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K channels in the membrane of murine pancreatic β-cells were studied using the patch-clamp technique. The delayed outward current was activated in whole-cell experiments by depolarizing voltage pulses to potentials between −30 mV and 0 mV. Forskolin blocked the current rapidly (<5 s) and reversibly with 50% inhibition at 13 μM. The inhibition did not depend on a stimulation of the adenylate cyclase since it occurred even in presence of 1 mM cAMP in the pipette solution which replaced the cytoplasm. Membrane permeant cAMP analogues and phosphodiesterase inhibitors did not influence the delayed outward current. In experiments on outside-out patches forskolin (100 μM) shortened the openings of a channel of about 10 pS conductance at 0 mV and a time course of activation and inactivation similar to the whole-cell current. Another smaller, slowly activating channel and the Ca- and ATP-dependent K channels were influenced only weakly or not at all. It is therefore concluded that the 10-pS channel generates most of the delayed outward K current in murine pancreatic β-cells. The Ca-independent part of the delayed outward current in bovine adrenal chromaffin cells was also blocked by forskolin (100 μM). [ABSTRACT FROM AUTHOR]

The oscillation of membrane potential in fibroblastic L cells is known to result from periodic stimulation of Ca2+-activated K+ channels due to the oscillatory increase in the intracellular Ca2+ concentration. These repeated hyperpolarizations were inhibited by putative calmodulin antagonists, trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and promethazine (PMZ), and the concentrations required for half-maximal inhibition were 25, 30 and 300 microM, respectively. These doses were lower than those for reducing the membrane resistance due to nonspecific cell damages. Another calmodulin antagonist, chlorpromazine (CPZ), was also effective, but CPZ-sulfoxide was not. Intracellular pressure injections of calmodulin-interacting divalent cations, Ca2+, Sr2+, Mn2+ and Ni2+, elicited slow hyperpolarizations, whereas Mg2+ and Ba2+, which are known to be essentially inert for calmodulin, failed to evoke any responses. The injection of purified calmodulin also brought about a similar hyperpolarization. Quinine, an inhibitor of Ca2+-activated K+ channels, abolished both Ca2+- and calmodulin-induced hyperpolarizations. TFP prevented Ca2+-induced hyperpolarizations. The TFP effect was partially reversed by the calmodulin injection. It is concluded that calmodulin is involved in the operation of Ca2+-activated K+ channels in fibroblasts. [ABSTRACT FROM AUTHOR]

Mouse leukemic lymphoblasts (L5178Y) brought into close contact by dielectrophoresis underwent cell fusion following the application of electrical pulses in the presence of electrolytes. The electrically fused cells became spherical after switching off the dielectrophoretic field. Fusion between a cell vitally stained with Janus Green and that with Neutral Red resulted in the homokaryon with a mixed color. Intracellular potentials simultaneously recorded from the two cells located on both sides of the homokaryon were identical. The fusion efficiency was remarkably dependent upon temperature, displaying a discontinuity at about 11 degrees C in the Arrhenius plot. The extracellular application of phospholipase-A2 or -C suppressed the fusion yield. Thus, it appears that the phospholipid domains play a crucial role in the electric pulse-induced cell fusion. Treatment of the cells with proteolytic enzymes markedly enhanced the fusion yield, presumably due to removing the glycocalix and/or giving rise to fusion-potent, protein-free lipid domains. The presence of millimolar concentrations of divalent cations (irrespective of Mg2+ or Ca2+) as well as of micromolar concentrations of Ca2+ (but not Mg2+) was prerequisite to the resealing of membranes suffered from electrical breakdown upon exposure to electric pulses. In addition, extracellular Ca2+ (but not Mg2+) ions at more than micromolar concentrations were indispensable for the cell fusion. [ABSTRACT FROM AUTHOR]

