Age-Dependent Activation of Purinergic Transmission Contributes to the Development of Epileptogenesis in ADSHE Model Rats.

To explore the developmental processes of epileptogenesis/ictogenesis, this study determined age-dependent functional abnormalities associated with purinergic transmission in a genetic rat model (S286L-TG) of autosomal-dominant sleep-related hypermotor epilepsy (ADSHE). The age-dependent fluctuations in the release of ATP and L-glutamate in the orbitofrontal cortex (OFC) were determined using microdialysis and ultra-high-performance liquid chromatography with mass spectrometry (UHPLC-MS). ATP release from cultured astrocytes was also determined using UHPLC-MS. The expressions of P2X7 receptor (P2X7R), connexin 43, phosphorylated-Akt and phosphorylated-Erk were determined using capillary immunoblotting. No functional abnormalities associated with purinergic transmission could be detected in the OFC of 4-week-old S286L-TG and cultured S286L-TG astrocytes. However, P2X7R expression, as well as basal and P2X7R agonist-induced ATP releases, was enhanced in S286L-TG OFC in the critical ADSHE seizure onset period (7-week-old). Long-term exposure to a modest level of P2X7R agonist, which could not increase astroglial ATP release, for 14 d increased the expressions of P2X7R and connexin 43 and the signaling of Akt and Erk in astrocytes, and it enhanced the sensitivity of P2X7R to its agonists. Akt but not Erk increased P2X7R expression, whereas both Akt and Erk increased connexin 43 expression. Functional abnormalities, enhanced ATP release and P2X7R expression were already seen before the onset of ADSHE seizure in S286L-TG. Additionally, long-term exposure to the P2X7R agonist mimicked the functional abnormalities associated with purinergic transmission in astrocytes, similar to those in S286L-TG OFC. Therefore, these results suggest that long-term modestly enhanced purinergic transmission and/or activated P2X7R are, at least partially, involved in the development of the epileptogenesis of ADSHE, rather than that of ictogenesis.

Age-Dependent Activation of Pannexin1 Function Contributes to the Development of Epileptogenesis in Autosomal Dominant Sleep-related Hypermotor Epilepsy Model Rats.

To explore the processes of epileptogenesis/ictogenesis, this study determined the age-dependent development of the functional abnormalities in astroglial transmission associated with pannexin1-hemichannel using a genetic rat model of autosomal dominant sleep-related hypermotor epilepsy (ADSHE) named 'S286L-TG'. Pannexin1 expression in the plasma membrane of primary cultured cortical astrocytes and the orbitofrontal cortex (OFC), which is an ADSHE focus region, were determined using capillary immunoblotting. Astroglial D-serine releases induced by artificial high-frequency oscillation (HFO)-evoked stimulation, the removal of extracellular Ca2+, and the P2X7 receptor agonist (BzATP) were determined using ultra-high performance liquid chromatography (UHPLC). The expressions of pannexin1 in the plasma membrane fraction of the OFC in S286L-TG at four weeks old were almost equivalent when compared to the wild type. The pannexin1 expression in the OFC of the wild type non-statistically decreased age-dependently, whereas that in S286L-TG significantly increased age-dependently, resulting in relatively increasing pannexin1 expression from the 7- (at the onset of interictal discharge) and 10-week-old (after the ADSHE seizure onset) S286L-TG compared to the wild type. However, no functional abnormalities of astroglial pannexin1 expression or D-serine release through the pannexin1-hemichannels from the cultured astrocytes of S286L-TG could be detected. Acutely HFO-evoked stimulation, such as physiological ripple burst (200 Hz) and epileptogenic fast ripple burst (500 Hz), frequency-dependently increased both pannexin1 expression in the astroglial plasma membrane and astroglial D-serine release. Neither the selective inhibitors of pannexin1-hemichannel (10PANX) nor connexin43-hemichannel (Gap19) affected astroglial D-serine release during the resting stage, whereas HFO-evoked D-serine release was suppressed by both inhibitors. The inhibitory effect of 10PANX on the ripple burst-evoked D-serine release was more predominant than that of Gap19, whereas fast ripple burst-evoked D-serine release was predominantly suppressed by Gap19 rather than 10PANX. Astroglial D-serine release induced by acute exposure to BzATP was suppressed by 10PANX but not by Gap19. These results suggest that physiological ripple burst during the sleep spindle plays important roles in the organization of some components of cognition in healthy individuals, but conversely, it contributes to the initial development of epileptogenesis/ictogenesis in individuals who have ADSHE vulnerability via activation of the astroglial excitatory transmission associated with pannexin1-hemichannels.

A Novel Gliotransmitter, L-ß-Aminoisobutyric Acid, Contributes to Pathophysiology of Clinical Efficacies and Adverse Reactions of Clozapine.

