments were commenced 18 h after recovery from isoflurane anaesthesia. The perfusion rate was set at 2 µL/min in all experiments, using modified Ringer’s solution (MRS) + + 2+ 2+ composed of the following (in mM): 145 Na , 2.7 K , 1.2 Ca , 1.0 Mg , and 154.4 Cl−, buffered with 2 mM phosphate buffer and 1.1 mM Tris buffer at pH 7.4, or 100 mM potassium containing MRS + + 2+ 2+ (HKMRS) composed of the following (in mM): 47.7 Na , 100.0 K , 1.2 Ca , 1.0 Mg , and 154.4 Cl−, buffered with 2 mM phosphate buffer and 1.1 mM Tris buffer at pH 7.4. The detailed experimental designs are described in the following section (in Section 2.3). Extracellular L-glutamate level was measured by ultra-high-performance liquid chromatography (UHPLC), 8 h after starting the MRS perfusion. When the coefficients of variation for L-glutamate reached < 5% over a period of 60 min (stabilization), control data were obtained over another 60 min period (pre-treatment period). This was followed by perfusion with MRS containing target agents. Following the microdialysis experiments, the rats were anesthetized using 1.8% isoflurane and their brains removed. The locations of the dialysis probes were verified by histological examination using 200 µm thick tissue slices (Vibratome 1000, Technical Products International, St. Louis, MO, USA).

2.3. Experimental Designs of Microdialysis Study Rats were randomly assigned to the treatment groups of each experiment. The three experimental designs were distinguished by the route of drug administration. Experiments were not started until three consecutive baseline transmitter measurements yielded a coeﬃcient of variation of less than 5%. Dialysates were then collected for 60 min (pretreatment period) followed by 180 min of sampling after the administration of experimental agents. The schematic experimental designs are shown in Figure1. Pharmaceuticals 2020, 13, 58 5 of 23

Pharmaceuticals 2020, 13, x FOR PEER REVIEW 5 of 23

FigureFigure 1. 1.Schematic Schematic experimental experimental designs designs of microdialysis of microdialysis experiments. experiments. Panels PanelsA, B, CAare, B represented, C are therepresented experimental the experimental designs of Study_1,designs of Study_2 Study_1, and Study_2 Study_3, and Study_3, respectively. respectively. MRS: modiﬁedMRS: modified Ringer’s solution,Ringer’s HKMRS:solution, HKMRS: 100 mM 100 potassium mM potassium containing containing MRS, RJR2403MRS, RJR2403 (100 µ (100M:selective μM: selectiveα4β2-nAChR α4β2- agonist):nAChR (E)-N-Methyl-4-(3-pyridinyl)-3-buten-1-amineagonist): (E)-N-Methyl-4-(3-pyridinyl)-3-buten-1-amine oxalate, AMPAoxalate, (100 AMPAµM: AMPA (100/glutamate μM: receptorAMPA/glutamate agonist): amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic receptor agonist): amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid, GAP19 (100 µM: selectiveacid, connexin43GAP19 (100 (Cx43) μM: selective hemichannel connexin43 blocker), (Cx43) MoTN: hemichanne motor thalamicl blocker), nuclei, MoTN: RTN: motor reticular thalamic thalamic nuclei, nucleus, M2C:RTN: secondary reticular thalamic motor cortex. nucleus, M2C: secondary motor cortex.

2.3.1.2.3.1. Study_1: Study_1: EEffectsffects of Subchronic Administrati Administrationon of of the the Therapeutic-Re Therapeutic-Relevantlevant Dose Dose of ZNS of ZNS on on GlutamatergicGlutamatergic Transmission Transmission inin thethe Thalamocortical Motor Motor Pathway. Pathway RatsRats werewere subchronically administrated administrated with with or without or without therapeutic-relevant therapeutic-relevant dose of dose ZNS of (40 ZNS (40mg/kg/day) mg/kg/day for) for 7 days 7 days [39,40,44] [39,40 ,using44] using a subcutaneous a subcutaneous osmotic osmotic pump pump(2ML_1, (2ML_1, Alzet, Cupertino, Alzet, Cupertino, CA, CA,USA). USA). To Toexplore explore the the effects effects of subchronic of subchronic administ administrationration of a of therapeutic-relevant a therapeutic-relevant dose dose of ZNS of ZNS on on glutamatergicglutamatergic transmission transmission in in the the thalamocortical thalamocortical motor motor pathway pathway (RTN-MoTN-M2C) (RTN-MoTN-M2C)of of wild-type wild-type and S286L-TG,and S286L-TG, the perfusion the perfusion medium medium in the in RTN the wasRTN replaced was replaced with MRSwith withMRS orwith without or without 100 µ M100 RJR2403 μM (αRJR24034β2-nAChR (α4β2-nAChR agonist) (Figureagonist)1A). (Figure The perfusates1A). The perfusates in the MoTN in the and MoTN M2C and were M2C commenced were commenced with MRS alonewith (FigureMRS alone1A). (Figure After stabilization 1A). After stabilization of L-glutamate of L-glutamate levels in the levels M2C, in thethe perfusateM2C, the perfusate in the MoTN in the was MoTN was switched from MRS to MRS containing 100 μM AMPA (AMPA/glutamate receptor switched from MRS to MRS containing 100 µM AMPA (AMPA/glutamate receptor agonist) for 180 min agonist) for 180 min (Figure 1A). During experiments, the perfusate in the M2C were maintained by (Figure1A). During experiments, the perfusate in the M2C were maintained by MRS alone (Figure1A). MRS alone (Figure 1A). 2.3.2. Study_2: Interaction between Subchronic Administration of the Therapeutic-Relevant Dose of ZNS2.3.2. and Study_2: Acute Interaction Local Administration between Subchronic of GAP19 Admi intonistration the M2C of on the Glutamatergic Therapeutic-Relevant Transmission Dose inof the ThalamocorticalZNS and Acute MotorLocal Administration Pathway of GAP19 into the M2C on Glutamatergic Transmission in the Thalamocortical Motor Pathway Rats were subchronically administrated either a ZNS-free or a therapeutic-relevant dose of ZNS (40 mgRats/kg /wereday) subchronically for 7 days. To administrated explore the interactioneither a ZNS-free between or a ZNStherapeutic-relevant and GAP19 on dose glutamatergic of ZNS transmission(40 mg/kg/day) in thefor thalamocortical7 days. To explore motor the inte pathwayraction (MoTN-M2C) between ZNS ofand wild-type GAP19 on and glutamatergic S286L-TG, the perfusiontransmission medium in the in thalamocortical the M2C was commenced motor pathway with (MoTN-M2C) MRS containing of wild-type with or without and S286L-TG, 100 µM GAP19the perfusion medium in the M2C was commenced with MRS containing with or without 100 μM GAP19 (Cx43 inhibitor) (Figure1B). The perfusate in the MoTN was commenced with MRS alone (Figure1B). (Cx43 inhibitor) (Figure 1B). The perfusate in the MoTN was commenced with MRS alone (Figure After the stabilization of L-glutamate levels in the M2C, the perfusate in the MoTN was switched from 1B). After the stabilization of L-glutamate levels in the M2C, the perfusate in the MoTN was switched MRS to MRS containing 100 µM AMPA (AMPA/glutamate receptor agonist) for 180 min (Figure1B). from MRS to MRS containing 100 μM AMPA (AMPA/glutamate receptor agonist) for 180 min 2.3.3.(Figure Study_3: 1B). Effects of Subchronic Administration of the Therapeutic-Relevant Dose of ZNS and Acute Local Administration of GAP19 into the M2C on Repetitive Potassium-Dependent L-Glutamate Release2.3.3. Study_3: in the M2C Effects of Subchronic Administration of the Therapeutic-Relevant Dose of ZNS and Acute Local Administration of GAP19 into the M2C on Repetitive Potassium-Dependent L- GlutamateStudy_3 Release was composed in the M2C. of two experimental designs, Study_3-1 and Study_3-2. Rats were subchronically administrated by either a ZNS-free or therapeutic-relevant dose of ZNS (40 mg/kg/day) for 7 daysStudy_3 (Study_3-1). was composed To explore of two the experimental effects of subchronic designs, Study_3-1 administration and Study_3-2. of therapeutic-relevant Rats were subchronically administrated by either a ZNS-free or therapeutic-relevant dose of ZNS (40 doses of ZNS on Cx43 associated transmission in the M2C induced by repetitive potassium-evoked

Pharmaceuticals 2020, 13, 58 6 of 23 stimulations of wild-type and S286L-TG, the perfusion medium in the M2C was commenced with MRS. After the stabilization of L-glutamate levels in the M2C, the perfusate in the M2C was switched to HKMRS for 20 min (1st potassium-evoked stimulation: HKMRS 1st) (Figure1C). After the 1 st potassium-evoked stimulation, the perfusate in the M2C was returned to MRS for 240 min (recovery) (Figure1C). After the recovery, perfusate in the M2C was switched to HKMRS (2 nd potassium-evoked stimulation: HKMRS 2nd) for 20 min (Figure1C). After the 2 nd potassium-evoked stimulation, the perfusate in the M2C was returned to MRS again (Figure1C). To explore the eﬀects of local administration of GAP19 (Cx43 inhibitor) ZNS on Cx43 associated transmission in the M2C induced by repetitive potassium-evoked stimulations of wild-type and S286L-TG (Study_3-2), the perfusion medium in the M2C was commenced MRS containing with or without 100 µM GAP19 (Figure1C). After the stabilization of L-glutamate levels in the M2C, the perfusate in the M2C was switched to HKMRS containing the same agent for 20 min (1st potassium-evoked stimulation) (Figure1C). After the 1 st potassium-evoked stimulation, the perfusate in the M2C was returned to MRS containing the same agent for 240 min (Figure1C). After the recovery, perfusate in the M2C was switched to HKMRS containing the same agent (2nd potassium-evoked stimulation) for 20 min (Figure1C). After the 2 nd potassium-evoked stimulation, the perfusate in the M2C was returned to MRS containing the same agent again (Figure1C).