The thalamic reticular nucleus (TRN) is a thin sheet of GABAergic neurons surrounding the thalamus, and it regulates the activity of thalamic relay neurons. The TRN has been reported to be involved in sensory gating, attentional regulation, and some other functions. However, little is known about the contribution of the TRN to sequence learning. In the present study, we examined whether the TRN is involved in reward-based learning of action sequence with no eliciting stimuli (operant conditioning), by analyzing the performance of male and female Avp-Vgat -/- mice (Vgat flox/flox mice crossed to an Avp-Cre driver line) on tasks conducted in an operant box having three levers. Our histological and electrophysiological data demonstrated that in adult Avp-Vgat -/- mice, vesicular GABA transporter (VGAT) was absent in most TRN neurons and the GABAergic transmission from the TRN to the thalamus was largely suppressed. The performance on a task in which mice needed to press an active lever for food reward showed that simple operant learning of lever pressing and learning of win-stay and lose-shift strategies are not affected in Avp-Vgat -/- mice. In contrast, the performance on a task in which mice needed to press three levers in a correct order for food reward showed that learning of the order of lever pressing (action sequence learning) was impaired in Avp-Vgat -/- mice. These results suggest that the TRN plays an important role in action sequence learning., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Neural circuits connecting the cerebellum with the cerebral cortex are important for both motor and cognitive functions. Therefore, assessment of cerebellar function is clinically important for patients with various motor and cognitive dysfunctions. Cerebellum-dependent motor learning has been studied using various tasks. The most widely used tasks are visuomotor adaptation tasks, in which subjects are required to make movements in two dimensions. Studies using simpler tasks of one-dimensional movement, which are easier for patients with motor problems to perform, have suggested that anticipatory responses in these tasks are useful to evaluate cerebellum-dependent motor control or motor learning. In this study, we examined whether the motor learning process can be evaluated in a simple loading task. Using space interface device for artificial reality (SPIDAR), a constant downward force was loaded to subjects' hands in a predictable condition, and the vertical movement of the hand was recorded. The hand deflection from the initial position was displayed on a screen for visual feedback information. We examined effects of repeated loading task training (90 times) on hand movements, by analyzing a small upward movement just before loading (anticipatory response) and a large downward movement after loading in each trial. We found that the repeated training lowered the time constant of upward movement and reduced the amplitude and time-to-peak of downward movement. These training effects were maintained into the next day. Furthermore, we found that loading task training with eyes closed was also effective, which indicates that proprioceptive information is enough for improvement of performance., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Beneficial effects of environmental enrichment (EE) on the central nervous system have been demonstrated. Although the effects of EE on spatial learning have been extensively studied, studies on reward-based motor learning are limited. In this study we examined the effects of EE on the performance of operant tasks using three levers (A-C). Mice were divided into two groups and housed either in the control condition or in the physical EE condition. The mice were trained in three types of operant tasks in sequence. First, mice were trained to press one of the active levers for a food reward (one-lever task). Second, mice were trained to press the three levers in the order of A, B, and C (three-lever task). Third, the lever order was reversed to C, B, and A (reverse three-lever task). We found some behavioral differences between control and EE mice. When all three levers were active in the one-lever task, mice tended to press the three levers equally at first, then shifted to press one lever preferentially. This behavioral shift from exploration to exploitation was delayed in EE mice. When only one lever was active, EE mice showed a higher lose-shift performance. In the three-lever and reverse three-lever tasks, EE mice pressed three levers more often and acquired more food rewards, compared to control mice, although the success rate in both tasks was not different between the two groups. These behavioral features observed in EE mice (higher lose-shift performance and higher trial and error activity) might be advantageous when circumstances are not stable., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Plastic changes of skeletal muscles, such as hypertrophy and atrophy, are dependent on physiological activities and regulated by a variety of signaling pathways, including cyclic adenosine monophosphate (cAMP) pathway. The cAMP inducing agents, such as the β2-adrenergic agonist clenbuterol, are known to induce muscle hypertrophy, and has been reported to induce slow-to-fast transitions in rat soleus muscle. Theobromine, one of the active components of cacao, functions as an inhibitor of phosphodiesterase and increases cAMP. This study hypothesized that theobromine, like clenbuterol, can induce muscle hypertrophy and influence contractile properties., Methods and Results: Male Wistar rats were fed a normal diet or a diet containing 0.05% theobromine for 20 weeks. Using biochemical, anatomical, and physiological techniques, effects of dietary theobromine on skeletal muscles (soleus, extensor digitorum longus, plantaris, and gastrocnemius) were examined. There were no significant differences in body weight, serum levels of proteins and lipids, muscle weights, dry/wet ratio of muscle weights, mitochondrial oxidation enzyme activity of muscles, isometric contractile properties of muscles, and muscle fatigue between control and theobromine-fed rats. Quantitative analysis of mRNA, however, revealed upregulation of myosin heavy chain 2x and myogenic differentiation 1, as previously reported in clenbuterol-treated muscles., Conclusion: The long-term theobromine (0.05%) diet in rats had no effect in inducing muscle hypertrophy and in changing contractile properties, although it had some similar effects of clenbuterol on muscle gene expression., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The endocannabinoid system modulates synaptic transmission, controls neuronal excitability, and is involved in various brain functions including learning and memory. 2-arachidonoylglycerol, a major endocannabinoid produced by diacylglycerol lipase-α (DGLα), is released from postsynaptic neurons, retrogradely activates presynaptic CB 1 cannabinoid receptors, and induces short-term or long-term synaptic plasticity. To examine whether and how the endocannabinoid system contributes to reward-based learning of a motor sequence, we subjected male CB 1 -knockout (KO) and DGLα-KO mice to three types of operant lever-press tasks. First, we trained mice to press one of three levers labeled A, B, and C for a food reward (one-lever task). Second, we trained mice to press the three levers in the order of A, B, and C (three-lever task). Third, the order of the levers was reversed to C, B, and A (reverse three-lever task). We found that CB 1 -KO mice and DGLα-KO mice exhibited essentially the same deficits in the operant lever-press tasks. In the one-lever task, both strains of knockout mice showed a slower rate of learning to press a lever for food. In the three-lever task, both strains of knockout mice showed a slower rate of learning of the motor sequence. In the reverse three-lever task, both strains of knockout mice needed more lever presses for reversal learning. These results suggest that the endocannabinoid system facilitates reward-based learning of a motor sequence by conferring the flexibility with which animals can switch between strategies., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