Clozapine is listed as one of the most effective antipsychotics and has been approved for treating treatment-resistant schizophrenia (TRS); however, several type A and B adverse reactions, including weight gain, metabolic complications, cardiotoxicity, convulsions, and discontinuation syndromes, exist. The critical mechanisms of clinical efficacy for schizophrenia, TRS, and adverse reactions of clozapine have not been elucidated. Recently, the GABA isomer L-ß-aminoisobutyric acid (L-BAIBA), a protective myokine in the peripheral organs, was identified as a candidate novel transmission modulator in the central nervous system (CNS). L-BAIBA activates adenosine monophosphate-activated protein kinase (AMPK) signalling in both the peripheral organs and CNS. Activated AMPK signalling in peripheral organs is an established major target for treating insulin-resistant diabetes, whereas activated AMPK signalling in the hypothalamus contributes to the pathophysiology of weight gain and metabolic disturbances. Clozapine increases L-BAIBA synthesis in the hypothalamus. In addition, the various functions of L-BAIBA in the CNS have recently been elucidated, including as an activator of GABA-B and group-III metabotropic glutamate (III-mGlu) receptors. Considering the expressions of GABA-B and III-mGlu receptors (localised in the presynaptic regions), the activation of GABA-B and III-mGlu receptors can explain the distinct therapeutic advantages of clozapine in schizophrenia or TRS associated with N-methyl-D-aspartate (NMDA) receptor disturbance compared with other atypical antipsychotics via the inhibition of the persistent tonic hyperactivation of thalamocortical glutamatergic transmission in the prefrontal cortex. L-BAIBA has also been identified as a gliotransmitter, and a detailed exploration of the function of L-BAIBA in tripartite synaptic transmission can further elucidate the pathophysiology of effectiveness for treating TRS and/or specific adverse reactions of clozapine.

Enhanced L-ß-Aminoisobutyric Acid Is Involved in the Pathophysiology of Effectiveness for Treatment-Resistant Schizophrenia and Adverse Reactions of Clozapine.

Clozapine is an effective antipsychotic for the treatment of antipsychotic-resistant schizophrenia; however, specific types of A/B adverse effects and clozapine-discontinuation syndromes are also well known. To date, both the critical mechanisms of clinical actions (effective for antipsychotic-resistant schizophrenia) and the adverse effects of clozapine remain to be elucidated. Recently, we demonstrated that clozapine increased the synthesis of L-ß-aminoisobutyric acid (L-BAIBA) in the hypothalamus. L-BAIBA is an activator of the adenosine monophosphate-activated protein kinase (AMPK), glycine receptor, GABAA receptor, and GABAB receptor (GABAB-R). These targets of L-BAIBA overlap as potential targets other than the monoamine receptors of clozapine. However, the direct binding of clozapine to these aminoacidic transmitter/modulator receptors remains to be clarified. Therefore, to explore the contribution of increased L-BAIBA on the clinical action of clozapine, this study determined the effects of clozapine and L-BAIBA on tripartite synaptic transmission, including GABAB-R and the group-III metabotropic glutamate receptor (III-mGluR) using cultured astrocytes, as well as on the thalamocortical hyper-glutamatergic transmission induced by impaired glutamate/NMDA receptors using microdialysis. Clozapine increased astroglial L-BAIBA synthesis in time/concentration-dependent manners. Increased L-BAIBA synthesis was observed until 3 days after clozapine discontinuation. Clozapine did not directly bind III-mGluR or GABAB-R, whereas L-BAIBA activated these receptors in the astrocytes. Local administration of MK801 into the reticular thalamic nucleus (RTN) increased L-glutamate release in the medial frontal cortex (mPFC) (MK801-evoked L-glutamate release). Local administration of L-BAIBA into the mPFC suppressed MK801-evoked L-glutamate release. These actions of L-BAIBA were inhibited by antagonists of III-mGluR and GABAB-R, similar to clozapine. These in vitro and in vivo analyses suggest that increased frontal L-BAIBA signaling likely plays an important role in the pharmacological actions of clozapine, such as improving the effectiveness of treating treatment-resistant schizophrenia and several clozapine discontinuation syndromes via the activation of III-mGluR and GABAB-R in the mPFC.

A Candidate Gliotransmitter, L-ß-Aminoisobutyrate, Contributes to Weight Gain and Metabolic Complication Induced by Atypical Antipsychotics.

Lurasidone and quetiapine are effective atypical mood-stabilizing antipsychotics, but lurasidone and quetiapine are listed as lower-risk and high-risk for weight gain/metabolic complications, respectively. The pathophysiology of the discrepancy of metabolic adverse reactions between these antipsychotics remains to be clarified. The GABA isomer, ß-aminoisobutyric acid (BAIBA) enantiomer, was recently re-discovered as myokine via an AMP-activated protein kinase activator (AMPK) enhancer and inhibitory gliotransmitter. Notably, activation of AMPK in peripheral organs improves, but in the hypothalamus, it aggravates metabolic disturbances. Therefore, we determined effects of chronic administration of lurasidone and quetiapine on intracellular and extracellular levels of the BAIBA enantiomer. L-BAIBA is a major BAIBA enantiomer in the hypothalamus and astrocytes, whereas L-BAIBA only accounted for about 5% of total plasma BAIBA enantiomers. Chronic lurasidone administration did not affect body weight but decreased the L-BAIBA level in hypothalamus and cultured astrocytes, whereas chronic quetiapine administration increased body weight and the L-BAIBA level in hypothalamus and astrocytes. Contrary, neither lurasidone nor quetiapine affected total plasma levels of the BAIBA enantiomer since D-BAIBA levels were not affected by these antipsychotics. These results suggest that activation of intracellular L-BAIBA signaling is, at least partially, involved in the pathophysiology of metabolic adverse reaction of quetiapine. Furthermore, this study also demonstrated that lurasidone and quetiapine suppressed and enhanced astroglial L-BAIBA release induced by ripple-burst stimulation (which physiologically contributes to cognitive memory integration during sleep), respectively. Therefore, L-BAIBA probably contributes to the pathophysiology of not only metabolic adverse reactions, but also a part of clinical action of lurasidone or quetiapine.

