Age-Dependent and Sleep/Seizure-Induced Pathomechanisms of Autosomal Dominant Sleep-Related Hypermotor Epilepsy

The loss-of-function S284L-mutant α4 subunit of the nicotinic acetylcholine receptor (nAChR) is considered to contribute to the pathomechanism of autosomal dominant sleep-related hypermotor epilepsy (ADSHE); however, the age-dependent and sleep-related pathomechanisms of ADSHE remain to be clarified. To explore the age-dependent and sleep-induced pathomechanism of ADSHE, the present study determined the glutamatergic transmission abnormalities associated with α4β2-nAChR and the astroglial hemichannel in the hyperdirect and corticostriatal pathways of ADSHE model transgenic rats (S286L-TG) bearing the rat S286L-mutant Chrna4 gene corresponding to the human S284L-mutant CHRNA4 gene of ADSHE, using multiprobe microdialysis and capillary immunoblotting analyses. This study could not detect glutamatergic transmission in the corticostriatal pathway from the orbitofrontal cortex (OFC) to the striatum. Before ADSHE onset (four weeks of age), functional abnormalities of glutamatergic transmission compared to the wild-type in the cortical hyperdirect pathway, from OFC to the subthalamic nucleus (STN) in S286L-TG, could not be detected. Conversely, after ADSHE onset (eight weeks of age), glutamatergic transmission in the hyperdirect pathway of S286L-TG was enhanced compared to the wild-type. Notably, enhanced glutamatergic transmission of S286L-TG was revealed by hemichannel activation in the OFC. Expression of connexin43 (Cx43) in the OFC of S286L-TG was upregulated after ADSHE onset but was almost equal to the wild-type prior to ADSHE onset. Differences in the expression of phosphorylated protein kinase B (pAkt) before ADSHE onset between the wild-type and S286L-TG were not observed; however, after ADSHE onset, pAkt was upregulated in S286L-TG. Conversely, the expression of phosphorylated extracellular signal-regulated kinase (pErk) was already upregulated before ADSHE onset compared to the wild-type. Both before and after ADSHE onset, subchronic nicotine administration decreased and did not affect the both expression of Cx43 and pErk of respective wild-type and S286L-TG, whereas the pAkt expression of both the wild-type and S286L-TG was increased by nicotine. Cx43 expression in the plasma membrane of the primary cultured astrocytes of the wild-type was increased by elevation of the extracellular K+ level (higher than 10 mM), and the increase in Cx43 expression in the plasma membrane required pErk functions. These observations indicate that a combination of functional abnormalities, GABAergic disinhibition, and upregulated pErk induced by the loss-of-function S286L-mutant α4β2-nAChR contribute to the age-dependent and sleep-induced pathomechanism of ADSHE via the upregulation/hyperactivation of the Cx43 hemichannels.

be clarified [25]. Therefore, to explore the mechanisms of upregulation of Cx43 in S286L-TG, the effects of the subchronic administration of nicotine on the expression of Cx43, phosphorylated Akt (pAkt), and phosphorylated Erk (pErk) were also explored, using a capillary immunoblotting system.