The endocannabinoid system regulates physiological and pathological conditions, including inflammation and cancer. Recently, emotional and physical stressors were observed to be involved in impairing the endocannabinoid system, which was concomitant with an increase in serum corticosteroids. However, the influence of corticosteroids on the endocannabinoid system has yet to be completely elucidated. The present study investigated the effects of corticosterone, one of the corticosteroids, on the endocannabinoid system in malignant glioblastoma cells in vitro . U-87 MG cells derived from malignant glioblastoma were subjected to corticosterone stimulation and their viability, signal transduction, and endocannabinoid-related gene expression were examined. Corticosterone decreased the mRNA and protein expressions of cyclooxygenase-2. Of note, although endocannabinoids decreased cell viability, corticosterone inhibited the cannabinoid receptor agonist-induced decrease in cell viability by downregulating the mRNA and protein expressions of cannabinoid receptor 1 (CB1) in glioblastoma cells. These results suggest that corticosteroids modify the endocannabinoid system in glioblastoma cells, and a reduction in the beneficial anti-tumor effects of endocannabinoids through downregulation of the CB1 receptor by corticosterone may promote the malignant phenotype of glioblastoma.

The endocannabinoid system plays an important role in the regulation of physiological and pathological conditions, including inflammation and cancer. Hypoxia is a fundamental phenomenon for the establishment and maintenance of the microenvironments in various physiological and pathological conditions. However, the influence of hypoxia on the endocannabinoid system is not fully understood. In the present study, we investigated the effects of hypoxia on the endocannabinoid system in malignant brain tumors. We subjected U-87 MG cells, derived from malignant glioblastoma, to hypoxia (1.5% O2) for 3 days, and evaluated their viability and expression of endocannabinoid-related genes. Hypoxia decreased the expression of cannabinoid receptor 1 and the astrocyte marker glial fibrillary acidic protein, and increased the expression of vascular endothelial growth factor and cyclooxygenase-2, the enzyme responsible for the metabolism of endocannabinoids, in U-87 MG cells. Although cannabinoid receptor (CB) engagement induces cell death in U-87 MG cells in normoxic conditions, CB agonist-induced death was attenuated in hypoxic conditions. These results suggest that hypoxia modifies the endocannabinoid system in glioblastoma cells. Hypoxia-induced inhibition of the endocannabinoid system may aid the development of glioblastoma.