2.4. Ultra-High-Performance Liquid-Chromatography (UHPLC) L-glutamate levels were determined using UHPLC equipped with xLC3185PU (Jasco, Tokyo, Japan) and fluorescence detection (xLC3120FP, Jasco, Tokyo, Japan) following dual derivatisation with isobutyryl-L-cysteine and o-phthalaldehyde [45,46]. Derivatised solutions were prepared by dissolving isobutyryl-L-cysteine (2 mg) and o-phthalaldehyde (2 mg) in 0.1 mL ethanol, followed by the addition of 0.9 mL sodium borate buffer (0.2 M, pH 9.0) [47]. Automated pre-column derivatisation was performed by drawing 5 µL aliquots of sample, standard, or blank solutions and 5 µL of derivatisation solution together into a reaction vial and incubating for 5 min before injection. The derivatised samples (5 µL aliquots) were injected by an auto sampler (xLC3059AS, Jasco). The analytical column (YMC Triat C18, particle 1.8 µm, 50 2.1 mm, YMC, Kyoto, Japan) was maintained at 45 C and flow rate × ◦ was set at 500 µL/min. A linear gradient elution program was performed over a period of 10 min with mobile phases A (0.05 M citrate buffer, pH 5.0) and B (0.05 M citrate buffer containing 30% acetonitrile and 30% methanol, pH 3.5). The excitation/emission wavelengths of the fluorescence detector were set at 280/455 nm.

2.5. Preparation of Primary Astrocyte Culture Astrocytes were prepared using a protocol adapted from previously described methods [47–49]. Pregnant Sprague-Dawley rats (SLC, Sizuoka, Japan) were housed individually in cages, kept in air-conditioned rooms (temperature, 22 2 C) set at 12 h light/dark cycle, with free access to food and ± ◦ water. Cultured astrocytes were prepared from cortical astrocyte cultures of neonatal Sprague-Dawley rats (N = 48) sacrificed by decapitation at 0–24 h of age. The cerebral hemispheres were removed under dissecting microscope. Tissue was chopped into fine pieces using scissors and then triturated briefly with a micropipette. Suspension was filtered using 70 µm nylon mesh (BD, Franklin Lakes, NJ, USA) and centrifuged. Pellets were then re-suspended in 10 mL Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (fDMEM), which was repeated three times. After culture for 14 days (DIV14), contaminating cells were removed by shaking in a standard incubator (BNA-111, Espec, Osaka, Japan) for 16 h at 200 rpm. On DIV21, astrocytes were removed from flasks by trypsinization and seeded directly onto translucent PET membrane (1.0 µm) with 24-well plates (BD) at a density of 1 105 cells/cm2 for experiments. From DIV21 to DIV28, the culture medium (fDMEM) was changed × twice a week, and nicotine (0, 1 and 3 µM) or ZNS (50 µM) were added for subchronic administrations (7 days). On DIV28, cultured astrocytes were washed out using ACSF, and this was repeated three times. The ACSF comprised NaCl 150.0 mM, KCl 3.0 mM, CaCl2 1.4 mM, MgCl2 0.8 mM, and glucose Pharmaceuticals 2020, 13, 58 7 of 23

5.5 mM, buﬀered to pH 7.3 with 20 mM HEPES buﬀer. The remaining adherent cells contained 95% GFAP-positive and A2B5-negative cells detected using immunohistochemical staining [47–49]. After the wash out, plasma membrane proteins of cultured astrocytes were extracted using Minute Plasma Membrane Protein Isolation Kit (Invent Biotechnologies, Plymouth, MN, USA).

2.6. Simple Western Analysis Total plasma membrane proteins of rat frontal cortex and primary cultured astrocytes were extracted using Minute Plasma Membrane Protein Isolation Kit (Invent Biotechnologies, Plymouth, MN, USA) [33]. Simple Western analyses were performed using Wes (ProteinSimple, Santa Clara, CA, USA) according to the ProteinSimple user manual [21,33]. Lysate of primary cultured astrocytes were mixed with a master mix (ProteinSimple, San Jose, CA, USA) to a final concentration of 1 sample × buffer, 1 fluorescent molecular weight marker and 40 mM dithiothreitol, then heated at 95 C for × ◦ 5 min. The samples, blocking reagent, primary antibodies, HRP-conjugated secondary antibodies, chemiluminescent substrate, 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, USA) and Cx43 (C6219, Sigma-Aldrich, St. Louis, MO) 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 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.

2.7. Data Analysis All experiments in this study were designed with equally sized animal groups (N = 6) without carrying out a formal power analysis, in keeping with previous studies [21–23,33,50–52]. All values are expressed as mean standard deviation (SD) and P < 0.05 (two-tailed) was considered statistically ± signiﬁcant for all tests. Drug levels in acutely local and subchronically systemic administrations were selected based on values in previous studies [21–24,33,50–52]. Where possible, we sought to randomize and blind the data. In particular, for the determination of extracellular transmitter levels, the sample order on the autosampler was determined by a random number table. Regional transmitter concentrations were analyzed by Mauchly’s sphericity test followed by (MANOVA) using BellCurve for Excel ver. 3.2 (Social Survey Research Information Co., Ltd., Tokyo, Japan). 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 genotype/drug/time factors of MANOVA was signiﬁcant, the data was analyzed by Tukey’s post hoc test. Transmitter level was expressed as the area under the curve between 20 and 180 min (AUC20–180) after perfusion of target agent. Protein expression of Cx43 in the M2C plasma membrane fraction was analysed by student T-test, one-way or two-way analysis of variance (ANOVA) with Tukey’s post hoc test using BellCurve for Excel. All statistical analyses complied with the recommendations on experimental design and analysis in pharmacology [53]. Pharmaceuticals 2020, 13, 58 8 of 23 Pharmaceuticals 2020, 13, x FOR PEER REVIEW 8 of 23

2.8. Nomenclature of Targets and Ligands 2.8. Nomenclature of Targets and Ligands Key protein targets and ligands in this article are hyperlinked to corresponding entries in Key protein targets and ligands in this article are hyperlinked to corresponding entries in http: http://www.guidetopharmacology.org, which is the common portal for data from the IUPHAR/BPS //www.guidetopharmacology.org, which is the common portal for data from the IUPHAR/BPS Guide Guide to PHARMACOLOGY [54] and are permanently archived in the Concise Guide to to PHARMACOLOGY [54] and are permanently archived in the Concise Guide to PHARMACOLOGY PHARMACOLOGY 2017/18 [55]. 2017/18 [55].

3.3. Results Results

3.1.3.1. Cx43 Cx43 Expression Expression in in the the M2C M2C Plasma Plasma Membrane Membrane Fraction Fraction of of S286L-TG S286L-TG and and Its Its Response Response to to Subchronic Subchronic NicotineNicotine Administration Administration In In Vivo Vivo AnAnin in vitro vitrostudy study demonstrated demonstrated that that Cx43 Cx43 expression expression in the in humanthe human umbilical umbilical vein endothelialvein endothelial cells wascells reduced was reduced by the subchronic by the subchronic administration administrati of nicotineon [of24 ].nicotine The present [24]. studyThe alsopresent demonstrated study also thatdemonstrated systemic subchronic that systemic administration subchronic administration of nicotine (10, of nicotine 25 and (10, 50 mg25 and/kg/ day)50 mg/kg/day) [41] for 7 [41] days, for dose-dependently7 days, dose-dependently decreased decreased Cx43 expression Cx43 expression level in the level M2C in (ADSHE the M2C focus) (ADSHE plasma focus) membrane plasma fractionmembrane of wild-type fraction of (F wild-typenicotine(3,23) (Fnicotine= 9.7(3,23) (P < =0.01)) 9.7 (P (Figure < 0.01))2A,B). (Figure Contrary 2A,B). toContrary wild-type, to wild-type, neither subchronicneither subchronic nicotine administrationnicotine administration of 25 nor of 50 25 mg nor/kg 50/day mg/kg/day could aﬀ couldect Cx43 affect expression Cx43 expression level in level the M2Cin the plasma M2C plasma membrane membrane fraction fraction of S286L-TG, of S286L-TG but Cx43, but expression Cx43 expression of S286L-TG of S286L-TG was larger was larger than comparedthan compared with that with of wild-typethat of wild-type (Fnicotine (3,23)(Fnicotine=(3,23)8.2 (P =< 8.20.01)) (P (Figure< 0.01))2 A,C).(Figure These 2A,C). results These suggest results thatsuggest activation that ofactivationα4β2-nAChR of α4 decreasesβ2-nAChR Cx43 decreases expression Cx43 in expression plasma membrane, in plasma whereas membrane, S286L-mutant whereas αS286L-mutant4β2-nAChR impairs α4β2-nAChR suppression impairs eﬀ ectsuppression on Cx43. effect on Cx43.

Figure 2. Dose-dependent effects of subcutaneous administration of subchronic doses of nicotine (0, 10,Figure 25 and 2. 50Dose-dependent mg/kg/day) for effects 7 days of onsubcutaneous Cx43 expression administration in the M2C of plasmasubchronic membrane doses of fraction nicotine of (0, wild-type10, 25 and and 50 S286L-TGmg/kg/day) (A )for and 7 days pseudo-gel on Cx43 images expre usingssion in Simple the M2C Western plasma using membrane anti-GAPDH fraction and of anti-Cx43wild-type antibody and S286L-TG for blotting (A) and of plasma pseudo-gel membrane images fractions using Simp of wild-typele Western (B) using and S286L-TG anti-GAPDH rats ( Cand). Inanti-Cx43 panel 2A, antibody ordinate: for mean blottingSD of (n plas= 6)ma of membrane relative protein fractions level of of wild-type Cx43. *P <(B0.05,) and ** S286L-TGP < 0.01 relative rats (C). ± toIn nicotine panel 2A, free ordinate: wild-type mean by one-way ± SD (n = analysis 6) of relative of variance protein (ANOVA) level of Cx43. with Tukey’s *P < 0.05, post **P hoc < 0.01 test. relative to nicotine free wild-type by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. 3.2. Effects of Subchronic Administration of Nicotine and ZNS on Cx43 Expression in Astroglial Plasma Membrane3.2. Effects Fractions of Subchronic of Wild-Type Administration Primary of Cultured Nicotine Astrocytes and ZNS on Cx43 Expression in Astroglial Plasma MembraneCx43 is Fractions the most of predominant Wild-Type Primary connexin Cultured isoform Astrocytes in astrocytes but not expressed in neurons [28,29]; however,Cx43α 4-nAChRis the most expresses predominant in neurons connexin but not isoform astrocytes in astrocytes [23]. Therefore, but not to expressed clarify the targetin neurons cell of[28,29]; the inhibitory however, eff ectα4-nAChR of nicotine expresses on Cx43 in expression neurons but in plasmanot astr membrane,ocytes [23]. the Therefore, effects of to subchronic clarify the administrationtarget cell of the of inhibitory nicotine (1 effect and 3ofµ nicotineM) for 7 on days Cx43 were expression examined in plasma in primary membrane, cultured the astrocytes effects of ofsubchronic wild-type administration rats. Cx43 expression of nicotine in (1 total and lysate 3 μM) of for the 7 days human were umbilical examined vein in endothelialprimary cultured cells wasastrocytes decreased of wild-type to 50% by rats. subchronic Cx43 expression administration in total of lysate 1 µM of nicotine the human over umbilical 5 days [24 vein]; however, endothelial in thecells present was decreased study, astroglial to 50% by Cx43 subchronic in the plasma administration membrane of fraction 1 μM nicotine was not over aff ected5 days by [24]; subchronic however, in the present study, astroglial Cx43 in the plasma membrane fraction was not affected by subchronic