Glutamatergic Transmission Abnormality in the Hyperdirect and Cirticostriatal Pathways Associated with the Hemichannel before and after ADSHE Onset
It is well-known that, during the resting stage, the hemichannel has low opening probability, but an extracellular cation condition, increased K + , and decreased Ca 2+ levels activates hemichannel activity [6,12,20,21,37]. According to previous demonstrations, to study the activated hemichannel activity on tripartite synaptic transmission in the OFC, the perfusion medium in the OFC was switched from modified Ringers solution (MRS) to Ca 2+ -free with 100 mM K + containing modified Ringer's solution (FCHK-MRS), for 20 min (FCHK-evoked stimulation) [6,8]. To explore the interaction between the effects of hemichannel and α4β2-nAChR on glutamatergic transmission in the cortical hyperdirect (OFC-STN) and corticostriatal (OFC-striatum) pathways of the wild-type and S286L-TG, before (four weeks of age) and after (eight weeks of age) ADSHE onset, the perfusion medium in the OFC began by using MRS with or without (control) 100 µM carbenoxolone (CBX: hemichannel inhibitor), 100 µM (E)-N-Methyl-4-(3-pyridinyl)-3-buten-1-amine oxalate (RJR2406: selective α4β2-nAChR agonist), or 100 µM CBX plus 100 µM RJR2406. The perfusates in the STN and striatum were maintained with MRS, alone, during the experiment. After the stabilization of the L-glutamate level in the STN or the striatum, the perfusate in the OFC was switched to MRS containing the same agent with 100 µM amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA) for 180 min (first AMPA-evoked stimulation). After the first AMPA-evoked stimulation, the perfusion medium in the OFC was switched to MRS. After the stabilization of the L-glutamate level in the STN or striatum, the perfusion medium in the OFC was switched to FCHK-MRS (Ca 2+ -free with 100 mM K + ) for 20 min (hemichannel activation). After the stabilization of the L-glutamate level in the STN or striatum, the perfusion medium in the OFC was switched to MRS containing the same agent with 100 µM AMPA, for 180 min again (second AMPA-evoked stimulation). The interval between the first and second AMPA-evoked stimulations was around 240 min. The detailed experimental designs are indicated in Section 4. 3.
Notably, CBX (non-selective hemichannel and gap-junction inhibitor) rapidly/reversibly inhibits these channels, and is widely used but offer lower permeability through the plasma membrane [38]. Therefore, CBX is a hemichannel blocker rather than a gap-junction inhibitor; however, 100 µM CBX inhibited the voltage-gated sodium channel [39]. In the present study, CBX was administered, using the reverse dialysis technique [40,41]. The estimated penetration ratio of CBX (molecular weight is 570.8) from the intra to extra dialysis probe was lower than 10% [42]. Therefore, the concentration of CBX in the brain tissue around the dialysis probe was lower than 10 µM.
Contrary to the STN, neither the L-glutamate level in the striatum of S286L-TG nor the wild-type was affected by perfusion with 100 µM AMPA and 100 µM RJR2403 into the OFC; however, the basal L-glutamate level in the striatum of S286L-TG was larger compared to that of the wild-type ( Figure 2). Therefore, the present study could not detect a corticostriatal (OFC-striatum) glutamatergic pathway, whereas glutamatergic neurons are present in the OFC project terminals to the STN (cortical hyperdirect pathway). The cortical hyperdirect glutamatergic transmission receives excitatory α4β2-nAChR in the OFC in the wild-type, whereas the S286L-mutant α4β2-nAChR in the OFC cannot affect cortical hyperdirect glutamatergic transmission. However, the cortical hyperdirect pathway in S286L-TG was regulated by the activated hemichannel in the OFC compared to that of the wild-type. Contrary to the STN, neither the L-glutamate level in the striatum of S286L-TG nor the wild-type was affected by perfusion with 100 μM AMPA and 100 μM RJR2403 into the OFC; however, the basal Lglutamate level in the striatum of S286L-TG was larger compared to that of the wild-type ( Figure 2). Therefore, the present study could not detect a corticostriatal (OFC-striatum) glutamatergic pathway, whereas glutamatergic neurons are present in the OFC project terminals to the STN (cortical hyperdirect pathway). The cortical hyperdirect glutamatergic transmission receives excitatory α4β2-nAChR in the OFC in the wild-type, whereas the S286L-mutant α4β2-nAChR in the OFC cannot affect cortical hyperdirect glutamatergic transmission. However, the cortical hyperdirect pathway in S286L-TG was regulated by the activated hemichannel in the OFC compared to that of the wild-type.  Contrary to after ADSHE onset (eight weeks of age), the basal extracellular L-glutamate level in the STN of S286L-TG prior to ADSHE seizure onset (four weeks of age) was almost equal to that of the wild-type ( Figure 3). Both the first and second AMPA-evoked stimulation into the OFC increased L-glutamate release in the STN ( Figure 3A,D). The first AMPA-evoked L-glutamate release in the STN of S286L-TG and the wild-type were also almost equal ( Figure 3C,F). Perfusion with 100 µM CBX into the OFC did not affect the first AMPA-evoked L-glutamate release but decreased the second AMPA-evoked L-glutamate release in the STN of both S286L-TG and the wild-type ( Figure 3C,F). Perfusion with 100 µM RJR2406 increased the first AMPA-evoked L-glutamate release in the STN of the wild-type but did not affect that of the S286L-TG; however, the second AMPA-evoked L-glutamate released in the STN of both S286L-TG and the wild-type were enhanced by 100 µM RJR2406 ( Figure 3C,F). Perfusion with 100 µM CBX suppressed the stimulatory effects of 100 µM RJR2406 on the second AMPA-evoked L-glutamate release in the STN of both genotypes ( Figure 3C,F). Therefore, before ADSHE onset, there were some functional abnormalities in L-glutamate release associated with the hemichannels, whereas the stimulatory effects of α4β2-nAChR on AMPA-evoked L-glutamate release were impaired in S286L-TG. Contrary to after ADSHE onset (eight weeks of age), the basal extracellular L-glutamate level in the STN of S286L-TG prior to ADSHE seizure onset (four weeks of age) was almost equal to that of the wild-type ( Figure 3). Both the first and second AMPA-evoked stimulation into the OFC increased L-glutamate release in the STN ( Figure 3A,D). The first AMPA-evoked L-glutamate release in the STN of S286L-TG and the wild-type were also almost equal ( Figure 3C,F). Perfusion with 100 μM CBX into the OFC did not affect the first AMPA-evoked L-glutamate release but decreased the second AMPA-evoked L-glutamate release in the STN of both S286L-TG and the wild-type ( Figure 3C,F). Perfusion with 100 μM RJR2406 increased the first AMPA-evoked L-glutamate release in the STN of the wild-type but did not affect that of the S286L-TG; however, the second AMPA-evoked Lglutamate released in the STN of both S286L-TG and the wild-type were enhanced by 100 μM RJR2406 ( Figure 3C,F). Perfusion with 100 μM CBX suppressed the stimulatory effects of 100 μM RJR2406 on the second AMPA-evoked L-glutamate release in the STN of both genotypes ( Figure  3C,F). Therefore, before ADSHE onset, there were some functional abnormalities in L-glutamate release associated with the hemichannels, whereas the stimulatory effects of α4β2-nAChR on AMPAevoked L-glutamate release were impaired in S286L-TG.  * p < 0.05, ** p < 0.01; relative to the first (first AMPA-evoked stimulation), @p < 0.05, @@p < 0.01; relative to the control, # p < 0.05, ## p < 0.01; relative to RJR by MANOVA with Tukey's multiple comparison. The F-values of the L-glutamate level in the STN, according to a multivariate analysis of variance (MANOVA), were F event (1,80 and F event*RJR*CBX*genotype (1,80)= 0.1 (p > 0.05).