Theobromine, which is a caffeine derivative, is the primary methylxanthine produced by Theobroma cacao. Theobromine works as a phosphodiesterase (PDE) inhibitor to increase intracellular cyclic adenosine monophosphate (cAMP). cAMP activates the cAMP-response element-binding protein (CREB), which is involved in a large variety of brain processes, including the induction of the brain-derived neurotrophic factor (BDNF). BDNF supports cell survival and neuronal functions, including learning and memory. Thus, cAMP/CREB/BDNF pathways play an important role in learning and memory. Here, we investigated whether orally administered theobromine could act as a PDE inhibitor centrally and affect cAMP/CREB/BDNF pathways and learning behavior in mice. The mice were divided into two groups. The control group (CN) was fed a normal diet, whereas the theobromine group (TB) was fed a diet supplemented with 0.05% theobromine for 30 days. We measured the levels of theobromine, phosphorylated vasodilator-stimulated phosphoprotein (p-VASP), phosphorylated CREB (p-CREB), and BDNF in the brain. p-VASP was used as an index of cAMP increases. Moreover, we analyzed the performance of the mice on a three-lever motor learning task. Theobromine was detectable in the brains of TB mice. The brain levels of p-VASP, p-CREB, and BDNF were higher in the TB mice compared with those in the CN mice. In addition, the TB mice performed better on the three-lever task than the CN mice did. These results strongly suggested that orally administered theobromine acted as a PDE inhibitor in the brain, and it augmented the cAMP/CREB/BDNF pathways and motor learning in mice., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The atypical antipsychotic clozapine is widely used for treatment-resistant schizophrenic patients. Clozapine and its major active metabolite, N-desmethylclozapine (NDMC), have complex pharmacological properties, and interact with various neurotransmitter receptors. There are several biochemical studies reporting that NDMC exhibits a partial agonist profile at the human recombinant M1 muscarinic receptors. However, direct electrophysiological evidence showing the ability of NDMC to activate native M1 receptors in intact neurons is poor. Using rat hippocampal neurons, we previously demonstrated that activation of muscarinic receptors by a muscarinic agonist, oxotremorine M (oxo-M), induces a decrease in outward K(+)current at -40mV. In the present study, using this muscarinic current response we assessed agonist and antagonist activities of clozapine and NDMC at native muscarinic receptors in intact hippocampal excitatory and inhibitory neurons. Suppression of the oxo-M-induced current response by the M1 antagonist pirenzepine was evident only in excitatory neurons, while the M3 antagonist darifenacin was effective in both types of neurons. Muscarinic agonist activity of NDMC was higher than that of clozapine, higher in excitatory neurons than in inhibitory neurons, sensitive to pirenzepine, and partially masked when co-applied with clozapine. Muscarinic antagonist activity of clozapine as well as NDMC was not different between excitatory and inhibitory neurons, but clozapine was more effective than NDMC. These results demonstrate that NDMC has the ability to activate native M1 receptors expressed in hippocampal excitatory neurons, but its agonist activity might be limited in clozapine-treated patients because of the presence of excessive clozapine with muscarinic antagonist activity., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Endocannabinoids are lipid-derived messengers, and both their synthesis and breakdown are under tight spatiotemporal regulation. As retrograde signalling molecules, endocannabinoids are synthesized postsynaptically but activate presynaptic cannabinoid receptor 1 (CB1) receptors to inhibit neurotransmitter release. In turn, CB1-expressing inhibitory and excitatory synapses act as strategically placed control points for activity-dependent regulation of dynamically changing normal and pathological oscillatory network activity. Here, we highlight emerging principles of cannabinoid circuit control and plasticity, and discuss their relevance for epilepsy and related comorbidities. New insights into cannabinoid signalling may facilitate the translation of the recent interest in cannabis-related substances as antiseizure medications to evidence-based treatment strategies.