Pharmaceuticals 2020, 13, x FOR PEER REVIEW 9 of 23

Pharmaceuticalsnicotine (12020 and, 13 3, 58μM) administration (Figure 3A,B). Therefore, the inhibitory effects of nicotine9 of 23 on Cx43 expression in astroglial plasma membrane is not a direct action on astroglial Cx43 metabolism, but is possibly mediated by neuronal α4β2-nAChR indirectly. nicotineContrary (1 and 3 µtoM) nicotine, administration ZNS directly (Figure decreases3A,B). Therefore, astroglial the Cx43 inhibitory expression e ﬀects in ofthe nicotine astroglial on plasma Cx43 expressionmembrane, in astroglialsince the plasmalowest membranetherapeutic-relevant is not a direct concentration action on astroglial of ZNS (50 Cx43 μM) metabolism, [38,40] decreased but is possiblyCx43 expression mediated byin the neuronal plasmaα 4meβ2-nAChRmbrane fraction indirectly. of primary cultured astrocytes (Figure 3A,B).

Figure 3. Concentration-dependent effects of subchronic administration of nicotine (0, 1 and 3 µM) and Figure 3. Concentration-dependent effects of subchronic administration of nicotine (0, 1 and 3 μM) lowest therapeutic-relevant concentration of zonisamide (ZNS) (50 µM) for 7 days on Cx43 expression and lowest therapeutic-relevant concentration of zonisamide (ZNS) (50 μM) for 7 days on Cx43 in the plasma membrane fraction of primary cultured astrocytes from wild-type rat (A) and pseudo-gel expression in the plasma membrane fraction of primary cultured astrocytes from wild-type rat (A) images using Simple Western results using anti-GAPDH and anti-Cx43 antibody for blotting of plasma and pseudo-gel images using Simple Western results using anti-GAPDH and anti-Cx43 antibody for membrane fractions of wild-type (B). In panel 3A, ordinate: mean SD (n = 6) of relative protein level blotting of plasma membrane fractions of wild-type (B). In panel± 3A, ordinate: mean ± SD (n = 6) of of Cx43. *: P < 0.05 relative to control by student T-test. Concentration-dependent effects of nicotine on relative protein level of Cx43. *: P < 0.05 relative to control by student T-test. Concentration-dependent Cx43 expression was not detected by one-way ANOVA. effects of nicotine on Cx43 expression was not detected by one-way ANOVA. Contrary to nicotine, ZNS directly decreases astroglial Cx43 expression in the astroglial plasma membrane,3.3. Effects since of Subchronic the lowest Administ therapeutic-relevantration of Therapeutic-Relevant concentration of Dose ZNS of (50 ZNSµM) on [Cx4338,40 ]Expression decreased in Cx43 the expressionM2C Plasma in the Membrane plasma of membrane Wild-Type fraction and S286L-TG of primary cultured astrocytes (Figure3A,B). Systemic subchronic administration of therapeutic-relevant doses of ZNS (40 mg/kg/day) for 7 3.3. Effects of Subchronic Administration of Therapeutic-Relevant Dose of ZNS on Cx43 Expression in the M2C Plasmadays Membrane[23,39,40,44] of Wild-Typedecreased and the S286L-TGCx43 expression levels in the M2C plasma membrane fraction of both wild-type and S286L-TG (Fgenotype(1,20) = 35.2 (P < 0.01), FZNS(1,20) = 21.6 (P < 0.01), Fgenotype*ZNS (1,20) = 1.2Systemic (P > 0.1)). subchronic The level of administration Cx43 in the M2C of therapeutic-relevant plasma membrane fraction doses of of ZNS S286L-TG (40 mg /waskg/day) larger for than 7 daysthat [23 of, 39wild-type,40,44] decreased rats (Figure the 4A). Cx43 Subchronic expression administration levels in the M2C of therapeutic-relevant plasma membrane fraction dose of of ZNS both (40 wild-typemg/kg/day) and for S286L-TG 7 days (Fdecreasedgenotype(1,20) Cx43= expression35.2 (P < 0.01), in the FZNS M2C(1,20) plasma= 21.6 membrane (P < 0.01), fraction Fgenotype*ZNS of both (1,20)wild-type= 1.2 (P and> 0.1)). S286L-TG The level (Figure of Cx43 4A,B). in theTherefore, M2C plasma in S286L-TG, membrane nicotine fraction lost of its S286L-TG suppression was effect larger on thanCx43 that expression of wild-type in astroglial rats (Figure plasma4A). Subchronic membrane administrationdue to the loss-of-function of therapeutic-relevant S286L-mutant dose α of4β2- ZNSnAChR, (40 mg but/kg /ZNSday) fordirectly 7 days decreased decreased Cx43 Cx43 levels expression in the in theastroglial M2C plasmaplasma membrane membrane fraction without ofmediating both wild-type neuronal and S286L-TGfunction. (Figure4A,B). Therefore, in S286L-TG, nicotine lost its suppression effect on Cx43 expression in astroglial plasma membrane due to the loss-of-function S286L-mutant α4β2-nAChR, but ZNS directly decreased Cx43 levels in the astroglial plasma membrane without mediating neuronal function.

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Figure 4. Eﬀects of systemic subchronic administration of ZNS (40 mg/kg/day for 7 days) on Cx43 expressionFigure in 4. the Effects M2C of plasma systemic membrane subchronic fraction administration of wild-type of ZNS and S286L-TG(40 mg/kg/day (A) and for pseudo-gel7 days) on Cx43 imagesexpression using Simple in the Western M2C resultsplasma using membrane anti-GAPDH fraction and of anti-Cx43wild-type antibody and S286L-TG for blotting (A) and of plasma pseudo-gel membrane fractions (B). In panel 4A, ordinate: mean SD (n = 6) of relative protein level of Cx43. images using Simple Western results using anti-G± APDH and anti-Cx43 antibody for blotting of *P < 0.05, **P < 0.01 vs. ZNS-free (non) and @@P < 0.01 vs. wild-type by two-way analysis of variance plasma membrane fractions (B). In panel 4A, ordinate: mean ± SD (n = 6) of relative protein level of (ANOVA) with Tukey’s post hoc test. Cx43. *P < 0.05, **P < 0.01 vs. ZNS-free (non) and @@P < 0.01 vs. wild-type by two-way analysis of 3.4. Microdialysisvariance Study (ANOVA) with Tukey’s post hoc test.

3.4.1.3.4. Eff ectsMicrodialysis of Local AdministrationStudy of RJR2403 into the RTN and Subchronic Administration of Therapeutic-Relevant Dose of ZNS on the L-Glutamate Release in the M2C Induced by Local Administration3.4.1. Effects of of AMPA Local intoAdministration the MoTN (Study_1) of RJR2403 into the RTN and Subchronic Administration of Study_1Therapeutic-Relevant demonstrated Dose varying of ZNS responses on the L-Gl betweenutamate wild-typeRelease in andthe M2C S286L-TG Induced to by the Local local administrationAdministration of 100 of µAMPAM RJR2403 into the (α 4MoTNβ2-nAChR (Study_1) agonist) into the RTN on L-glutamate release in the M2CStudy_1 that was demonstrated induced by MoTN varying AMPA-evoked responses betw stimulationeen wild-type (Fgenotype and(1,20) S286L-TG= 102.2 (P to< 0.01the), local Fagentadministration(1,20) = 1.1 (P of> 1000.1), μMFgenotype*agent RJR2403 (α4(1,20)β2-nAChR=11.2 agonist) (P < 0.01), into F thetime RTN(3.1,62.1) on L-glutamate= 110.9 (P release< 0.01), in the Fgenotype*agent*timeM2C that was(3.1,62.1) induced= 6.7 by ( PMoTN< 0.01)) AMPA-evoked (Figure5A–C). stimulation Perfusion with(Fgenotype 100(1,20)µM AMPA= 102.2 into (P the< 0.01), MoTNFagent increased(1,20) = extracellular1.1 (P > 0.1), L-glutamate Fgenotype*agent levels(1,20)=11.2 in the M2C(P < (AMPA-evoked0.01), Ftime(3.1,62.1) L-glutamate = 110.9 release)(P < 0.01), of bothFgenotype*agent*time wild-type and(3.1,62.1) S286L-TG = 6.7 [(21P <,22 0.01))] (Figure (Figure5A–C). 5A–C). In concurrence Perfusion with with 100 previous μM AMPA studies, into basalthe MoTN extracellularincreased L-glutamate extracellular inthe L-glutamate M2C of S286L-TG levels in was the larger M2C than(AMPA-evoked that of wild-type L-glutamate (Figure5 C)release) [ 21–23 of]. both Neitherwild-type basal extracellular and S286L-TG L-glutamate [21,22] levels(Figure in the5A–C). M2C ofIn wild-typeconcurrence nor S286L-TGwith previous were affstudies,ected by basal perfusionextracellular with 100 L-glutamateµM RJR2403 in into the theM2 RTNC of S286L-TG (Figure5A–C). was larger Contrary than to th basalat of levels,wild-type perfusion (Figure with 5C) [21– 100 µ23].M RJR2403 Neither into basal the extracellular RTN decreased L-glutamate AMPA-evoked levels L-glutamate in the M2C release of wild-type in the M2C nor of S286L-TG wild-type were rats; however,affected by in perfusion S286L-TG, with local 100 administration μM RJR2403 ofinto RJR2403 the RTN into (Figure the RTN 5A–C). increased Contrary AMPA-evoked to basal levels, L-glutamateperfusion release with in100 the μM M2C RJR2403 of S286L-TG into the (Figure RTN 5decreasedA–C). AMPA-evoked L-glutamate release in the Study_1M2C of wild-type also revealed rats; the however, statistically in S286L-TG, significant local eff ectsadministration of systemic of subchronic RJR2403 into administration the RTN increased of therapeutic-relevantAMPA-evoked dosesL-glutamate of ZNS (40release mg/ kgin /thdaye M2C for 7 of days) S286L-TG on L-glutamate (Figure 5A–C). release in the M2C induced by MoTNStudy_1 AMPA-evoked also revealed stimulation the statistically (Fgenotype (1,20)significant= 58.6 effects (P < 0.01), of systemic Fagent(1,20) subchronic= 30.7 (administrationP < 0.01), Fgenotype*agentof therapeutic-relevant(1,20) = 21.1 (P doses< 0.01), of ZNS Ftime (40(3.2,64.1) mg/kg/da= 82.7y for (P 7< days)0.01), on F genotype*agent*timeL-glutamate release(3.2,64.1) in the= M2C 6.7 (Pinduced< 0.01)) by (Figure MoTN5A–C). AMPA-evoked Subchronic stimulation administration (Fgenotype of(1,20) therapeutic-relevant = 58.6 (P < 0.01), doses Fagent of(1,20) ZNS = did30.7 (P < not a0.01),ffect basalFgenotype*agent extracellular(1,20) = 21.1 L-glutamate (P < 0.01), levelsFtime(3.2,64.1) in the = M2C 82.7 of(P wild-type< 0.01), Fgenotype*agent*time but decreased(3.2,64.1 that )= of 6.7 (P S286L-TG< 0.01)) (Figure (Figure5A–C). 5A–C). Subchronic Subchronic administration administration of of therapeutic-relevant therapeutic-relevant doses doses of of ZNS ZNS inhibited did not affect the AMPA-evokedbasal extracellular L-glutamate L-glutamate release levels in the in M2Cthe M2C of S286L-TG of wild-type without but decreased affecting that that of of wild-type S286L-TG rats (Figure (Figure5A–C).5A–C). Subchronic administration of therapeutic-relevant doses of ZNS inhibited the AMPA-evoked L-glutamate release in the M2C of S286L-TG without affecting that of wild-type rats (Figure 5A–C).