Opposing effects of clozapine and brexpiprazole on ß-aminoisobutyric acid: Pathophysiology of antipsychotics-induced weight gain.

Clozapine is one of the most effective antipsychotics and has the highest risk of weight gain and metabolic complications; however, the detailed pathophysiology of its clinical action and adverse reactions remains to be clarified. Therefore, the present study determined the chronic effects of clozapine (high risk of weight gain) and brexpiprazole (relatively low risk of weight gain) on intracellular and extracellular levels of ß-aminoisobutyric acid (BAIBA) enantiomers, which are endogenous activators of AMP-activated protein kinase (AMPK). L-BAIBA is the dominant BAIBA enantiomer in the rat hypothalamus and cultured astrocytes, whereas L-BAIBA accounts for only approximately 5% of the total plasma BAIBA enantiomers. L-BAIBA displayed GABAB receptor agonistic action in the extracellular space and was released through activated astroglial hemichannels, whereas in the intracellular space, L-BAIBA activated AMPK signalling. Chronic administration of the effective doses of clozapine increased intracellular and extracellular levels of L-BAIBA in the hypothalamus and cultured astrocytes, whereas that of brexpiprazole decreased them. These results suggest that enhancing hypothalamic AMPK signalling by increasing intracellular L-BAIBA levels is, at least partially, involved in the pathophysiology of clozapine-induced weight gain and metabolic complications.

Therapeutic Potential and Limitation of Serotonin Type 7 Receptor Modulation.

Although a number of mood-stabilising atypical antipsychotics and antidepressants modulate serotonin type 7 receptor (5-HT7), the detailed contributions of 5-HT7 function to clinical efficacy and pathophysiology have not been fully understood. The mood-stabilising antipsychotic agent, lurasidone, and the serotonin partial agonist reuptake inhibitor, vortioxetine, exhibit higher binding affinity to 5-HT7 than other conventional antipsychotics and antidepressants. To date, the initially expected rapid onset of antidepressant effects-in comparison with conventional antidepressants or mood-stabilising antipsychotics-due to 5-HT7 inhibition has not been observed with lurasidone and vortioxetine; however, several clinical studies suggest that 5-HT7 inhibition likely contributes to quality of life of patients with schizophrenia and mood disorders via the improvement of cognition. Furthermore, recent preclinical studies reported that 5-HT7 inhibition might mitigate antipsychotic-induced weight gain and metabolic complication by blocking other monoamine receptors. Further preclinical studies for the development of 5-HT7 modulation against neurodevelopmental disorders and neurodegenerative diseases have been ongoing. To date, various findings from various preclinical studies indicate the possibility that 5-HT7 modifications can provide two independent strategies. The first is that 5-HT7 inhibition ameliorates the dysfunction of inter-neuronal transmission in mature networks. The other is that activation of 5-HT7 can improve transmission dysfunction due to microstructure abnormality in the neurotransmission network-which could be unaffected by conventional therapeutic agents-via modulating intracellular signalling during the neurodevelopmental stage or via loss of neural networks with aging. This review attempts to describe the current and novel clinical applications of 5-HT7 modulation based on preclinical findings.

Dose-Dependent Biphasic Action of Quetiapine on AMPK Signalling via 5-HT7 Receptor: Exploring Pathophysiology of Clinical and Adverse Effects of Quetiapine.