One of the two major endocannabinoids, 2-arachidonoylglycerol (2-AG), serves as a retrograde messenger at various types of synapses throughout the brain. Upon postsynaptic activation, 2-AG is released immediately after de novo synthesis, activates presynaptic CB1 cannabinoid receptors, and transiently suppresses neurotransmitter release. When CB1 receptor activation is combined with some other factors such as presynaptic activity, the suppression is converted to a long-lasting form. Whereas 2-AG primarily transmits a rapid, transient, point-to-point retrograde signal, the other major endocannabinoid, anandamide, may function as a relatively slow retrograde or non-retrograde signal or as an agonist of the vanilloid receptor. The endocannabinoid system can be up- or down-regulated by a variety of physiological and environmental factors including stress, which might be clinically important., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) produced by diacylglycerol lipase α (DGLα) is one of the best-characterized retrograde messengers at central synapses. It has been thought that 2-AG is produced 'on demand' upon activation of postsynaptic neurons. However, recent studies propose that 2-AG is pre-synthesized by DGLα and stored in neurons, and that 2-AG is released from such 'pre-formed pools' without the participation of DGLα. To address whether the 2-AG source for retrograde signalling is the on-demand biosynthesis by DGLα or the mobilization from pre-formed pools, we examined the effects of acute pharmacological inhibition of DGL by a novel potent DGL inhibitor, OMDM-188, on retrograde eCB signalling triggered by Ca(2+) elevation, Gq/11 protein-coupled receptor activation or synergy of these two stimuli in postsynaptic neurons. We found that pretreatment for 1 h with OMDM-188 effectively blocked depolarization-induced suppression of inhibition (DSI), a purely Ca(2+)-dependent form of eCB signalling, in slices from the hippocampus, striatum and cerebellum. We also found that at parallel fibre-Purkinje cell synapses in the cerebellum OMDM-188 abolished synaptically induced retrograde eCB signalling, which is known to be caused by the synergy of postsynaptic Ca(2+) elevation and group I metabotropic glutamate receptor (I-mGluR) activation. Moreover, brief OMDM-188 treatments for several minutes were sufficient to suppress both DSI and the I-mGluR-induced retrograde eCB signalling in cultured hippocampal neurons. These results are consistent with the hypothesis that 2-AG for synaptic retrograde signalling is supplied as a result of on-demand biosynthesis by DGLα rather than mobilization from presumptive pre-formed pools.

Signaling pathways involving phospholipase C (PLC) are involved in various neural functions. Understanding how these pathways are regulated will lead to a better understanding of their roles in neural functions. Previous studies demonstrated that receptor-driven PLCβ activation depends on intracellular Ca(2+) concentration ([Ca(2+)]i), suggesting the possibility that PLCβ-dependent cellular responses are basically Ca(2+) dependent. To test this possibility, we examined whether modulations of ion channels driven by PLC-coupled metabotropic receptors are sensitive to [Ca(2+)]i using cultured hippocampal neurons. Muscarinic activation triggered an inward current at -100 mV (the equilibrium potential for K(+)) in a subpopulation of neurons. This current response was suppressed by pirenzepine (an M1-preferring antagonist), PLC inhibitor, non-selective cation channel blocker, and lowering [Ca(2+)]i. Using the neurons showing no response at -100 mV, effects of muscarinic activation on K(+) channels were examined at -40 mV. Muscarinic activation induced a transient decrease of the holding outward current. This current response was mimicked and occluded by XE991, an M-current K(+) channel blocker, suppressed by pirenzepine, PLC inhibitor and lowering [Ca(2+)]i, and enhanced by elevating [Ca(2+)]i. Similar results were obtained when group I metabotropic glutamate receptors were activated instead of muscarinic receptors. These results clearly show that ion channel modulations driven by PLC-coupled metabotropic receptors are dependent on [Ca(2+)]i, supporting the hypothesis that cellular responses induced by receptor-driven PLCβ activation are basically Ca(2+) dependent., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Nuclear factor (NF)-κB is the key transcription factor involved in the inflammatory responses, and its activation aggravates tumors. Peptidoglycan (PGN), a main cell wall component of Gram-positive bacteria, stimulates Toll-like receptor 2 (TLR-2) and activates a number of inflammatory pathways, including NF-κB. Cannabinoids have been reported to exert anti-inflammatory and antitumor effects. The mechanisms underlying these actions, however, are largely unknown. The purpose of this study was to investigate whether cannabinoids can suppress the PGN-induced activation of NF-κB and cell growth via cannabinoid receptors in U87MG human malignant glioma cells. PGN treatment induced the phosphorylation of NF-κB and cell proliferation in a concentration-dependent manner. The main endocannabinoid, 2-arachidonoylglycerol, prevented the PGN-induced phosphorylation of NF-κB, which was reversed by the CB1 cannabinoid receptor antagonist, AM281. The synthetic cannabinoid, WIN55,212-2, abolished the PGN-activated cell growth, and this effect was reversed by AM281. The preferential expression of CB1 rather than CB2 receptors in these cells was confirmed by reverse transcription-mediated polymerase chain reaction experiments and the observation that the WIN55,212-2-induced morphological changes were completely reversed by AM281 but not by the CB2 antagonist, AM630. Our finding that cannabinoids suppress the NF-κB inflammatory pathway and cell growth via CB1 receptors in glioma cells provides evidence for the therapeutic potential of targeting cannabinoid receptors for the treatment of inflammation-dependent tumor progression.