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FigureFigure 5. E5.ff Effectsects of of local local administration administration of of RJR2403 RJR2403 ((αα44ββ2-nAChR agonist) agonist) into into the the reticular reticular thalamic thalamic nucleusnucleus (RTN) (RTN) and and subchronic subchronic administrationadministration of of th therapeutic-relevanterapeutic-relevant dose dose of ZNS of ZNS (40 mg/kg/day (40 mg/kg for/day for7 7days) days) on on the the L-glutamate L-glutamate release release in in the the secondary secondary motor motor cortex cortex (M2C) (M2C) induced induced by by local local administrationadministration of of 100 100µ MμM amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid acid (AMPA) (AMPA) into into thethe motor motor thalamic thalamic nuclei nuclei (MoTN) (MoTN) of ofwild-type wild-type ((AA)) andand S286L-TG ( (BB).). Rats Rats were were subchronically subchronically administeredadministered a therapeutic-relevanta therapeutic-relevant dose dose of of ZNSZNS (chronic(chronic ZNS) or or ZNS-fr ZNS-freeee (control (control or or AMPA+RJR) AMPA+RJR) usingusing a subcutaneousa subcutaneous osmotic osmotic pump. pump. InIn ZNSZNS non-treatednon-treated rats, perfusion perfusion medium medium in in the the RTN RTN was was commencedcommenced with with modified modified Ringer’s Ringer’s solution solution (MRS) (MRS with) with (AMPA (AMPA+RJR:+RJR: blue circles)blue circles) or without or without (control: opened(control: circles) opened 100 circles)µM RJR2403 100 μM (blue RJR2403 bars). (blue After bars). the stabilizationAfter the stabilization of L-glutamate of L-glutamate levels in levels the M2C, in thethe perfusion M2C, the medium perfusion in themedium MoTN in wasthe MoTN switched was from switched MRS from to MRS MRS containing to MRS containing 100 µM AMPA 100 μM for 180AMPA min (gray for 180 bars). min In (gray ZNS bars). treated In ratsZNS (chronic treated rats ZNS: (chronic red circles), ZNS: perfusionred circles), medium perfusion in themedium RTN wasin commencedthe RTN withwas commenced MRS containing with 100 MRSµM containing RJR2403. After100 μ theM stabilizationRJR2403. After of L-glutamatethe stabilization levels of in L- the M2C,glutamate the perfusion levels in medium the M2C, in thethe MoTNperfusion was medi switchedum in fromthe MoTN MRS towas MRS switched containing from 100MRSµ Mto AMPAMRS forcontaining 180 min (gray 100 μ bars).M AMPA Ordinates for 180 min indicate (gray mean bars). extracellularOrdinates indicate L-glutamate mean extracellular level (µM) L-glutamate (N = 6), and level (μM) (N = 6), and abscissas indicate time after AMPA-evoked stimulation (min). Blue bars abscissas indicate time after AMPA-evoked stimulation (min). Blue bars indicate the perfusion with indicate the perfusion with 100 μM RJR2403 into the RTN. Gray bars indicate the perfusion with 100 100 µM RJR2403 into the RTN. Gray bars indicate the perfusion with 100 µM AMPA into the MoTN. (C) μM AMPA into the MoTN. (C) indicates the area under curve value (AUC) value of extracellular L- indicates the area under curve value (AUC) value of extracellular L-glutamate level (nmol) before (basal glutamate level (nmol) before (basal extracellular L-glutamate level) and during perfusion with extracellular L-glutamate level) and during perfusion with AMPA (from 20 to 180 min) of Figure5A AMPA (from 20 to 180 min) of Figure 5A and 5B. Gray columns in Figure 5C indicate the AUC values and 5B. Gray columns in Figure5C indicate the AUC values of basal extracellular levels of L-glutamate of basal extracellular levels of L-glutamate in Figure 5A and 5B (during −60 to 0 min). **P < 0.01; in Figure5A,B (during 60 to 0 min). **P < 0.01; relative to wild-type, @P < 0.05, @@P < 0.01; relative relative to wild-type,− @P < 0.05, @@P < 0.01; relative to control, ##P < 0.01; relative to AMPA+RJR by to control, ##P < 0.01; relative to AMPA+RJR by multivariate analysis of variance (MANOVA) with multivariate analysis of variance (MANOVA) with Tukey’s post hoc test. Tukey’s post hoc test. 3.4.2. Interaction between Local Administration of GAP19 into the M2C and the Subchronic 3.4.2. Interaction between Local Administration of GAP19 into the M2C and the Subchronic AdministrationAdministration of of Therapeutic-Relevant Therapeutic-Relevant Dose Dose of of ZNS ZNS onon AMPA-EvokedAMPA-Evoked L-Glutamate Release Release in in the M2Cthe (Study_2) M2C (Study_2) Study_2Study_2 demonstrated demonstrated thethe didifferentfferent responsesresponses betweenbetween wild-type wild-type and and S286L-TG S286L-TG to tolocal local administrationadministration of of GAP19 GAP19 (Cx43 (Cx43 inhibitor) inhibitor) intointo thethe M2CM2C on L-glutamate release release in in the the M2C M2C induced induced by MoTN AMPA-evoked stimulation (Fgenotype(1,20) = 60.1 (P < 0.01), Fagent(1,20) = 12.6 (P < 0.01), by MoTN AMPA-evoked stimulation (Fgenotype(1,20) = 60.1 (P < 0.01), Fagent(1,20) = 12.6 (P < 0.01), Fgenotype*agent(1,20) = 10.1 (P < 0.01), Ftime(6.5,130.0) = 351.4 (P < 0.01), Fgenotype*agent*time (6.5,130.0) = 5.9 (P < Fgenotype*agent(1,20) = 10.1 (P < 0.01), Ftime(6.5,130.0) = 351.4 (P < 0.01), Fgenotype*agent*time (6.5,130.0) 0.01)) (Figure 6A–C). Perfusion with 100 μM GAP19 into the M2C decreased both the basal = 5.9 (P < 0.01)) (Figure6A–C). Perfusion with 100 µM GAP19 into the M2C decreased both the extracellular L-glutamate level and AMPA-evoked L-glutamate release in the M2C of S286L-TG, basal extracellular L-glutamate level and AMPA-evoked L-glutamate release in the M2C of S286L-TG, whereas neither AMPA-evoked L-glutamate release nor basal extracellular L-glutamate levels in the whereas neither AMPA-evoked L-glutamate release nor basal extracellular L-glutamate levels in the M2C of wild-type were affected by perfusion with 100 μM GAP19 into the M2C (Figure 6A–C). M2C of wild-type were affected by perfusion with 100 µM GAP19 into the M2C (Figure6A–C).

Pharmaceuticals 2020, 13, 58 12 of 23 Pharmaceuticals 2020, 13, x FOR PEER REVIEW 12 of 23