Tetrodotoxin (TTX) and CBX-Sensitive Basal L-Glutamate Release in the OFC of S286L-TG after Hemichannel Activation (Study_3)
To clarify the mechanisms of enhanced glutamatergic transmission in the cortical hyperdirect pathway induced by hemichannel activation in the OFC of ADSHE onset S286L-TG after FCHK-evoked stimulation in the OFC (after the Study_1), the perfusion medium in the OFC was switched to MRS containing 1 µM TTX (voltage-dependent sodium channel inhibitor) [43] or 100 µM CBX (hemichannel inhibitor). The increased basal L-glutamate release in the OFC after FCHK-evoked stimulation was decreased by CBX but not affected by TTX ( Figure 4). Therefore, the increased basal L-glutamate release in the STN of S286L-TG induced by FCHK-evoked stimulation was considered to be of astroglial hemichannel origin but not of neuronal exocytosis origin. To clarify the mechanisms of enhanced glutamatergic transmission in the cortical hyperdirect pathway induced by hemichannel activation in the OFC of ADSHE onset S286L-TG after FCHKevoked stimulation in the OFC (after the Study_1), the perfusion medium in the OFC was switched to MRS containing 1 μM TTX (voltage-dependent sodium channel inhibitor) [43] or 100 μM CBX (hemichannel inhibitor). The increased basal L-glutamate release in the OFC after FCHK-evoked stimulation was decreased by CBX but not affected by TTX ( Figure 4). Therefore, the increased basal L-glutamate release in the STN of S286L-TG induced by FCHK-evoked stimulation was considered to be of astroglial hemichannel origin but not of neuronal exocytosis origin.  . Effects of the local administration of 1 µM tetrodotoxin (TTX) and 100 µM CBX into the OFC on basal L-glutamate release in the OFC of S286L-TG after FCHK-evoked stimulation. The perfusion medium in the OFC was switched to MRS, with or without (control: post-stimulation: gray column) 1 µM TTX (red column) or 100 µM CBX (blue column). The ordinate AUC of the extracellular L-glutamate level (nmol) for 60 min during perfusion of MRS with or without TTX or CBX is shown. ** p < 0.01, relative to the control (pre-stimulation), and @@ p < 0.01, relative to the control (post-stimulation) by a one-way analysis of variance (ANOVA) with Tukey's multiple comparison. The F-values of the L-glutamate level in the OFC, according to the one-way ANOVA, are F(3,20) = 9.6 (p < 0.01).

Effects of Subchronic Nicotine Administration on Akt and Erk Expression in the OFC
At four weeks of age (before ADSHE onset), phosphorylated Akt (pAkt) expression in the OFC plasma membrane fraction of S286L-TG was almost equal to that of the wild-type; however, at 12 weeks of age (after ADSHE onset), pAkt expression in the OFC plasma membrane fraction of S286L-TG was larger than that of the wild-type ( Figure 6A  Effects of systemic subchronic administration of nicotine (50 mg/kg/day for seven days) on Cx43 expression in the OFC plasma membrane fraction before four weeks of age (A), and after 12 weeks of age (B), autosomal dominant sleep-related hypermotor epilepsy (ADSHE) onset of the wild-type and S286L-TG and pseudo-gel images from the capillary immunoblotting results, using anti-GAPDH and anti-connexin43 antibodies for blotting of the plasma membrane fractions. Ordinate: mean ± SD (n = 6) of the relative protein level of Cx43. ** p < 0.01 vs. the wild-type, and @ p < 0.05, @@ p < 0.01 vs. nicotine-free (non) based on a two-way ANOVA with Tukey's multiple comparison.