Recent pharmacological studies indicated that the modulation of tripartite-synaptic transmission plays important roles in the pathophysiology of schizophrenia, mood disorders and adverse reactions. Therefore, to explore the mechanisms underlying the clinical and adverse reactions to atypical antipsychotics, the present study determined the effects of the sub-chronic administration of quetiapine (QTP: 3~30 µM) on the protein expression of 5-HT7 receptor (5-HT7R), connexin43 (Cx43), cAMP level and intracellular signalling, Akt, Erk and adenosine monophosphate-activated protein kinase (AMPK) in cultured astrocytes and the rat hypothalamus, using ultra-high-pressure liquid chromatography with mass spectrometry and capillary immunoblotting systems. QTP biphasically increased physiological ripple-burst evoked astroglial D-serine release in a concentration-dependent manner, peaking at 10 µM. QTP enhanced the astroglial signalling of Erk concentration-dependently, whereas both Akt and AMPK signalling's were biphasically enhanced by QTP, peaking at 10 µM and 3 µM, respectively. QTP downregulated astroglial 5-HT7R in the plasma membrane concentration-dependently. Protein expression of Cx43 in astroglial cytosol and intracellular cAMP levels were decreased and increased by QTP also biphasically, peaking at 3 µM. The dose-dependent effects of QTP on the protein expression of 5-HT7R and Cx43, AMPK signalling and intracellular cAMP levels in the hypothalamus were similar to those in astrocytes. These results suggest several complicated pharmacological features of QTP. A therapeutically relevant concentration/dose of QTP activates Akt, Erk and AMPK signalling, whereas a higher concentration/dose of QTP suppresses AMPK signalling via its low-affinity 5-HT7R inverse agonistic action. Therefore, 5-HT7R inverse agonistic action probably plays important roles in the prevention of a part of adverse reactions of QTP, such as weight gain and metabolic complications.

Brexpiprazole Reduces 5-HT7 Receptor Function on Astroglial Transmission Systems.

Several atypical antipsychotics exert mood-stabilising effects via the modulation of various monoamine receptors and intracellular signallings. Recent pharmacodynamic studies suggested that tripartite synaptic transmission can contribute to the pathophysiology of schizophrenia and mood disorders, their associated cognitive impairment, and several adverse reactions to atypical antipsychotics. Therefore, to explore the mechanisms underlying the antidepressive mood-stabilising and antipsychotic effects of brexpiprazole (Brex), we determined the effects of subchronic administration of therapeutically relevant concentrations/doses of Brex on the protein expression of 5-HT receptors, connexin43, cAMP levels, and intracellular signalling in cultured astrocytes and rat hypothalamus using ultra-high-pressure liquid chromatography with mass spectrometry and capillary immunoblotting systems. Subchronic administration of a therapeutically relevant concentration of Brex (300 nM) downregulated both 5-HT1A (5-HT1AR) and 5-HT7 (5-HT7R) receptors, in addition to phosphorylated Erk (pErk), without affecting phosphorylated Akt in the astroglial plasma membrane. Subchronic administration of 300 nM Brex decreased and increased phosphorylated AMPK and connexin43, respectively, in the astroglial cytosol fraction. A therapeutically relevant concentration of Brex acutely decreased the astroglial cAMP level, whereas, under the inhibition of 5-HT1AR, Brex did not affect astroglial cAMP levels. However, the 5-HT7R-agonist-induced increased astroglial cAMP level was inhibited by Brex. In contrast to the in vitro study, systemic subchronic administration of effective doses of Brex (3 and 10 mg/kg/day for 14 days) increased the cAMP level but did not affect phosphorylated AMPK in the rat hypothalamus. These results suggest several complicated pharmacological features of Brex. Partial 5-HT1AR agonistic action predominates in the low range of therapeutically relevant concentrations of Brex, whereas in the high range, 5-HT7R inverse agonist-like action is overlapped on the 5-HT1A agonistic action. These unique suppressive effects of Brex on 5-HT7R play important roles in the clinical features of Brex regarding its antidepressive mood-stabilising actions.

Brivaracetam and Levetiracetam Suppress Astroglial L-Glutamate Release through Hemichannel via Inhibition of Synaptic Vesicle Protein.

To explore the pathophysiological mechanisms of antiseizure and adverse behavioural/psychiatric effects of brivaracetam and levetiracetam, in the present study, we determined the effects of brivaracetam and levetiracetam on astroglial L-glutamate release induced by artificial high-frequency oscillation (HFO) bursts using ultra-high-performance liquid chromatography. Additionally, the effects of brivaracetam and levetiracetam on protein expressions of connexin43 (Cx43) and synaptic vesicle protein 2A (SV2A) in the plasma membrane of primary cultured rat astrocytes were determined using a capillary immunoblotting system. Acutely artificial fast-ripple HFO (500 Hz) burst stimulation use-dependently increased L-glutamate release through Cx43-containing hemichannels without affecting the expression of Cx43 or SV2A in the plasma membrane, whereas acute physiological ripple HFO (200 Hz) stimulation did not affect astroglial L-glutamate release or expression of Cx43 or SV2A. Contrarily, subchronic ripple HFO and acute pathological fast-ripple HFO (500 Hz) stimulations use-dependently increased L-glutamate release through Cx43-containing hemichannels and Cx43 expression in the plasma membrane. Subchronic fast-ripple HFO-evoked stimulation produced ectopic expression of SV2A in the plasma membrane, but subchronic ripple HFO stimulation did not generate ectopic SV2A. Subchronic administration of brivaracetam and levetiracetam concentration-dependently suppressed fast-ripple HFO-induced astroglial L-glutamate release and expression of Cx43 and SV2A in the plasma membrane. In contrast, subchronic ripple HFO-evoked stimulation induced astroglial L-glutamate release, and Cx43 expression in the plasma membrane was inhibited by subchronic levetiracetam administration, but was not affected by brivaracetam. These results suggest that brivaracetam and levetiracetam inhibit epileptogenic fast-ripple HFO-induced activated astroglial transmission associated with hemichannels. In contrast, the inhibitory effect of therapeutic-relevant concentrations of levetiracetam on physiological ripple HFO-induced astroglial responses probably contributes to the adverse behavioural/psychiatric effects of levetiracetam.