FigureFigure 6. 6.Interaction Interaction between between local local administrationadministration of GAP19 (Cx43 (Cx43 inhibitor) inhibitor) into into the the M2C M2C and and subchronicsubchronic administration administration ofof therapeutic-relevant therapeutic-relevant dose dose of ZNS of ZNS (40 mg/kg/day (40 mg/kg for/day 7 days) for on 7 days)AMPA- on AMPA-evokedevoked L-glutamate L-glutamate release releasein the M2C in theof wild-type M2C of (A wild-type) and S286L-TG (A) and (B). S286L-TGRats were subchronically (B). Rats were subchronicallyadministered administered a therapeutic-relevant a therapeutic-relevant dose of ZNS (chronic dose of ZNS ZNS (chronicor Chronic ZNS ZNS+GAP19) or Chronic ZNSor ZNS+GAP19) free or ZNS(control free or (control AMPA + or GAP19) AMPA using+ GAP19) a subcutaneous using asubcutaneous osmotic pump. osmotic Perfusion pump. medium Perfusion in the M2C medium was in thecommenced M2C was with commenced MRS containing with MRS with containing(AMPA+GAP with19 and (AMPA chronic+GAP19 ZNS+GAP19) and chronic or without ZNS +(controlGAP19) or withoutand chronic (control ZNS) and 100 chronic μM GAP19. ZNS) 100AfterµM the GAP19. stabilization After theof L-glutamate stabilization level of L-glutamate in the M2C, level the in theperfusion M2C, the medium perfusion in the medium MoTN in was the switched MoTN wasfrom switched MRS to MRS from containing MRS to MRS 100 μ containingM AMPA for 100 180µM AMPAmin. for Ordinates 180 min. indicate Ordinates mean indicate extracellular mean extracellularL-glutamate level L-glutamate (μM) (N level= 6), and (µM) abscissas (N = 6), indicate and abscissas time indicateafter AMPA-evoked time after AMPA-evoked stimulation (min). stimulation Blue bars (min). indicate Blue the bars perfusion indicate with the 100 perfusion μM GAP19 with into 100 theµM GAP19M2C. into Gray the bars M2C. indicate Gray bars the indicateperfusion the with perfusion 100 μM with AMPA 100 intoµM AMPAthe MoTN. into the(C) MoTN.indicates (C the) indicates AUC thevalue AUC valueof extracellular of extracellular L-glutamate L-glutamate level (nmol) level (nmol) before before(basal (basalextracellular extracellular L-glutamate L-glutamate level) and level) andduring during perfusion perfusion with with AMPA AMPA (from (from 20 to 20 180 to min) 180 min) of Figure of Figure 6A and6A,B. 6B. GrayGray columns in in Figure Figure 6C6C indicate the AUC values of basal extracellular levels of L-glutamate in Figure 6A and 6B. **P < 0.01; indicate the AUC values of basal extracellular levels of L-glutamate in Figure6A,B. ** P < 0.01; relative relative to wild-type, @P < 0.05, @@P < 0.01; relative to control by MANOVA with Tukey’s post hoc test. to wild-type, @P < 0.05, @@P < 0.01; relative to control by MANOVA with Tukey’s post hoc test.

SystemicSystemic subchronic subchronic administration administration ofof therapeutic-relevanttherapeutic-relevant doses doses of of ZNS ZNS (40 (40 mg/kg/day mg/kg/day for for 7 7 days) decreased AMPA-evoked L-glutamate release without affecting basal extracellular L-glutamate days) decreased AMPA-evoked L-glutamate release without affecting basal extracellular L-glutamate level in the M2C of wild-type, whereas subchronic ZNS administration decreased both AMPA- level in the M2C of wild-type, whereas subchronic ZNS administration decreased both AMPA-evoked evoked L-glutamate release and basal extracellular L-glutamate level in the M2C of S286L-TG L-glutamate release and basal extracellular L-glutamate level in the M2C of S286L-TG (Fgenotype(1,20) = (Fgenotype(1,20) = 52.2 (P < 0.01), Fagent(1,20) = 72.3 (P < 0.01), Fgenotype*agent(1,20) = 13.3 (P < 0.01), 52.2 (P < 0.01), Fagent(1,20) = 72.3 (P < 0.01), Fgenotype*agent(1,20) = 13.3 (P < 0.01), Ftime(6.7,132.9) = 238.1 Ftime(6.7,132.9) = 238.1 (P < 0.01), Fgenotype*agent*time (6.7,132.9) = 3.7 (P < 0.01)) (Figure 6A–C). (P 0.01), F (6.7,132.9) 3.7 (P 0.01)) (Figure6A–C). < In bothgenotype*agent*time ZNS treated wild-type and= S286L-TG,< perfusion with 100 μM GAP19 into the M2C did µ notIn affect both ZNSeither treated AMPA-evoked wild-type L-glutamate and S286L-TG, release perfusion or basal withextracellular 100 M L- GAP19glutamate into levels the M2C in the did notM2C affect (Figure either 6A–C). AMPA-evoked L-glutamate release or basal extracellular L-glutamate levels in the M2C (Figure6A–C). 3.5. Effects of Subchronic Administration of Therapeutic-Relevant Dose of ZNS on Repetitive Potassium- 3.5.Evoked Effects L-Glutamate of Subchronic Release Administration in the M2C of (Study_3-1) Therapeutic-Relevant Dose of ZNS on Repetitive Potassium-Evoked L-Glutamate Release in the M2C (Study_3-1) Repetitive potassium-evoked stimulation in the M2C use-dependently increased basal extracellularRepetitive potassium-evokedL-glutamate level an stimulationd potassium-evoked in the M2C L-gl use-dependentlyutamate release increased in the M2C basal of extracellular wild-type L-glutamate level and potassium-evoked L-glutamate release in the M2C of wild-type and S286L-TG and S286L-TG (Fgenotype(1,20) = 11.5 (P < 0.01), Fpotassium(1,20) = 7.4 (P < 0.05), Fgenotype*potassium(1,20) = 0.1 (P (Fgenotype> 0.5), (1,20)Ftime(3.4,67.4)= 11.5 (=P 162.0< 0.01), (P < F potassium0.01), Fgenotype*potassium*time(1,20) = 7.4 (P <(3.4,67.4)0.05), F genotype*potassium= 2.1 (P > 0.1)) (Figure(1,20) 7A–C).= 0.1 (P Basal> 0.5), Ftimeextracellular(3.4,67.4) = L-glutam162.0 (P ate< 0.01), level F(beforegenotype*potassium*time 1st potassium-evoked(3.4,67.4) stimulation)= 2.1 (P > 0.1))in the (Figure M2C of7A–C). S286L-TG Basal st extracellularwas higher L-glutamate than that of wild-type level (before (Figure 1 7A–C).potassium-evoked First potassium-evoked stimulation) stimulation in the M2C (perfusion of S286L-TG with wasHKMRS higher thaninto the that M2C) of wild-type increased (Figure extracellular7A–C). L-glutamate First potassium-evoked level in the M2C stimulation (1st potassium-evoked (perfusion with L- HKMRSglutamate into release) the M2C) of both increased wild-type extracellular and S286L- L-glutamateTG, whereas level potassium-evo in the M2Cked (1 stL-glutamatepotassium-evoked release

Pharmaceuticals 2020, 13, 58 13 of 23

L-glutamate release) of both wild-type and S286L-TG, whereas potassium-evoked L-glutamate release was larger than that of wild-type (Figure7A–C). After the 240 min recovery from the 1 st potassium-evoked stimulation, the basal extracellular L-glutamate levels were higher than basal L-glutamate level before the 1st potassium-evoked stimulation of wild-type and S286L-TG (1st vs. 2nd in Figure7A–C). Second potassium-evoked stimulation also increased L-glutamate release of both wild-type and S286L-TG (Figure7A–C). Especially, the peak elevation of the 2 nd potassium-evoked L-glutamate release was lower than those of the 1st potassium-evoked release, but the extracellular L-glutamate levels 180 min after the 2nd potassium-evoked stimulation was larger than those after the st 1 potassium-evokedPharmaceuticals 2020, 13, stimulationx FOR PEER REVIEW of both wild-type and S286L-TG (Figure7A–C). 14 of 23

FigureFigure 7. E 7.ﬀ ectsEffects of subchronicof subchronic administration administration of of therapeutic-relevant therapeutic-relevant dose dose of of ZNSZNS (40(40 mgmg/kg/day/kg/day for 7 days)7 days) on repetitive on repetitive potassium-evoked potassium-evoked L-glutamate L-glutamate release release in the in M2C the M2C of wild-type of wild-type (A) and (A) S286L-TG and S286L- (B). RatsTG were (B).subchronically Rats were subchronically administered administered with therapeutic-relevant with therapeutic-relevant dose of ZNS dose or of ZNS ZNS free. or ZNS Perfusion free. mediumPerfusion in the medium M2C was in the commenced M2C was commenced with MRS. After with theMRS. stabilization After the stabilization of L-glutamate of L-glutamate level in the level M2C, the perfusionin the M2C, medium the perfusion was switched medium from was MRS switched to HKMRS from forMRS 20 minto HKMRS (1st potassium-evoked for 20 min (1st potassium- stimulation) (ZNSevoked non-treated stimulation) with (ZNS 1st stimulation: non-treated openedwith 1st stimulation: circles, ZNS opened administered circles, ZNS with administered 1st stimulation: with 1 redst st circles).stimulation: After thered 1circles).st potassium-evoked After the 1 potassium-evoked stimulation, the stimulation, perfusate wasthe perfusate returned was to MRSreturned for 240to minMRS (recovery). for 240 min Following (recovery). recovery, Following the recovery, perfusate the was perfusate switched was to switched HKMRS to for HKMRS 20 min for again 20 min (2nd nd nd potassium-evokedagain (2 potassium-evoked stimulation). stimulation). After the 2nd Afterpotassium-evoked the 2 potassium-evoked stimulation, stimulation, perfusatewas perfusate returned was to returned to MRS. Ordinates indicate mean extracellular L-glutamate level (μM) (N = 6), and abscissas MRS. Ordinates indicate mean extracellular L-glutamate level (µM) (N = 6), and abscissas indicate time indicate time after 1st or 2nd potassium-evoked stimulations (min). Black bars indicate the perfusion after 1st or 2nd potassium-evoked stimulations (min). Black bars indicate the perfusion with HKMRS with HKMRS (1st and 2nd potassium-evoked stimulation). (C) indicates the AUC value of extracellular (1st and 2nd potassium-evoked stimulation). (C) indicates the AUC value of extracellular L-glutamate L-glutamate level (nmol) before (basal extracellular L-glutamate level) and after potassium-evoked level (nmol) before (basal extracellular L-glutamate level) and after potassium-evoked stimulation stimulation (from 20 to 180 min) of Figure 7A and 7B. Gray columns in Figure 7C indicate the AUC (from 20 to 180 min) of Figure7A,B. Gray columns in Figure7C indicate the AUC values of basal values of basal extracellular levels of L-glutamate in Figure 7A and 7B. *P < 0.05, **P < 0.01; relative to extracellular levels of L-glutamate in Figure7A,B. * P < 0.05, **P < 0.01; relative to wild-type, @P < 0.05, wild-type, @P < 0.05, @@P < 0.01; relative to HKMRS (1st), $$P < 0.01 relative to HKMRS (2nd) by @@P < 0.01; relative to HKMRS (1st), $$P < 0.01 relative to HKMRS (2nd) by MANOVA with Tukey’s MANOVA with Tukey’s post hoc test. post hoc test. 3.6. Effects of Local Administration of GAP19 on Repetitive Potassium-Evoked L-Glutamate Release in the The subchronic administration of therapeutic-relevant doses of ZNS (40 mg/kg/day for 7 days) M2C (Study_3-2) inhibited 1st potassium-evoked L-glutamate release in the M2C of both wild-type and S286L-TG μ st (FgenotypeAcute(1,20) local= 9.5 administration (P < 0.01), Fpotassium of 100(1,20) M GAP19= 20.4 into (P < the0.01), M2C Fgenotype*potassium inhibited 1 potassium-evoked(1,20) = 1.7 (P > 0.1),L- glutamate release in the M2C of S286L-TG without affecting that of wild-type (Fgenotype(1,20) = 11.5 (P Ftime(3.8,76.8) = 230.5 (P < 0.01), Fgenotype*potassium*time(3.8,76.8) = 2.0 (P > 0.1)) (Figure7A–C). Subchronic administration< 0.01), Fpotassium of(1,20) therapeutic-relevant = 7.4 (P < 0.05), Fgenotype*potassium doses of(1,20) ZNS = decreased 0.1 (P > 0.1), basal Ftime(3.4,67.4) extracellular = 162.0 L-glutamate (P < 0.01), levelFgenotype*potassium*time (before 1st potassium-evoked(3.4,67.4) = 2.1 (P > stimulation)0.1)) (Figure 8A–C). in the Local M2C administration of S286L-TG of without 100 μM a GAP19ﬀecting into that st of wild-typethe M2C decreased (Figure7A–C). basal Subchronicextracellular administration L-glutamate level of therapeutic-relevant(before 1 potassium-evoked dose of stimulation) ZNS decreased in the M2C of S286L-TG without affecting that of wild-type (Figure 8A–C). Local administration of 1st potassium-evoked L-glutamate release of both wild-type and S286L-TG (Figure7A–C). After the GAP19 also decreased 1st potassium-evoked L-glutamate release of S286L-TG without affecting that subchronic administration of therapeutic-relevant dose of ZNS, the basal extracellular L-glutamate of wild-type (Figure 8A–C). Local administration of GAP19 into the M2C inhibited 2nd potassium-evoked L-glutamate release in the M2C of both wild-type and S286L-TG (Fgenotype(1,20) = 8.1 (P < 0.01), Fpotassium(1,20) = 3.7 (P > 0.05), Fgenotype*potassium(1,20) = 0.1 (P > 0.1), Ftime(2.6,51.1)=243.4 (P < 0.01), Fgenotype*potassium*time(2.6,51.1) = 0.3 (P > 0.1)) (Figure 8A–C). Local administration of GAP19 into the M2C decreased basal extracellular L-glutamate level after 1st potassium-evoked stimulation in the M2C of both wild-type and S286L-TG, whereas the basal extracellular L-glutamate release after 1st potassium-evoked stimulation of S286L-TG was larger than that of wild-type (Figure 8A–C). Local administration of GAP19 into the M2C also decreased 2nd potassium-evoked L-glutamate release of both wild-type and S286L-TG (Figure 8A–C).