Effect of Erk and Extracellular K + Level on Astroglial Cx43 Expression
After culturing for 28 days (DIV28) to study the effects of the extracellular K + level on Cx43 expression in the plasma membrane, the cultured medium was changed from a Dulbecco's modified Eagle's medium containing 10% fetal calf serum (fDMEM) to N-fDMEM (control: fDMEM plus 4.6 mM NaCl: 5.4 mM K + ), MK-fDMEM (fDMEM plus 2.1 mM KCl and 2.5 mM NaCl: 7.5 mM K + ), and HK-fDMEM (fDMEM plus 4.6 mM KCl: 10.0 mM K + ) for 6 h (around the half-life of Cx43 [44]). To study the effects of Akt and Erk on Cx43 expression in the plasma membrane, the cultured medium was changed to HK-fDMEM containing 20 μM FR180204 (Erk inhibitor) or 10 μM 10-DEBC (Akt inhibitor) for 6 h.
Cx43 expression in the plasma membrane fraction of the wild-type primary cultured astrocytes increased the extracellular K + level concentration-dependently (F(2,15) =27.2 (p < 0.01)) ( Figure 7A). MK-fDMEM (7.5 mM K + ) did not affect Cx43 expression, whereas HK-fDMEM (10.0 mM K + ) increased Cx43 expression in the plasma membrane fraction ( Figure 6A). The concentrationdependent expression of the extracellular K + of Cx43 in the plasma membrane was suppressed by the inhibitor of both Erk (FR180204) and Akt (10-DEBC) (p < 0.01) ( Figure 7B). Figure 6. Effects of subchronic nicotine administration on the expression of phosphorylated protein kinase B (pAkt) and phosphorylated extracellular signal-regulated kinase (pErk) in the plasma membrane fraction of OFC. Effects of the systemic subchronic administration of nicotine (50 mg/kg/day for seven days) on pAkt and pErk expression in the OFC plasma membrane fraction before four week of age (A,C) and after 12 week of age (B,D), ADSHE onset of the wild-type and S286L-TG and pseudo-gel images, using capillary immunoblotting. Ordinate: mean ± SD (n = 6) of the relative protein level of pErk and pAkt. * p < 0.05, ** p < 0.01 vs. wild-type, and @ p < 0.05, @@ p < 0.01 vs. nicotine-free (non) by two-way ANOVA with Tukey's multiple comparison.

Effect of Erk and Extracellular K + Level on Astroglial Cx43 Expression
After culturing for 28 days (DIV28) to study the effects of the extracellular K + level on Cx43 expression in the plasma membrane, the cultured medium was changed from a Dulbecco's modified Eagle's medium containing 10% fetal calf serum (fDMEM) to N-fDMEM (control: fDMEM plus 4.6 mM NaCl: 5.4 mM K + ), MK-fDMEM (fDMEM plus 2.1 mM KCl and 2.5 mM NaCl: 7.5 mM K + ), and HK-fDMEM (fDMEM plus 4.6 mM KCl: 10.0 mM K + ) for 6 h (around the half-life of Cx43 [44]). To study the effects of Akt and Erk on Cx43 expression in the plasma membrane, the cultured medium was changed to HK-fDMEM containing 20 µM FR180204 (Erk inhibitor) or 10 µM 10-DEBC (Akt inhibitor) for 6 h.

Figure 7.
Effects of subacute administration of an increase in the extracellular K + level on Cx43 expression in the plasma membrane fraction of primary cultured astrocytes (A). Effects of the inhibitor of Erk (FR180204) and Akt (10-DEBC) on K + -dependent Cx43 expression in the plasma membrane (B). Ordinate: mean ± SD (n = 6) of the relative protein level of Cx43. Concentrationdependent effects of extracellular K + on Cx43 expression in the plasma membrane fraction of the primary cultured astrocytes were analyzed by a one-way ANOVA (@@ p < 0.01 vs. control). The effects of 20 μM FR180204 and 10 μM 10-DEBC on Cx43 expression in the plasma membrane fraction were analyzed by a Student's t-test (** p < 0.01 vs. 10 mM K + ).

Mechanisms of Upregulation of Cx43 of S286-TG.
We have already demonstrated that a combination of attenuated intrathalamic GABAergic transmission and upregulated/hyperactivated Cx43 in the secondary motor cortex and thalamus of S286L-TG plays important roles in the pathomechanisms of ADSHE [6][7][8]12]. The propagation of epileptic discharge in the thalamocortical cognitive (MDTN-OFC) pathway was highly dependent upon Cx43 upregulation/hyperactivation in the thalamus compared to that in the thalamocortical motor pathway (from the motor thalamic nuclei to the secondary motor cortex) [8,12]. The present study also detected the upregulation of Cx43 in the OFC plasma membrane of S286L-TG.
According to our expectations, before ADSHE onset (four weeks of age), Cx43 expression in the plasma membrane of S286L-TG OFC was almost equal to that of the wild-type; however, after ADSHE onset (12 weeks of age), Cx43 expression in the plasma membrane of S286L-TG was upregulated compared to that of the wild-type. Subchronic nicotine administration reduced Cx43 expression of the wild-type at both 4 and 12 weeks of age, whereas nicotine did not affect the Cx43 expression of S286L-TG before and after ADSHE onset. These results suggest that the loss-of-function Ordinate: mean ± SD (n = 6) of the relative protein level of Cx43. Concentration-dependent effects of extracellular K + on Cx43 expression in the plasma membrane fraction of the primary cultured astrocytes were analyzed by a one-way ANOVA (@@ p < 0.01 vs. control). The effects of 20 µM FR180204 and 10 µM 10-DEBC on Cx43 expression in the plasma membrane fraction were analyzed by a Student's t-test (** p < 0.01 vs. 10 mM K + ).