High frequency oscillations play important roles in development of epileptogenesis/ictogenesis via activation of astroglial signallings.

To explore developmental processes of epileptogenesis/ictogenesis and pathophysiology of carbamazepine-resistant epilepsy, we determined effects of high-frequency-oscillation (HFO) on glutamatergic tripartite-synaptic transmission, astroglial expression of connexin43, and intracellular Erk- and Akt-signalling, using genetic rat model (S286L-TG) of autosomal-dominant sleep-related hypermotor epilepsy(ADSHE), which bears rat S286L-mutant Chrna4(corresponding to human S284L-mutant CHRNA4). Artificial physiological ripple- and pathological fast-ripple-burst stimulations use-dependently increased L-glutamate release through connexin43-containing hemichannels by enhancing Erk-signalling alone or both ERK- and Akt-signalling together, respectively. Stimulatory effects of HFO-bursts on astroglial L-glutamate release were enhanced by increasing extracellular K+ levels, Akt- and Erk-signalling-dependently. HFO-bursts also activated connexin43 expression and Akt- and Erk-signallings use-dependently. Extracellular pH elevation enhanced HFO-burst-evoked astroglial L-glutamate release, which was suppressed by therapeutically-relevant concentration of zonisamide via possible carbonic-anhydrase inhibition, but not by that of carbamazepine. Unexpectedly, these responses of S286L-TG to HFO-bursts were almost equal to those of wild-type astrocytes. These results indicated that candidate pathomechanism/pathophysiology of carbamazepine-resistant ADSHE, which enhanced HFO-bursts in S286L-TG neurons may contribute to epileptogenesis/ictogenesis development via activation of connexin43-associated astroglial transmission, which was directly unaffected by mutation, and induced through activated Erk-signalling, followed by Akt-signalling. Therefore, suppression of overexpressed Erk-signalling probably prevents ADSHE onset via indirect inhibition of mutant CHRNA4-associated pathomechanistic developments.

Impact of 5-HT7 receptor inverse agonism of lurasidone on monoaminergic tripartite synaptic transmission and pathophysiology of lower risk of weight gain.

A part of atypical antipsychotics exert mood-stabilising effects via modulation of various monoamine receptors and intracellular signalling. Recent pharmacodynamic studies suggested that tripartite-synaptic transmission can be involved in pathophysiology of mood-disorders, schizophrenia, their associated cognitive impairments, and several adverse-reactions to atypical antipsychotics. Therefore, to explore mechanisms underlying antidepressive mood-stabilising and antipsychotic effects of lurasidone, we determined concentration-dependent effects of acute and subchronic lurasidone administrations on astroglial L-glutamate release, and expression of connexin43, ERK, AKT, adenosine monophosphate activated protein kinase (AMPK), 5-HT1A (5-HT1AR) and 5-HT7 (5-HT7R) receptors in cultured astrocytes using ultra-high-pressure liquid-chromatography with mass-spectrometry and capillary-immunoblotting systems. Therapeutically-relevant lurasidone concentration suppressed astroglial L-glutamate release through activated connexin43-containing hemichannel by decreasing connexin43 expression in plasma-membrane. Subchronic lurasidone administration downregulated 5-HT1AR and 5-HT7R in astroglial plasma-membrane concentration-dependently. Subchronic lurasidone administration attenuated ERK and AMPK signallings concentration-dependently without affecting AKT signalling. These results suggest that effects of subchronic lurasidone administration on astroglial L-glutamate release, 5-HT receptor, and intracellular signalling are similar to vortioxetine and different from mood-stabilising atypical antipsychotics, clozapine. Therefore, inhibitory effects of subchronic lurasidone administration on astroglial L-glutamate release through activated connexin43-containing hemichannel probably contribute to pathophysiology of antidepressive mood-stabilising effects of lurasidone. Furthermore, inhibitory effects of subchronic lurasidone administration on ERK and AMPK activities (without affecting AKT activity) induced by downregulation of 5-HT7R could result in clinical advantages of lurasidone, lower risk of weight gain.

Effects of an Atypical Antipsychotic, Zotepine, on Astroglial L-Glutamate Release through Hemichannels: Exploring the Mechanism of Mood-Stabilising Antipsychotic Actions and Antipsychotic-Induced Convulsion.