Pharmaceuticals 2020, 13, 58 14 of 23 level and 1st potassium-evoked L-glutamate release in the M2C between wild-type and S286L-TG became almost equal (Figure7A–C). The subchronic administration of therapeutic-relevant doses of ZNS inhibited 2nd potassium-evoked L-glutamate release in the M2C of both wild-type and S286L-TG (Fgenotype(1,20) = 6.5 (P < 0.05), Fpotassium(1,20) = 28.0 (P < 0.01), Fgenotype*potassium(1,20) = 1.5 (P > 0.1), Ftime(5.2,104.6) = 78.6 (P < 0.01), Fgenotype*potassium*time(5.2,104.6) = 1.4 (P > 0.1)) (Figure7A–C). Subchronic administration of therapeutic-relevant doses of ZNS decreased basal extracellular L-glutamate level after 1st potassium-evoked stimulation in the M2C of both wild-type and S286L-TG, whereas the basal extracellular L-glutamate release after 1st potassium-evoked stimulation of S286L-TG was larger than that of wild-type (Figure7A–C). Subchronic administration of therapeutic-relevant doses of ZNS also decreased 2nd potassium-evoked L-glutamate release of both wild-type and S286L-TG (Figure7A–C). Subchronic administration of therapeutic-relevant doses of ZNS inhibited repetitive potassium-evoked L-glutamate release in the M2C (Fgenotype(1,20) = 3.9 (P > 0.05), Fpotassium(1,20) = 3.1 (P > 0.05), Fgenotype*potassium(1,20) = 0.1 (P > 0.5), Ftime(4.0,80.3) = 111.8 (P < 0.01), Fgenotype*potassium*time(4.0,80.3) = 0.8 (P > 0.1)) (Figure7A–C). There were no di ﬀerences in basal extracellular L-glutamate levels or potassium-evoked L-glutamate releases in wild-type or S286L-TG between 1st and 2nd potassium-evoked stimulation (Figure7A–C).

3.6. Effects of Local Administration of GAP19 on Repetitive Potassium-Evoked L-Glutamate Release in the M2C (Study_3-2) Acute local administration of 100 µM GAP19 into the M2C inhibited 1st potassium-evoked L-glutamate release in the M2C of S286L-TG without affecting that of wild-type (Fgenotype(1,20) = 11.5 (P < 0.01), Fpotassium(1,20) = 7.4 (P < 0.05), Fgenotype*potassium(1,20) = 0.1 (P > 0.1), Ftime(3.4,67.4) = 162.0 (P < 0.01), Fgenotype*potassium*time(3.4,67.4) = 2.1 (P > 0.1)) (Figure8A–C). Local administration of 100 µM GAP19 into the M2C decreased basal extracellular L-glutamate level (before 1st potassium-evoked stimulation) in the M2C of S286L-TG without affecting that of wild-type (Figure8A–C). Local administration of GAP19 also decreased 1st potassium-evoked L-glutamate release of S286L-TG without affecting that of wild-type (Figure8A–C). Local administration of GAP19 into the M2C inhibited 2nd potassium-evoked L-glutamate release in the M2C of both wild-type and S286L-TG (Fgenotype(1,20) = 8.1 (P < 0.01), Fpotassium(1,20) = 3.7 (P > 0.05), Fgenotype*potassium(1,20) = 0.1 (P > 0.1), Ftime(2.6,51.1)=243.4 (P < 0.01), Fgenotype*potassium*time(2.6,51.1) = 0.3 (P > 0.1)) (Figure8A–C). Local administration of GAP19 into the M2C decreased basal extracellular L-glutamate level after 1st potassium-evoked stimulation in the M2C of both wild-type and S286L-TG, whereas the basal extracellular L-glutamate release after 1st potassium-evoked stimulation of S286L-TG was larger than that of wild-type (Figure8A–C). Local administration of GAP19 into the M2C also decreased 2nd potassium-evoked L-glutamate release of both wild-type and S286L-TG (Figure8A–C). Local administration of GAP19 into the M2C inhibited repetitive potassium-evoked L-glutamate release in the M2C (Fgenotype(1,20) = 8.3 (P < 0.01), Fpotassium(1,20) = 12.3 (P < 0.01), Fgenotype*potassium(1,20) = 0.7 (P < 0.1), Ftime(4.5,88.9) = 117.3 (P < 0.01), Fgenotype*potassium*time(4.5,88.9) = 0.8 (P < 0.1)) (Figure8A–C). There were no di fferences of basal extracellular L-glutamate levels or potassium-evoked L-glutamate releases in wild-type and S286L-TG between 1st and 2nd potassium-evoked stimulation (Figure8A–C). Pharmaceuticals 2020, 13, x FOR PEER REVIEW 15 of 23

Local administration of GAP19 into the M2C inhibited repetitive potassium-evoked L-glutamate release in the M2C (Fgenotype(1,20) = 8.3 (P < 0.01), Fpotassium(1,20) = 12.3 (P < 0.01), Fgenotype*potassium(1,20) = 0.7 (P < 0.1), Ftime(4.5,88.9) = 117.3 (P < 0.01), Fgenotype*potassium*time(4.5,88.9) = 0.8 (P < 0.1)) (Figure 8A–C).

PharmaceuticalsThere were 2020no differences, 13, 58 of basal extracellular L-glutamate levels or potassium-evoked L-glutamate15 of 23 releases in wild-type and S286L-TG between 1st and 2nd potassium-evoked stimulation (Figure 8A–C).

FigureFigure 8.8. EEffectsﬀects ofof locallocal administrationadministration ofof GAP19GAP19 (Cx43(Cx43 inhibitor)inhibitor) intointo thethe M2CM2C onon repetitiverepetitive potassium-evokedpotassium-evoked L-glutamateL-glutamate releaserelease inin thethe M2CM2C ofof wild-typewild-type (A(A)) and and S286L-TG S286L-TG ( B(B).). Perfusion Perfusion mediummedium inin thethe M2CM2C was commenced with with MRS MRS containing containing with with or or without without 100 100 μMµM GAP19. GAP19. After After the thestabilization stabilization of ofL-glutamate L-glutamate level level in inthe the M2C, M2C, the the perfusion perfusion medium medium was switched fromfrom MRSMRS toto st HKMRSHKMRS containing containing the the same same agent agent for for 20 20 min min (1 (1st potassium-evokedpotassium-evokedstimulation) stimulation) (GAP19 (GAP19 non-treated non-treated st st withwith 1 1st stimulation:stimulation: opened circles, circles, GAP19 GAP19 administered administered with with 1 1st stimulation:stimulation: red red circles). circles). After After the st the1st 1potassium-evokedpotassium-evoked stimulation, stimulation, the theperfusate perfusate was was returned returned to MRS to MRS containing containing the the same same agent agent for for240 240 min min (recovery). (recovery). Following Following recovery, recovery, the theperfusat perfusatee was wasswitched switched to HKMRS to HKMRS containing containing the same the nd nd sameagent agent for for20 20min min again again (2 (2nd potassium-evokedpotassium-evoked stimulation). stimulation). After After thethe 22nd potassium-evokedpotassium-evoked stimulation,stimulation, perfusateperfusate waswas returned to to MRS MRS cont containingaining the the same same agent agent (GAP19 (GAP19 non-treated non-treated with with 2nd nd nd 2stimulation:stimulation: blue blue circles, circles, GAP19 GAP19 administered administered with with2nd stimulation: 2 stimulation: green circles). green circles). Ordinates Ordinates indicate st indicatemean extracellular mean extracellular L-glutamate L-glutamate level (μ levelM) (N (µM) = (N6), =and6), andabscissas abscissas indicate indicate time time after after 1st 1or or2nd nd st 2potassium-evokedpotassium-evoked stimulations stimulations (min). (min). Black Black bars bars indicate indicate the theperfusion perfusion with with HKMRS HKMRS (1st (1 andand 2nd nd 2potassium-evokedpotassium-evoked stimulation). stimulation). Blue Blue bars barsindicate indicate the perfusion the perfusion with with100 μ 100M GAP19µM GAP19 into the into M2C. the M2C.(C) indicates (C) indicates the AUC the AUC value value of extracellular of extracellular L-glutam L-glutamateate level level (nmol) (nmol) before before (basal (basal extracellular extracellular L- L-glutamateglutamate level) level) and and after after potassium-evoked potassium-evoked stimul stimulationation (from (from 20 to 180 min) ofof FigureFigure8 A,B.8A and Gray 8B. columnsGray columns in Figure in8 CFigure indicate 8C theindicate AUC valuesthe AUC of basal values extracellular of basal extracellular levels of L-glutamate levels of inL-glutamate Figure8A,B. in st *FigureP < 0.05, 8A **andP 8B.< 0.01; * P < relative0.05, ** P to < 0.01; wild-type, relative @ toP