Mechanisms of Upregulation of Cx43 of S286-TG.
We have already demonstrated that a combination of attenuated intrathalamic GABAergic transmission and upregulated/hyperactivated Cx43 in the secondary motor cortex and thalamus of S286L-TG plays important roles in the pathomechanisms of ADSHE [6][7][8]12]. The propagation of epileptic discharge in the thalamocortical cognitive (MDTN-OFC) pathway was highly dependent upon Cx43 upregulation/hyperactivation in the thalamus compared to that in the thalamocortical motor pathway (from the motor thalamic nuclei to the secondary motor cortex) [8,12]. The present study also detected the upregulation of Cx43 in the OFC plasma membrane of S286L-TG.
According to our expectations, before ADSHE onset (four weeks of age), Cx43 expression in the plasma membrane of S286L-TG OFC was almost equal to that of the wild-type; however, after ADSHE onset (12 weeks of age), Cx43 expression in the plasma membrane of S286L-TG was upregulated compared to that of the wild-type. Subchronic nicotine administration reduced Cx43 expression of the wild-type at both 4 and 12 weeks of age, whereas nicotine did not affect the Cx43 expression of S286L-TG before and after ADSHE onset. These results suggest that the loss-of-function S286L-mutant α4β2-nAChR plays important roles in the pathomechanisms of ADSHE via the upregulation of astroglial Cx43.
Contrary to Cx43 expression, the pErk of S286L-TG was already upregulated before ADSHE onset compared to that of the wild-type. Subchronic nicotine administration reduced the pErk of the wild-type at both 4 and 12 weeks of age, whereas nicotine did not affect the pErk of S286L-TG before and after ADSHE onset. Furthermore, our previous study already demonstrated that furosemide, which prevents the ADSHE onset of S284L-TG [10], inhibits MAPK/Erk signaling [29]. Thus, considering our previous demonstration, the upregulation of pErk preceding Cx43 upregulation in the OFC of S286L-TG suggests the possible pathomechanisms of ADSHE though which the upregulation of MAPK/Erk signaling induced by the loss-of-function S286L-mutant α4β2-nAChR plays key roles in the development of the epileptogenesis of ADSHE.
Differences between pAkt expression of the wild-type and S286L-TG were not observed before the ADSHE onset periods; however, after ADSHE onset, the pAkt of S286L-TG was upregulated compared to that of the wild-type. Conversely, sub-chronic nicotine administration increased the pAkt of both wild-type and S286L-TG before and after the ADSHE onset periods. This nicotine-induced upregulation of pAkt is generated by the nicotine-induced activation of α7-nAChR, which is consistent with a previous demonstration [24,25,35,36]. Whether the upregulation of pAkt after ADSHE onset is pathomechanism or a result of ADSHE seizure is a fundamental neuroscientific issue. It is well-known that enhanced IP3K/Akt signaling plays a key role in the development of the epileptogenesis and ictogenesis of various epileptic syndromes via suppression of the tuberous sclerosis complex (TSC) and activation of mammalian target of rapamycin (mTOR) signaling [47]. In other line aspects, epileptic seizure also upregulates PI3K/Akt/mTOR signaling [48]. Based on these previous findings, the present study cannot assert that pAkt is not involved in the pathomechanism of ADSHE, but the upregulation of pErk may play a more important role in the development of the epileptogenesis of ADSHE than in pAkt signaling.

Impact of the Upregulation/Activation of Cx43 in ADSHE.
During the resting stage, the astroglial hemichannel exhibits a low opening probability [6,12,20,21] but is activated by the depolarization of membrane potential and specific fluctuations in the extracellular and intracellular cation levels [6,12,21]. A functional analysis study using S284L-TG demonstrated that impaired GABAergic inhibition and interictal discharge onset are observable at four and six weeks of age (both before ADSHE onset), respectively [10,11]. Electrophysiological studies of Xenopus oocytes, using voltage-clamps, showed that the S284L-mutant α4β2-nAChR enhances ACh-sensitivity and desensitization [49]. Therefore, the enhanced irritability (repetitive/persistent excitability) induced by impaired GABAergic inhibition via the loss-of-function S286L-mutant α4β2-nAChR can activate hemichannel functions [6,12]. In our previous microdialysis studies, 25 mM K + -evoked stimulation that could increase neurotransmitter release [50], but gliotransmitter release required greater than 100 mM extracellular K + levels [12,37]. However, elevation of the extracellular K + level around 10-12 mM plays an important role in the generation of hypersynchronous neuronal excitability, and epileptic discharge also increases the extracellular K + level to over 10 mM [51]. Based on these previous findings, to explore the repetitive/persistent elevation of the extracellular K + level (over 10 mM) on astroglial hemichannel activity, the effects of subacute (for 6 h, longer than the half-life of Cx43 [44]) exposure to the 10 mM extracellular K + level on Cx43 expression in the plasma membrane of the wild-type primary cultured astrocytes were studied. According to our expectations, astroglial Cx43 expression was increased by elevation of the extracellular K + level (7.5 mM K + did not affect Cx43 expression, but the threshold level of 10 mM K + increased). Taken together with our previous findings, the present demonstrations suggest multiple age-dependent and event-induced (sleep and epileptic seizure) stages of pathomechanisms in ADSHE.
Therefore, the congenital loss-of-function S286L-mutant α4β2-nAChR generates two functional abnormalities in S286L-TG, GABAergic disinhibition [10,11] and upregulation of the MAPK/Erk signaling pathway. Impaired GABAergic inhibition leads to the relative enhancement of glutamatergic transmission in the thalamocortical and hyperdirect pathways in α4β2-nAChR predominant regions [6][7][8]12], resulting in the generation of interictal discharge before ADSHE onset [11]. A combination of hyperglutamatergic transmission (repetitive/persistent propagation of discharges) and an upregulated MAPK/Erk signaling pathway contributes to the development of epileptogenesis/ictogenesis via the upregulation of astroglial Cx43. It is well-known that the majority of ADSHEs (50-60%) can be controlled by a relatively low dose of carbamazepine, whereas ADSHE with an S284L mutation has a carbamazepine-resistant feature and requires other antiepileptic drugs, such as zonisamide [4,41,52,53]. A therapeutically relevant concentration of zonisamide inhibits both the activity and expression of Cx43 in the astroglial plasma membrane; however, a therapeutically relevant concentration of carbamazepine does not affect these factors [23,54]. Therefore, the activated hemichannel in the OFC likely also contributes to the pathophysiology/ictogenesis of carbamazepine-resistant/zonisamide-sensitive ADSHE seizures with S284L-mutations.