Accumulating neuropsychopharmacological evidence has suggested that functional abnormalities of astroglial transmission and protein kinase B (Akt) contribute to the pathophysiology and/or pathomechanisms of several neuropsychiatric disorders, such as epilepsy, schizophrenia, affective disorders and antipsychotic-induced convulsions. Therefore, to explore the pathophysiology of mood-stabilising antipsychotics and the proconvulsive actions of atypical antipsychotics, the present study determined the effects of a mood-stabilising, atypical, antipsychotic agent, zotepine (ZTP), on astroglial L-glutamate release and the expression of connexin43 (Cx43) protein in cortical, primary, cultured astrocytes using ultra-high-pressure liquid chromatography and capillary immunoblotting systems. Both acute and subchronic administrations of therapeutically relevant concentrations of ZTP did not affect astroglial L-glutamate release or Cx43 expression in plasma membranes; however, chronic administration of a therapeutically relevant concentration of ZTP increased astroglial L-glutamate release and Cx43 expression in the plasma membrane. Subchronic administrations of a supratherapeutic concentration of ZTP enhanced astroglial L-glutamate release and Cx43 expression in the plasma membrane, whereas acute administration of a supratherapeutic concentration of ZTP enhanced astroglial L-glutamate release without affecting Cx43 expression. These stimulatory effects of ZTP on astroglial L-glutamate release through activated hemichannels and Cx43 trafficking to the astroglial plasma membrane were suppressed by the Akt inhibitor. These results suggest that ZTP enhances astroglial L-glutamate release in a concentration-dependent and time-dependent manner due to the enhanced function of astroglial hemichannels, probably via activation of Akt signalling. Therefore, the enhanced astroglial L-glutamatergic transmission induced by ZTP is, at least partially, involved in the mood-stabilising antipsychotic and proconvulsive actions of ZTP.

Determining What Changed Japanese Suicide Mortality in 2020 Using Governmental Database.

The pandemic of 2019 novel coronavirus disease (COVID-19) caused both COVID-19-related health hazards and the deterioration of socioeconomic and sociopsychological status due to governmental restrictions. There were concerns that suicide mortality would increase during the COVID-19 pandemic; however, a recent study reported that suicide mortality did not increase in 21 countries during the early pandemic period. In Japan, suicide mortality was reduced from 2009 to 2019, but both the annual number of suicide victims and the national suicide mortality rates in 2020 increased compared to that in 2019. To clarify the discrepancy of suicide mortality between the first and second half of 2020 in Japan, the present study determines annual and monthly suicide mortality disaggregated by prefectures, gender, age, means, motive, and household factors during the COVID-19 pandemic and pre-pandemic periods using a linear mixed-effects model. Furthermore, the relationship between suicide mortality and COVID-19 data (the infection rate, mortality, and duration of the pandemic) was analysed using hierarchal linear regression with a robust standard error. The average of monthly suicide mortality of both males and females in all 47 prefectures decreased during the first stay-home order (April-May) (females: from 10.1-10.2 to 7.8-7.9; males: from 24.0-24.9 to 21.6 per 100,000 people), but increased after the end of the first stay-home order (July-December) (females: from 7.5-9.5 to 10.3-14.5; males: from 19.9-23.0 to 21.1-26.7 per 100,000 people). Increasing COVID-19-infected patients and victims indicated a tendency of suppression, but the prolongation of the pandemic indicated a tendency of increasing female suicide mortality without affecting that of males. Contrary to the national pattern, in metropolitan regions, decreasing suicide mortality during the first stay-home order was not observed. Decreasing suicide mortality during the first stay-home order was not observed in populations younger than 30 years old, whereas increasing suicide mortality of populations younger than 30 years old after the end of the first stay-home order was predominant. A decrease in suicide mortality of one-person household residents during the first stay-home order was not observed. The hanging suicide mortality of males and females was decreased and increased during and after the end of the first stay-home orders, respectively; however, there was no decrease in metropolitan regions. These results suggest that the suicide mortality in 2020 of females, younger populations, urban residents, and one-person household residents increased compared to those of males, the elderly, rural residents, and multiple-person household residents. Therefore, the unexpected drastic fluctuations of suicide mortality during the COVID-19 pandemic in Japan were probably composed of complicated reasons among various identified factors in this study, and other unknown factors.

Distinct Effects of Escitalopram and Vortioxetine on Astroglial L-Glutamate Release Associated with Connexin43.