GABAergic pathway (RTN-MoTN) via enhanced desensitization of S286L-mutant α4β2-nAChR contributes to the pathomechanisms of all three major typical ADSHE seizures, ‘nocturnal paroxysmal dystonia’, ‘nocturnal paroxysmal arousal’ and ‘episodic nocturnal wandering’ (Figure9B) [ 21,22]. The hyperactivation of the glutamatergic transmission in thalamocortical motor pathway (MoTN-M2C) induced by GABAergic disinhibition in the MoTN via impaired S286L-mutant α4β2-nAChR are integrated in the M2C resulting in the generation of ADSHE focus of episodic nocturnal wandering and nocturnal paroxysmal arousal [21] (Figure9B). The hyperactivation of glutamatergic transmission in the thalamic hyperdirect pathway (MoTN-STN), also induced by the S286L-mutant α4β2-nAChR-associated GABAergic disinhibition, contributes to the pathomechanism of the other phenotype, nocturnal paroxysmal dystonia [22]. Therefore, the loss-of-functional abnormality of S286L-mutant α4β2-nAChR induced by a combination of enhanced sensitivity and desensitization to ACh [56,57] in the RTN plays fundamental roles in the ictogenesis of ADSHE patients with the S284L-mutation [21,22]. In spite of these efforts, the mechanisms of focus generation and increased seizure frequency following initial seizure in the same night remains to be clarified. In our previous study, 50 mM potassium-evoked stimulation in the M2C increased L-glutamate release in both wild-type and S286L-TG, whereas the 50 mM potassium-evoked L-glutamate release of S286L-TG was almost equal to that of wild-type [21]. However, in the present study, 100 mM potassium-evoked L-glutamate release was larger compared with wild-type. These different responses of S286L-TG between 50 mM and 100 mM potassium-evoked stimulations suggest that S286L-TG does not acquire electrophysiological hypersensitivity in focus regions (M2C) [21], but acquires abnormality in extracellular potassium-sensitive L-glutamate release regulation systems. The neurotransmitter exocytosis is activated by 25 mM potassium-evoked stimulation [58], whereas the generation of astroglial gliotransmitter release requires higher than 50 mM potassium-evoked stimulation [47,49,59]. Additionally, astroglial L-glutamate release through astroglial connexin hemichannel is also activated by 100 mM potassium-evoked stimulation [33,34]. These demonstrations suggest the functional abnormalities of astroglial gliotransmitter release and/or astroglial hemichannel abnormalities of S286L-TG. Abnormalities of several connexin isoforms associated with epilepsy have been reported [26]. Indeed, Cx43 expression is upregulated in the human epileptic tissue [60,61]. Recently, we have also demonstrated that the combination between increased Cx43 expression in the plasma membrane and extracellular potassium levels contributes to antipsychotic-induced convulsion [33]. Furthermore, subchronic administration of nicotine decreases Cx43 protein in the plasma membrane without affecting Cx43 mRNA in the human endothelial cells induced by the activation of posttranscriptional proteasomal protein degradation via α4β2-nAChR [24]. Based on clinical and preclinical evidence, to clarify the pathogenesis of ADSHE seizure associated with S284L-mutation, the present study determined Cx43-associated transmission in the thalamocortical motor pathway and focus regions in S286L-TG. Confirming our hypothesis, the Cx43 expression in the M2C plasma membrane fraction of S286L-TG was upregulated compared with that of wild-type (Figure9B). Interestingly, subchronic administration of nicotine dose-dependently reduced the Cx43 expression in the M2C plasma membrane of wild-type, but did not affect that of S286L-TG (Figure9A,B). The upregulated Cx43 in focus region of S286L-TG provides at least two functional abnormalities of S286L-TG; increased basal L-glutamate level during resting stage, and enhanced glutamatergic transmission in thalamocortical motor pathway. Indeed, inhibition of Cx43 in the M2C by selective Cx43 inhibitor, GAP19 decreased basal extracellular L-glutamate level in the M2C of S286L-TG without affecting that of wild-type. Furthermore, local administration of GAP19 into the M2C also inhibited L-glutamate release induced by AMPA-evoke stimulation in the MoTN of S286L-TG without affecting that of wild-type. These results indicate that upregulated Cx43 in focus regions of S286L-TG is possibly induced by impaired inhibitory regulation of α4β2-nAChR (loss-of-functional S286L-mutant α4β2-nAChR). Therefore, the hypersensitive Cx43 Pharmaceuticals 2020, 13, 58 17 of 23 hemichannel in the M2C plays important roles in the focus generation mechanism of S286L-TG

(FigurePharmaceuticals9B). 2020, 13, x FOR PEER REVIEW 17 of 23

FigureFigure 9. 9.Proposed Proposed hypothesis hypothesis of of pathomechanisms pathomechanisms andand pathophysiologypathophysiology of S286L-TG. Proposed Proposed hypothesishypothesis for for functional functional abnormalities abnormalities of glutamatergic of glutamatergic transmission transmission in thethalamocortical in the thalamocortical pathways inpathways wild-type in (panel wild-typeA), S286L-TG(panel A), (panelS286L-TGB) and(panel pathophysiology B) and pathophysiology of ZNS-sensitive of ZNS-sensitive ADSHE ADSHE seizure (panelseizureC). (panel RTN mainlyC). RTN projects mainly projects GABAergic GABAergic terminals terminals to MoTN. to MoTN. Activation Activation of α 4ofβ 2-nAChRα4β2-nAChR in thein RTNthe enhancesRTN enhances GABAergic GABAergic inhibition inhibition in thein the RTN-MoTN RTN-MoTN pathways pathways of of wild-type wild-type (panel(panel A). MoTN MoTN projectproject glutamatergic glutamatergic terminals terminals to to the the M2C. M2C. In In the the MoTN,MoTN, bothboth αα44ββ2-nAChR and AMPA/glutamate AMPA/glutamate receptorreceptor activate activate glutamatergic glutamatergic transmission transmission to the to M2C the (panel M2CA ).(panel Wild-type A). Wild-typeα4β2-nAChR α4β suppresses2-nAChR thesuppresses Cx43 expression the Cx43 inexpression plasma membranein plasma membrane (panel A). (panel Contrary A). Contrary to wild-type, to wild-type, loss-of-function loss-of- S286L-mutantfunction S286L-mutantα4β2-nAChR α4β attenuates2-nAChR attenuates the inhibition the inhibition of Cx43 expression of Cx43 expression in the plasma in the membrane plasma (panelmembrane B). Additionally, (panel B). Additionally, loss-of-function loss-of-function of S286L-mutant of S286L-mutantα4β2-nAChRs α4β in2-nAChRs RTN generates in RTN GABAergic generates disinhibitionGABAergic indisinhibition the MoTN, in leading the MoTN, to enhancement leading to enhancement of glutamatergic of glutamatergi transmissionc transmission in M2C (panel in M2CB). Enhanced(panel B). glutamatergic Enhanced glutamatergic transmission tran insmission the M2C in stimulates the M2C stimulates both glutamatergic both glutamatergic pyramidal pyramidal neurons andneurons astrocytes and resultingastrocytes in resulting activation in of activation Cx43 (panel of Cx43B). A combination(panel B). A ofcombination enhanced glutamatergicof enhanced transmissionglutamatergic and transmission Cx43 expression and Cx43 in the expression M2C are candidatein the M2C mechanisms are candidate of focus mechanisms generation of focus in the M2Cgeneration (panel Bin). the ZNS M2C reduced (panel Cx43 B). ZNS expression reduced in Cx43 the M2Cexpression (panel inC the). ZNS M2C also (panel compensates C). ZNS also the enhancedcompensates glutamatergic the enhanced transmission glutamatergic in the thalamocortical transmission glutamatergicin the thalamocortical transmission glutamatergic via activation oftransmission II-mGluR [22 via] in activation the MoTN of and II-mGluR GABA [22] release in the [39 MoTN,62] in and the GABA M2C [ 21release]. [39,62] in the M2C [21].