Neural Circuits Associated with Dystonia Posturing in ADSHE Seizures
In our previous study, hyperactivated glutamatergic transmission in the thalamic hyperdirect pathway (from motor thalamic nuclei to the STN), but not the glutamatergic transmission abnormalities in the cortical hyperdirect pathway (from the secondary motor cortex to the STN), provided the pathomechanism of nocturnal paroxysmal dystonia [6]. The cortical connectivity to the STN is sparser compared to that to the striatum, but the OFC exhibits higher tract strength for the STN relative to the striatum [17]. A clinical study observed OFC seizures during nocturnal paroxysmal dystonia and episodic nocturnal wandering featuring stereotypical dystonic posturing [13]. In another ADSHE model, S280F-knockin mice strain also exhibited a dystonic/arousal complex, which is a complex between the nocturnal paroxysmal dystonia and nocturnal paroxysmal arousal of ADSHE seizures [55]. These clinical and preclinical findings suggest that hyperactivity in the basal ganglia is possibly involved in ADSHE seizures. Therefore, to explore the pathomechanisms of ADSHE seizures, the present study determined glutamatergic transmission abnormalities in the cortical hyperdirect (OFC-STN) and corticostriatal (OFC-striatum) pathways.
According to our expectations, after ADSHE onset (eight weeks of age), activation of the postsynaptic AMPA/glutamate receptor in the OFC increased L-glutamate release in the STN (cortical hyperdirect pathway) without affecting that in the striatum (corticostriatal pathway) in both wild-type and S286L-TG. Hemichannel activation in the OFC enhanced L-glutamate release in the STN of both wild-type and S286L-TG, but sensitivity to the hemichannel activation of S286L-TG was predominant rather than that of the wild-type. Interestingly, the glutamatergic transmission in the cortical hyperdirect pathway associated with the astroglial hemichannel was observed prior to hemichannel activation in S286L-TG, since CBX inhibited AMPA-evoked L-glutamate release before FCHK-evoked stimulation. Taken together with the upregulation of Cx43 expression in the OFC of S286L-TG, at eight weeks of age, the upregulated Cx43 hemichannel of S286L-TG was probably weakly activated during the interictal stages. Contrary to the hemichannel, in the wild-type, α4β2-nAChR enhanced AMPA-evoked glutamate release in the cortical hyperdirect pathway under conditions of both hemichannel resting and activation in the OFC, whereas the AMPA-evoked L-glutamate release in the STN of S286L-TG was insensitive to α4β2-nAChR before hemichannel activation. These discrepancies in responses to the α4β2-nAChR of glutamatergic transmission in the hyperdirect pathway between S286L-TG and the wild-type suggest that the loss-of-function S286L-mutant α4β2-nAChR likely does not directly contribute to the generation of epileptic ADSHE focus in the OFC, but indirectly contributes to the generation of focus via the activation/upregulation of Cx43 hemichannel hyperactivation.
Similar to the observations at eight weeks of age, the activation of α4β2-nAChR enhanced AMPA-evoked glutamate release in the cortical hyperdirect pathway under conditions of both hemichannel resting and activation in the OFC of the wild-type, whereas before hemichannel activation, the AMPA-evoked L-glutamate release of S286L-TG was insensitive to α4β2-nAChR. In contrast to α4β2-nAChR, the sensitivity levels to the activated hemichannel of glutamatergic transmission in the cortical hyperdirect pathway of S286L-TG showed similar features to the wild-type. Therefore, the differences in the sensitivity of glutamatergic transmission to activated hemichannels before (four weeks of age) and after (eight weeks of age) ADSHE seizure onset suggest that the activation of upregulated Cx43 in the OFC of S286L-TG likely contributes to the development of ictogenesis through the hyperactivation of tripartite synaptic transmission. In other words, the hypersensitivity of hemichannel activity in the OFC to the propagation of discharges (physiological sleep spindle, interictal and ictal discharges) plays important roles in clinical ADSHE features, such as the common occurrence of ADSHE seizures during non-REM sleep phases and once ADSHE seizure leading to subsequent frequent seizures during the same night [2,13].
All compounds were prepared on the day of the experiment. In the microdialysis study, AMPA, CBX, TTX, and RJR2403 were dissolved in a modified ringer solution (MRS) composed of the following (in mM): 145 Na + , 2.7 K + , 1.2 Ca 2+ , 1.0 Mg 2+ , and 154.4 Cl − , buffered with 2 mM phosphate buffer and 1.1 mM Tris buffer at pH 7.4 [56][57][58][59][60]. In the primary cultured astrocyte study, FR180204 was initially made as 10 mM stocks in dimethyl sulfoxide and then diluted to Dulbecco's modified Eagle's medium (D6546: Sigma-Aldrich, St. Louis, MO) containing 10% fetal calf serum (fDMEM). The 10-DEBC was dissolved in fDMEM directly. To study the effects of subchronic nicotine administration on the expression of Cx43, pErk/Erk, and pAkt/Akt in the OFC plasma membrane, rats were subchronically administered with nicotine ditartrate (50 mg/kg/day for 7 days), using a subcutaneous osmotic pump (2ML_1, Alzet, Cupertino, CA; the nominal pumping rate and duration were 10 µL/h over 7 days).