It has been established that enhancement of serotonergic transmission contributes to improvement of major depression; however, several post-mortem studies and experimental depression rodent models suggest that functional abnormalities of astrocytes play important roles in the pathomechanisms/pathophysiology of mood disorders. Direct effects of serotonin (5-HT) transporter inhibiting antidepressants on astroglial transmission systems has never been assessed in this context. Therefore, to explore the effects of antidepressants on transmission associated with astrocytes, the present study determined the effects of the selective 5-HT transporter inhibitor, escitalopram, and the 5-HT partial agonist reuptake inhibitor, vortioxetine, on astroglial L-glutamate release through activated hemichannels, and the expression of connexin43 (Cx43), type 1A (5-HT1AR) and type 7 (5-HT7R) 5-HT receptor subtypes, and extracellular signal-regulated kinase (ERK) in astrocytes using primary cultured rat cortical astrocytes in a 5-HT-free environment. Both escitalopram and 5-HT1AR antagonist (WAY100635) did not affect basal astroglial L-glutamate release or L-glutamate release through activated hemichannels. Subchronic (for seven days) administrations of vortioxetine and the 5-HT7R inverse agonist (SB269970) suppressed both basal L-glutamate release and L-glutamate release through activated hemichannels, whereas 5-HT1AR agonist (BP554) inhibited L-glutamate release through activated hemichannels, but did not affect basal L-glutamate release. In particular, WAY100635 did not affect the inhibitory effects of vortioxetine on L-glutamate release. Subchronic administration of vortioxetine, BP554 and SB269970 downregulated 5-HT1AR, 5-HT7R and phosphorylated ERK in the plasma membrane fraction, but escitalopram and WAY100635 did not affect them. Subchronic administration of SB269970 decreased Cx43 expression in the plasma membrane but did not affect the cytosol; however, subchronic administration of BP554 increased Cx43 expression in the cytosol but did not affect the plasma membrane. Subchronic vortioxetine administration increased Cx43 expression in the cytosol and decreased it in the plasma membrane. WAY100635 prevented an increased Cx43 expression in the cytosol induced by vortioxetine without affecting the reduced Cx43 expression in the plasma membrane. These results suggest that 5-HT1AR downregulation probably increases Cx43 synthesis, but 5-HT7R downregulation suppresses Cx43 trafficking to the plasma membrane. These results also suggest that the subchronic administration of therapeutic-relevant concentrations of vortioxetine inhibits both astroglial L-glutamate and Cx43 expression in the plasma membrane via 5-HT7R downregulation but enhances Cx43 synthesis in the cytosol via 5-HT1AR downregulation. This combination of the downregulation of 5-HT1AR, 5-HT7R and Cx43 in the astroglial plasma membrane induced by subchronic vortioxetine administration suggest that astrocytes is possibly involved in the pathophysiology of depression.

Effects of Financial Expenditure of Prefectures/Municipalities on Regional Suicide Mortality in Japan.

In Japan, suicide mortality has been improving from 2009; however, suicide remains one of the leading causes of death. Although previous studies identified solid relationships between governmental financial support for social welfare systems and suicide mortality, little attention is paid to how specific regional policies, designed according to regional cultural, economic, and social welfare situations, affect suicide mortality. Therefore, the present study analyses the relationships between the regional governmental expenditure of six major divisions and suicide mortality across the 47 prefectures in Japan from 2009 to 2018 using fixed-effect analysis of hierarchal linear regression with robust standard error. The expenditure in "public health", "police", "ambulance/fire services", "welfare" and "education" is associated with reduction in suicide mortality, at least in some statistical indicators, whereas expenditure of "public works" indicated the influence of increasing suicide mortality or had no effect. Welfare expenditure was the most predominantly effective among the six major divisions of regional governmental expenditure. In the welfare subdivisions, expenditure of "child welfare" and "social welfare" was effective in a reduction in suicide mortality, but expenditure of "elderly welfare" surprisingly contributed to increasing suicide mortality. Child welfare expenditure negatively impacted suicide mortality in wide-ranging generations of both males and females; the positive effects of elderly welfare expenditure reached were limited as working-age populations increased, but unexpectedly did not affect the suicide mortality of elderly populations. The relatively increasing expenditure of elderly welfare with the relatively decreasing child welfare are unavoidable due to the Japanese social issues associated with a declining birth rate and ageing population. Furthermore, the budget of that regional government that can modify its expenditure structure by making its own policies is limited since most regional governmental expenditure is composed of essential expenditure for maintaining and operating regional social welfare systems. Although severe social situations in Japan are still unoptimised, the present results suggest that scientific-evidence-based redistributions of welfare expenditure in regional governments can at least partially improve Japanese society and welfare systems.

Effects of Atypical Antipsychotics, Clozapine, Quetiapine and Brexpiprazole on Astroglial Transmission Associated with Connexin43.

Recently, accumulating preclinical findings suggest the possibility that functional abnormalities of tripartite synaptic transmission play important roles in the pathophysiology of schizophrenia and affective disorder. Therefore, to explore the novel mechanisms of mood-stabilizing effects associated with tripartite synaptic transmission, the present study determined the effects of mood-stabilizing antipsychotics, clozapine (CLZ), quetiapine (QTP) and brexpiprazole (BPZ), on the astroglial l-glutamate release and expression of connexin43 (Cx43) in the astroglial plasma membrane using cortical primary cultured astrocytes. Neither acute (for 120 min) nor subchronic (for 7 days) administrations of CLZ, QTP and BPZ affected basal astroglial l-glutamate release, whereas both acute and subchronic administration of CLZ, QTP and BPZ concentration-dependently enhanced astroglial l-glutamate release through activated hemichannels. Subchronic administration of therapeutic-relevant concentration of valproate (VPA), a histone deacetylase inhibiting mood-stabilizing antiepileptic drug, enhanced the stimulatory effects of therapeutic-relevant concentration of CLZ, QTP and BPZ on astroglial l-glutamate release through activated hemichannel. Subchronic administration of therapeutic-relevant concentration of CLZ, QTP and BPZ did not affect Cx43 protein expression in the plasma membrane during resting stage. After subchronic administration of VPA, acute and subchronic administration of therapeutic-relevant concentrations of CLZ increased Cx43 protein expression in the plasma membrane. Both acute administrations of therapeutic-relevant concentrations of QTP and BPZ did not affect, but subchronic administrations enhanced Cx43 protein expression in the astroglial plasma membrane. Furthermore, protein kinase B (Akt) inhibitor suppressed the stimulatory effects of CLZ and QTP, but did not affect Cx43 protein expression in the astroglial plasma membrane. These results suggest that three mood-stabilizing atypical antipsychotics, CLZ, QTP and BPZ enhance tripartite synaptic glutamatergic transmission due to enhancement of astroglial Cx43 containing hemichannel activities; however, the Cx43 activating mechanisms of these three mood-stabilizing antipsychotics were not identical. The enhanced astroglial glutamatergic transmission induced by CLZ, QTP and BPZ is, at least partially, involved in the actions of these three mood-stabilizing antipsychotics.