TheConfirming present study our hypothesis, reveals possible the Cx43 Cx43-associated expression in mechanisms the M2C plasma involved membrane in the frequency fraction of of ADSHES286L-TG seizures was upregulated following the compared initial seizure with that during of wild-type a single (Figure night. Repetitive9B). Interestingly, potassium-evoked subchronic stimulation,administration of which of nicotine intervals dose-dependently between 1st and 2 ndrepotassium-evokedduced the Cx43 expression stimulation in was the set M2C at 4 h,plasma which ismembrane longer than of the wild-type, half-life but of Cx43 did not (about affect 2–3 that h) of [63 S286L-TG], use-dependently (Figure 9A,B). enhanced The upregulated Cx43 hemichannel Cx43 in activityfocus region and thereby of S286L-TG resulted provides in increased at least two basal functional and depolarization-induced abnormalities of S286L-TG; L-glutamate increased releases. basal TheseL-glutamate results suggest level during that ADSHEresting stage, seizure and activates enhanced glutamatergic glutamatergic transmission transmission associated in thalamocortical with the Cx43motor hemichannel pathway. Indeed, and maintains inhibition the of epileptogenic Cx43 in the M2C condition by selective over several Cx43 inhibitor, hours. This GAP19 hypothesis decreased can basal extracellular L-glutamate level in the M2C of S286L-TG without affecting that of wild-type. also explain the frequent occurrence of ADSHE seizures during the non-rapid eye movement phase Furthermore, local administration of GAP19 into the M2C also inhibited L-glutamate release induced of sleep, since the sleep spindle, which is formed in the RTN and produces benign bursts, propagate by AMPA-evoke stimulation in the MoTN of S286L-TG without affecting that of wild-type. These through various cortical regions via intra-thalamic and thalamocortical pathways [64]. Therefore, sleep results indicate that upregulated Cx43 in focus regions of S286L-TG is possibly induced by impaired spindle burst during the non-rapid eye movement phase of sleep can be triggered in ADSHE seizures inhibitory regulation of α4β2-nAChR (loss-of-functional S286L-mutant α4β2-nAChR). Therefore, the via the activation of upregulated Cx43 hemichannel activity. hypersensitive Cx43 hemichannel in the M2C plays important roles in the focus generation mechanism of S286L-TG (Figure 9B). The present study reveals possible Cx43-associated mechanisms involved in the frequency of ADSHE seizures following the initial seizure during a single night. Repetitive potassium-evoked stimulation, of which intervals between 1st and 2nd potassium-evoked stimulation was set at 4 h,

Pharmaceuticals 2020, 13, 58 18 of 23

The present study cannot explain the mechanisms by which neuron-specific α4β2-nAChR affects astrocyte specific Cx43 expression. Translated Cx43 folds in the endoplasmic reticulum and traffics to the plasma membrane as hemichannels or Gap-junctions following oligomerization in the Golgi. Ubiquitylation of Cx43 at the plasma membrane signals the internalization of Cx43 [28]. Internalization of Cx43 leads to annular Gap-junction or hemichannel endocytosis resulting in lysosome or autophagy degradation. Deubiquitylation of Cx43 rescues the protein from lysosomal degradation, allowing its recycling shift to the plasma membrane [28]. In future studies we will investigate the mechanisms of inhibitory regulation of α4β2-nAChR on metabolism and cellular localisation of astroglial Cx43 using immunohistochemistry. It is well known that single neuronal connexon docks associate with a connexon on an adjacent cell to form a Gap-junction channels [25,28]. Gap-junctions allow the passive diffusion of various intracellular molecules, including mRNA, transmitters, second messengers, ions and other regulatory substances involved in cell growth and differentiation [25,28]. Upregulated astroglial Cx43 S286L-TG should generate a GAP-junction with adjacent neuronal connexons resulting in the enhancement of communication between neurons and astrocytes via upregulated Gap-junctions. Exploring the detailed regulatory mechanisms of the suppressive function of neuron-specific α4β2-nAChR on the metabolism of astroglial specific Cx43 through neuro-astroglial communication is an important issue for understanding epileptogenesis. Furthermore, in this study, we used anti-GAPDH antibody for normalization of Cx43 expression level in the plasma membrane fraction. To study the detailed mechanisms of the suppressive effects of α4β2-nAChR on Cx43 metabolism in the plasma membrane, we shall use anti Na+/K+-ATPase antibody as a housekeeping protein of plasma membrane [65].

4.2. Pathophysiology of ADSHE Seizures Associated with ZNS The present study demonstrated that subchronic administration of therapeutic-relevant dose of ZNS suppressed the enhanced glutamatergic transmission associated with Cx43 hemichannel of S286L-TG via reduction of Cx43 expression in the M2C plasma membrane. The present results suggest the loss-of-functional S286L-mutant nAChR impairs the inhibition of Cx43 degradation system, since subchronic administration of nicotine to S286L-TG could not decrease Cx43 expression (Figure9B). A previous study reported that ZNS increased basal ACh release [44]; however, the enhancement of ACh release induced by ZNS cannot provide compensation of loss-of-functional S286L-mutant α4β2-nAChR due to two possibilities. The first, the enhanced ACh release probably cannot activate S286L-mutant α4β2-nAChR, since this mutation has enhanced sensitivity and desensitization to ACh [56,57]. Indeed, in the present study, application of nicotine did not affect Cx43 expression of S286L-TG. The second, CBZ, which cannot prevent ADSHE seizure of patients with S284L-mutation, increases basal ACh release, similar to ZNS [44,66]. Therefore, ZNS probably compensates the Cx43 metabolism dysfunction through an α4β2-nAChR independent system. Furthermore, the present study demonstrated that ZNS directly reduced Cx43 expression, since ZNS decreased Cx43 expression in plasma membrane fractions of primary cultured astrocytes. It has been well established that posttranscriptional processes, including phosphorylation, acetylation, nitrosylation, sumoylation and ubiquitylation, play important roles in the transport to the plasma membrane, intracellular communication and degradation of Cx43 [28]. ZNS suppressed endoplasmic reticulum stress induced cell death via increased human ubiquitin ligase 1 [67]. Taken together with these previous findings, subchronic administration of therapeutic-relevant dose and concentration of ZNS compensate the lack of inhibitory regulation systems associated by S286L mutant α4β2-nAChR of S286L-TG due to the activation of ubiquitin ligase (Figure9C). Although the present study did not determine the effects of ZNS on Cx43 hemichannel activity, established pharmacological profiles of ZNS suggest that ZNS possibly inhibits Cx43 hemichannel activity. Hemichannel activity is regulated by membrane potential, potassium, calcium ion concentration and intracellular pH. Depolarization, increased extracellular potassium ion levels, decreased extracellular calcium ion levels open hemichannel and enhanced gliotransmitter releases through hemichannel [33,34,68], whereas complete or partial closure of hemichannel can be triggered Pharmaceuticals 2020, 13, 58 19 of 23 by low intracellular pH [69]. Both voltage-sensitive sodium channel and carbonic anhydrase inhibition is known to be one of the major mechanisms of anticonvulsive action of ZNS [62]. Inhibition of carbonic anhydrase reduces intracellular pH, and ZNS-induced acidosis and urolithiasis have been clinically reported [70]. Therefore, taken together, these pharmacological profiles of ZNS acutely and chronically inhibit L-glutamate release associated with upregulated and hyperactivated Cx43 hemichannel. Subchronic administration of therapeutic-relevant dose of ZNS reduced Cx43 expression in the M2C plasma membrane and possibly inhibits Cx43 hemichannel activity (Figure9C). The combination of these inhibitory effects of ZNS on Cx43 associated functions contributes to the compensation of functional abnormality of glutamatergic transmission of S286L-TG. The L-glutamate level in the M2C during resting stage and hyperactivated glutamatergic transmission in thalamocortical motor pathway were inhibited by ZNS (Figure9C). These two inhibitions probably prevent the focus generation via a combination of reduced basal L-glutamate level with L-glutamate release induced by the hyperactivation of the focus generator and thalamocortical motor pathway activity. Furthermore, prolonged hyperactivation of Cx43 hemichannel activity induced by hyperactivation around focus regions were also prevented by the inhibition of various transmission regulation systems, including voltage-sensitive calcium ion channel, voltage-dependent sodium ion channel, calcium-induced calcium releasing system [37–39,44,58,71], induced by ZNS. This inhibition of secondarily activated Cx43 hemichannel activity by ZNS can explain the pathomechanisms of ADSHE seizures during the non-rapid eye movement phase of sleep and the reoccurrence of seizures during a single night [4]. The various antiepileptic/anticonvulsive mechanisms of ZNS have been established, including exocytosis monoamine, L-glutamate and GABA via direct/indirect modulation of voltage-dependent sodium channel, voltage-sensitive calcium channel, calcium-induced calcium releasing system, carbonic anhydrase, redox system, GABA receptor and metabotropic glutamate receptors [37,38,40,44,48,58,62,70–72]. In these various targets of ZNS, voltage-dependent sodium channel inhibition has been considered to be the major mechanism of antiepileptic/anticonvulsive actions of ZNS, similar to CBZ. The pathophysiology of CBZ-resistant/ZNS-sensitive ADSHE seizure associated with S284L-mutation indicates the interesting possibility that voltage-dependent sodium channel inhibition cannot contribute to antiepileptic/anticonvulsive action of ZNS against ADSHE seizures of S284L-mutation. Taken together with our previous findings, the present study provides several indirect compensations of loss-of-functional S284L-mutant α4β2-nAChR by ZNS via the activation of group II metabotropic glutamate receptors in M2C, MoTN and subthalamic nucleus [21,22,48], and reduced Cx43 expression in plasma membrane (Figure9C). These two mechanisms possibly identify sodium channel inhibition-independent antiepileptic drugs as a potential therapeutic category. Furthermore, the present study suggests that ZNS can suppress Cx43 hemichannel activity. The detailed mechanisms of inhibitory effects of ZNS on Cx43 hemichannel should be determined in further studies, whereas inhibitory effects of ZNS on carbonic anhydrase and calcium-induced calcium releasing system are speculated to be candidate targets [37,62].

5. Conclusions This study provided the primary pathomechanism and secondary pathophysiology of ADSHE seizures of ADSHE patients with S284L-mutation using S286L-TG as a valid ADSHE rat model. Loss-of-function of S286L-mutant α4β2-nAChR in the RTN produces paradoxical functional abnormalities of glutamatergic transmission in the thalamocortical motor pathway (RTN-MoTN-M2C). In the thalamocortical motor pathway of S286L-TG, intra-thalamic (RTN-MoTN) GABAergic inhibition is reversed to GABAergic disinhibition by S286L-mutant α4β2-nAChR in the RTN. The functional conversion of intra-thalamic inhibitory system leads to hyperactivation of the thalamocortical motor pathway. Additionally, Cx43 expression in the M2C plasma membrane of S286L-TG is upregulated and induced by a lack of inhibitory regulation of loss-of-functional S286L-mutant α4β2-nAChR. The upregulated Cx43 hemichannel probably contributes to the generation of ADSHE focus and unique ADSHE phenotypes of multiple seizures during the same night. Therapeutic-relevant doses of ZNS Pharmaceuticals 2020, 13, 58 20 of 23 reduced Cx43 expression in the astroglial plasma membrane of both wild-type and S286L-TG. These compensations of ZNS on upregulated Cx43 expression in the ADSHE focus region play important roles in the CBZ-resistant/ZNS-sensitive ADSHE seizures of patients with S284L-mutation.

Author Contributions: Conceptualization, M.O.; Data curation, K.F., M.F. and M.O.; Formal analysis, K.F. and M.O.; Funding acquisition, M.O.; Methodology, M.F. and M.O.; Project administration; M.O., Validation, R.O. and M.O.; Writing original draft, M.O.; Writing review and editing, M.O. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by Japan Society for the Promotion of Science (15H04892 and 19K08073). Conﬂicts of Interest: The authors state no conﬂict of interest.

References

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