Experimental Animals
Animal care, experimental procedures, and protocols for the animal experiments were approved by the Animal Research Ethics Committee of the Mie University School of Medicine (No. 24-37-R3, 7 March 2018). All studies involving animals were reported in accordance with the ARRIVE guidelines for reporting experiments involving animals. A total 126 rats, wild-type littermates (n = 90), and S286L-TG rats (n = 84) [7,61] (Sprague Dawley strain background, SLC, Shizuoka, Japan) were maintained in a controlled environment (22 ± 1 • C) on a 12 h dark/light cycle and used in the experiments as described. Rats were randomly assigned to the treatment groups of each experiment. 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 [6][7][8]12]. Where possible, we sought to randomize and blind the data. In particular, for the determination of L-glutamate and protein levels, the sample order was determined by a random number table.
For the microdialysis study, to activate the hemichannel function in the OFC, the perfusion medium in the OFC was switched to Ca 2+ -free with 100 mM K + containing MRS (FCHK-MRS) for 20 min (FCHK-MRS activation) [6,8] (Figure 1). To explore the effects of the hemichannel and α4β2-nAChR on glutamatergic transmission in the cortical hyperdirect (OFC-STN) and corticostriatal (OFC-striatum) pathways of the wild-type and S286L-TG, the perfusion medium in the OFC began with MRS with or without (control) 100 µM CBX (non-selective hemichannel inhibitor), 100 µM RJR2406 (selective α4β2-nAChR agonist), or 100 µM CBX plus 100 µM RJR2406. The perfusates in the STN and striatum were maintained with MRS alone during the experiment. After stabilization of the L-glutamate level in the STN or striatum, the perfusate in the OFC was switched to MRS containing the same agent with 100 µM AMPA for 180 min (1st AMPA-evoked stimulation). After the 1st AMPA-evoked stimulation, the perfusion medium in the OFC was switched to MRS. After stabilization of the L-glutamate level in the STN or striatum, the perfusion medium in the OFC was switched to FCHK-MRS (Ca 2+ -free with 100 mM K + ) for 20 min (hemichannel activation). After stabilization of the L-glutamate level in the STN or striatum, the perfusion medium in the OFC was switched to MRS containing the same agent with 100 µM AMPA for 180 min again (2nd AMPA-evoked stimulation) (Figure 1). The time between the 1st and 2nd AMPA-evoked stimulations was around 240 min.

Capillary Immunoblotting Analysis
The capillary immunoblotting analysis was performed, using Wes (ProteinSimple, Santa Clara, CA, USA), according to the ProteinSimple user manual [6][7][8]12]. The lysates of the primary cultured astrocytes were mixed with a master mix (ProteinSimple) to a final concentration of 1 × sample buffer, 1 × fluorescent molecular weight marker, and 40 mM dithiothreitol and then heated at 95 • C for 5 min. The samples, blocking reagents, primary antibodies, HRP-conjugated secondary antibodies, chemiluminescent substrate (SuperSignal West Femto: Thermo Fisher Scientific, Waltham, MA, USA), and separation and stacking matrices were also dispensed to the 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. A capillary immunoblotting analysis was carried out at room temperature, and the instrument's default settings were used. Capillaries were first filled with a separation matrix followed by a stacking matrix, with about 40 nL of the sample used for loading. During electrophoresis, the proteins were separated by 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. The capillaries were next incubated with a blocking reagent for 15 min, and the target proteins were immunoprobed with primary antibodies followed by HRP-conjugated secondary antibodies (Anti-Rabbit IgG HRP, A00098, 10 µg/mL, GenScript, Piscataway, NJ). The antibodies of GAPDH (NB300-322, 1:100, Novus Biologicals, Littleton, CO, USA), Cx43 (C6219, 1:100, Sigma-Aldrich, St. Louis, MO, USA), Erk (AF1576, 10 µg/mL, R&D systems, Minneapolis, MN, USA), pErk (AF1018, 5 µg/mL, R&D systems), Akt (AF1775, 1 µg/mL, R&D systems), and pAkt (AF877, 5 µg/mL, R&D systems) were diluted in an antibody diluent (ProteinSimple).