Since the first reports of endocannabinoid-mediated retrograde signaling in 2001, great advances have been made toward understanding the molecular basis and functions of the endocannabinoid system. Electrophysiological studies have revealed that the endocannabinoid system is functional at various types of synapses throughout the brain. Basic mechanisms have been clarified as to how endocannabinoids are produced and released from postsynaptic neurons and regulate neurotransmitter release through activating presynaptic cannabinoid CB(1) receptors, although there remain unsolved questions and some discrepancies. In addition to this major function, recent studies suggest diverse functions of endocannabinoids, including control of other endocannabinoid-independent forms of synaptic plasticity, regulation of neuronal excitability, stimulation of glia-neuron interaction, and induction of CB(1)R-independent plasticity. Using recently developed pharmacological and genetic tools, behavioral studies have elucidated the roles of the endocannabinoid system in various aspects of neural functions. In this review, we make a brief overview of molecular mechanisms underlying the endocannabinoid-mediated synaptic modulation and also summarize recent findings, which shed new light on a diversity of functional roles of endocannabinoids.

The heat shock response has been extensively studied by a number of investigators to understand the molecular mechanism underlying the cellular response to severe heat stress (higher than 42°C). But, body or tissue temperature increases by only a few degrees Celsius during physiological events. Therefore, the physiological cellular response to mild heat stress rather than severe heat stress is likely to be more important. Repeated exposure to hyperthermia for consecutive 5 days induces heat acclimation which is an adaptive physiological process in humans and animals. However, thus far, the effect of continuous exposure to heat stress on cells has not been fully evaluated. In this study, we investigated an adaptive physiological process that is induced in culture cells by continuous exposure to mild heat stress for 5 days. Exposure to heat activated p38-mitogen-activated protein kinase; inhibited cell growth without apoptosis; and increased the levels of HSPs and HSF-1 in mouse fibroblast cells. Interestingly, exposure to heat regulated the expression of aquaporins and induced morphological change. In a physiological sense, these results suggested that continuous exposure to mild heat stress for 5 days, in which heat acclimation is attained in humans and animals, might induce molecular adaptation to heat in cells., (Copyright © 2012 S. Karger AG, Basel.)

Clozapine is the first atypical antipsychotic, and improves positive and negative symptoms of many patients with schizophrenia resistant to treatment with other antipsychotic agents. Clozapine induces minimal extrapyramidal side effects, but is more often associated with seizures. A large number of studies have been conducted to elucidate pharmacological profiles of clozapine and its major active metabolite, N-desmethylclozapine (NDMC). However, there are only a limited number of electrophysiological studies examining their effects on synaptic transmission. In this study, we examined effects of clozapine and NDMC on synaptic transmission by measuring inhibitory and excitatory postsynaptic currents in rat cultured hippocampal neurons. We found that clozapine and NDMC have qualitatively similar actions. They depressed the inhibitory transmission at 1-30 μM, and the excitatory transmission at 30 μM, the former being much more sensitive. The depression of IPSCs by 30 μM of these drugs was associated with an increase in the paired-pulse ratio. The GABA-induced currents were suppressed by these drugs, but less sensitive than IPSCs. The AMPA-induced currents were slightly potentiated by these drugs at 30 μM. At 30 μM, clozapine and NDMC slightly suppressed Ca(2+) and Na(+) channels. These results strongly suggest that clozapine and NMDC depress the inhibitory synaptic transmission mainly by antagonizing postsynaptic GABA(A) receptors, but at higher concentrations additionally by acting on presynaptic site, possibly in part through inhibition of presynaptic Ca(2+) and Na(+) channels. Preferential depression of inhibitory synaptic transmission by clozapine and NDMC might contribute to therapeutic actions and/or side-effects of clozapine., (Copyright © 2011 Elsevier B.V. All rights reserved.)