Chronic Administrations of Guanfacine on Mesocortical Catecholaminergic and Thalamocortical Glutamatergic Transmissions.

It has been established that the selective α2A adrenoceptor agonist guanfacine reduces hyperactivity and improves cognitive impairment in patients with attention-deficit/hyperactivity disorder (ADHD). The major mechanisms of guanfacine are considered to involve the activation of the postsynaptic α2A adrenoceptor of glutamatergic pyramidal neurons in the frontal cortex, but the effects of chronic guanfacine administration on catecholaminergic and glutamatergic transmissions associated with the orbitofrontal cortex (OFC) are yet to be clarified. The actions of guanfacine on catecholaminergic transmission, the effects of acutely local and systemically chronic (for 7 days) administrations of guanfacine on catecholamine release in pathways from the locus coeruleus (LC) to OFC, the ventral tegmental area (VTA) and reticular thalamic-nucleus (RTN), from VTA to OFC, from RTN to the mediodorsal thalamic-nucleus (MDTN), and from MDTN to OFC were determined using multi-probe microdialysis with ultra-high performance liquid chromatography. Additionally, the effects of chronic guanfacine administration on the expression of the α2A adrenoceptor in the plasma membrane fraction of OFC, VTA and LC were examined using a capillary immunoblotting system. The acute local administration of therapeutically relevant concentrations of guanfacine into the LC decreased norepinephrine release in the OFC, VTA and RTN without affecting dopamine release in the OFC. Systemically, chronic administration of therapeutically relevant doses of guanfacine for 14 days increased the basal release of norepinephrine in the OFC, VTA, RTN, and dopamine release in the OFC via the downregulation of the α2A adrenoceptor in the LC, OFC and VTA. Furthermore, systemically, chronic guanfacine administration did not affect intrathalamic GABAergic transmission, but it phasically enhanced thalamocortical glutamatergic transmission. The present study demonstrated the dual actions of guanfacine on catecholaminergic transmission-acute attenuation of noradrenergic transmission and chronic enhancement of noradrenergic transmission and thalamocortical glutamatergic transmission. These dual actions of guanfacine probably contribute to the clinical effects of guanfacine against ADHD.

Current Limitations and Candidate Potential of 5-HT7 Receptor Antagonism in Psychiatric Pharmacotherapy.

Several mood-stabilizing atypical antipsychotics and antidepressants weakly block serotonin (5-HT) receptor type-7 (5-HT7R); however, the contributions of 5-HT7R antagonism to clinical efficacy and pathophysiology are yet to be clarified. A novel mood-stabilizing antipsychotic agent, lurasidone exhibits predominant binding affinity to 5-HT7R when compared with other monoamine receptors. To date, we have failed to discover the superior clinical efficacy of lurasidone on schizophrenia, mood, or anxiety disorders when compared with conventional mood-stabilizing atypical antipsychotics; however, numerous preclinical findings have indicated the possible potential of 5-HT7R antagonism against several neuropsychiatric disorders, as well as the generation of novel therapeutic options that could not be expected with conventional atypical antipsychotics. Traditional experimental techniques, electrophysiology, and microdialysis have demonstrated that the effects of 5-HT receptor type-1A (5-HT1AR) and 5-HT7R on neurotransmission are in contrast, but the effect of 5-HT1AR is more predominant than that of 5-HT7R, resulting in an insufficient understanding of the 5-HT7R function in the field of psychopharmacology. Accumulating knowledge regarding the pharmacodynamic profiles of 5-HT7R suggests that 5-HT7R is one of the key players in the establishment and remodeling of neural development and cytoarchitecture during the early developmental stage to the mature brain, and dysfunction or modulation of 5-HT7R is linked to the pathogenesis/pathophysiology of neuropsychiatric and neurodevelopmental disorders. In this review, to explore candidate novel applications for the treatment of several neuropsychiatric disorders, including mood disorders, schizophrenia, and other cognitive disturbance disorders, we discuss perspectives of psychopharmacology regarding the effects of 5-HT7R antagonism on transmission and intracellular signaling systems, based on preclinical findings.