Primary Cultured Astrocytes
Astrocytes were prepared, using a protocol adapted from previously described methods [22,54,68]. Pregnant Sprague-Dawley rats (SLC, Sizuoka, Japan) were housed individually in cages and kept in air-conditioned rooms (temperature, 22 ± 2 • C), with a 12 h light/dark cycle and free access to food and water. Cultured astrocytes were prepared from the cortical astrocyte cultures of neonatal Sprague-Dawley rats (n = 6) sacrificed by decapitation at 0-24 h of age. Cerebral hemispheres were removed under a dissecting microscope. Tissues were chopped into fine pieces, using scissors and then triturated briefly with a micropipette. The suspension was filtered, using a 70 µm nylon mesh (BD, Franklin Lakes, NJ, USA), and centrifuged. Pellets were then re-suspended in fDMEM, which was repeated three times. After culturing for 14 days (DIV14), the contaminating cells were removed via shaking in a standard incubator (BNA-111, Espec, Osaka, Japan) for 16 h at 200 rpm. On DIV21, the astrocytes were removed from the flasks by trypsinization and seeded directly onto a translucent PET membrane (1.0 µm) with 24 well plates (BD) at a density of 1 × 105 cells/cm 2 for the experiments. From DIV21 to DIV27, the culture medium (fDMEM) was changed twice a week for 7 days. On DIV27, to study the effects of the extracellular K + level on Cx43 expression in the plasma membrane, the cultured medium was changed (for 6 h) to N-fDMEM (control: fDMEM plus 4.6 mM NaCl: 5.4 mM K + ), MK-fDMEM (fDMEM plus 2.1 mM KCl and 2.5 mM NaCl: 7.5 mM K + ), and HK-fDMEM (fDMEM plus 4.6 mM KCl = 10.0 mM K + ). The composition of NaCl and KCl in fDMEM was modified to maintain isotonicity and ionic strength. To study the effects of Erk and Akt on Cx43 expression in the plasma membrane, the medium was changed (for 6 h) to N-fDMEM containing 20 µM FR180204 (Erk inhibitor) or 10 µM 10-DEBC (Akt inhibitor) for 6 h. On DIV 28, the cultured astrocytes were washed out, using artificial-CSF, and then the total plasma membrane proteins were extracted, using a Minute Plasma Membrane Protein Isolation Kit (Invent Biotechnologies) [6].

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. All values are expressed as the mean ± SD, and p < 0.05 (two-tailed) was considered statistically significant for all tests. Drug levels in acute local and subchronically systemic administrations were selected based on values in previous studies. Where possible, we sought to randomize and blind the data. In particular, for the determination of transmitter levels and protein expression, the sample order on the autosampler and Wes were determined by a random number table.
The regional transmitter concentrations in the STN and striatum were analyzed via a Mauchly's sphericity test followed by a multivariate analysis of variance (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 the 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 were analyzed by a Tukey's multiple comparison test. The transmitter level was expressed as the area under the curve between 20 and 180 min (AUC  ) after the perfusion of AMPA containing MRS. The effects of perfusion with TTX and CBX into the basal L-glutamate release were analyzed, using a one-way analysis of variance (ANOVA) with Tukey's multiple comparison. The protein expression of Cx43, pErk, and pAkt in the plasma membrane fraction was analyzed by a two-way ANOVA with Tukey's multiple comparison, using BellCurve for Excel.

Conclusions
In conclusion, this study provided evidence for the age-dependent and sleep/seizure-induced multi-stage pathomechanisms of ADSHE with S284L-mutations, using the genetic ADSHE model (S286L-TG). Congenital functional abnormalities and the loss-of-function S286L-mutant α4β2-nAChR produce GABAergic disinhibition, resulting in enhanced glutamatergic transmission and an upregulated MAPK/Erk signaling pathway ( Figure 8). Wild-type α4β2-nAChR suppresses pErk, but the S286L-mutant α4β2-nAChR impairs the inhibitory function of pErk, resulting in the upregulation of Cx43 expression. Under functional abnormalities, the propagation of physiological (sleep spindle bursts) and pathological (interictal/ictal discharges) discharges to the OFC leads to an event-related enhancement in the function of upregulated Cx43, resulting in enhanced excitatory tripartite synaptic transmission ( Figure 8). Therefore, a combination of secondary functional abnormalities induced by the loss-of-function S286L-mutant α4β2-nAChR, GABAergic disinhibition, and Cx43 upregulation contributes to the pathomechanism of ADSHE. Interestingly, even if ADSHE seizures are controlled, once a patient experiences an ADSHE seizure, he or she will experience many more ADSHE seizures during that same night. enhancement in the function of upregulated Cx43, resulting in enhanced excitatory tripartite synaptic transmission ( Figure 8). Therefore, a combination of secondary functional abnormalities induced by the loss-of-function S286L-mutant α4β2-nAChR, GABAergic disinhibition, and Cx43 upregulation contributes to the pathomechanism of ADSHE. Interestingly, even if ADSHE seizures are controlled, once a patient experiences an ADSHE seizure, he or she will experience many more ADSHE seizures during that same night.  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.