Evaluation of motor learning in predictable loading task using a force sense presentation device.

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.

Effects of long-term treatment with dietary theobromine on rat skeletal muscles.

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 ß2-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.

Weeding out bad waves: towards selective cannabinoid circuit control in epilepsy.

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.

Acute inhibition of diacylglycerol lipase blocks endocannabinoid-mediated retrograde signalling: evidence for on-demand biosynthesis of 2-arachidonoylglycerol.

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.

Action Sequence Learning Is Impaired in Genetically Modified Mice with the Suppressed GABAergic Transmission from the Thalamic Reticular Nucleus to the Thalamus.

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 (Vgatflox/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.

Neuronal protease-activated receptor 1 drives synaptic retrograde signaling mediated by the endocannabinoid 2-arachidonoylglycerol.

Protease-activated receptor 1 (PAR1) is a member of the G-protein coupled receptors that are proteolytically activated by serine proteases. Recent studies suggest a definite contribution of PAR1 to brain functions, including learning and memory. However, cellular mechanisms by which PAR1 activation influences neuronal activity are not well understood. Here we show that PAR1 activation drives retrograde endocannabinoid signaling and thereby regulates synaptic transmission. In cultured hippocampal neurons from rat, PAR1 activation by thrombin or PAR1-specific peptide agonists transiently suppressed inhibitory transmission at cannabinoid-sensitive, but not cannabinoid-insensitive, synapses. The PAR1-induced suppression of synaptic transmission was accompanied by an increase in paired-pulse ratio, and was blocked by a cannabinoid CB(1) receptor antagonist. The PAR1-induced suppression was blocked by pharmacological inhibition of postsynaptic diacylglycerol lipase (DGL), a key enzyme for biosynthesis of the major endocannabinoid 2-arachidonoylglycerol (2-AG), and was absent in knock-out mice lacking the α isoform of DGL. The PAR1-induced IPSC suppression remained intact under the blockade of metabotropic glutamate receptors and was largely resistant to the treatment that blocked Ca(2+) elevation in glial cells following PAR1 activation, which excludes the major contribution of glial PAR1 in IPSC suppression. We conclude that activation of neuronal PAR1 triggers retrograde signaling mediated by 2-AG, which activates presynaptic CB(1) receptors and suppresses transmitter release at hippocampal inhibitory synapses.

Cellular heat acclimation regulates cell growth, cell morphology, mitogen-activated protein kinase activation, and expression of aquaporins in mouse fibroblast cells.

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.

Effects of environmental enrichment on exploratory behavior, win-stay and lose-shift performance, motor sequence learning, and reversal learning during the three-lever operant task in mice.

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.

Phospholipase Cbeta serves as a coincidence detector through its Ca2+ dependency for triggering retrograde endocannabinoid signal.

Endocannabinoids mediate retrograde signal and modulate transmission efficacy at various central synapses. Although endocannabinoid release is induced by either depolarization or activation of G(q/11)-coupled receptors, it is markedly enhanced by the coincidence of depolarization and receptor activation. Here we report that this coincidence is detected by phospholipase Cbeta1 (PLCbeta1) in hippocampal neurons. By measuring cannabinoid-sensitive synaptic currents, we found that the receptor-driven endocannabinoid release was dependent on physiological levels of intracellular Ca(2+) concentration ([Ca(2+)](i)), and markedly enhanced by depolarization-induced [Ca(2+)](i) elevation. Furthermore, we measured PLC activity in intact neurons by using exogenous TRPC6 channel as a biosensor for the PLC product diacylglycerol and found that the receptor-driven PLC activation exhibited similar [Ca(2+)](i) dependence to that of endocannabinoid release. Neither endocannabinoid release nor PLC activation was induced by receptor activation in PLCbeta1 knockout mice. We therefore conclude that PLCbeta1 serves as a coincidence detector through its Ca(2+) dependency for endocannabinoid release in hippocampal neurons.

Ca(2+)-assisted receptor-driven endocannabinoid release: mechanisms that associate presynaptic and postsynaptic activities.

Endogenous cannabinoids (endocannabinoids) serve as retrograde messengers at synapses in various regions of the brain. They are released from postsynaptic neurons and cause transient and long-lasting reduction of neurotransmitter release through activation of presynaptic cannabinoid receptors. Endocannabinoid release is induced either by increased postsynaptic Ca(2+) levels or by activation of G(q/11)-coupled receptors. When these two stimuli coincide, endocannabinoid release is markedly enhanced, which is attributed to the Ca(2+) dependency of phospholipase Cbeta (PLCbeta). This Ca(2+)-assisted receptor-driven endocannabinoid release is suggested to participate in various forms of synaptic plasticity, including short-term associative plasticity in the cerebellum and spike-timing-dependent long-term depression in the somatosensory cortex. In these forms of plasticity, PLCbeta seems to function as a coincident detector of presynaptic and postsynaptic activities.

Corticosterone inhibits the expression of cannabinoid receptor-1 and cannabinoid receptor agonist-induced decrease in cell viability in glioblastoma cells.

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.

Endocannabinoid Signaling from 2-Arachidonoylglycerol to CB1 Cannabinoid Receptor Facilitates Reward-based Learning of Motor Sequence.

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 CB1 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 CB1-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 CB1-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.

Presynaptic monoacylglycerol lipase activity determines basal endocannabinoid tone and terminates retrograde endocannabinoid signaling in the hippocampus.

Endocannabinoids function as retrograde messengers and modulate synaptic transmission through presynaptic cannabinoid CB1 receptors. The magnitude and time course of endocannabinoid signaling are thought to depend on the balance between the production and degradation of endocannabinoids. The major endocannabinoid 2-arachidonoylglycerol (2-AG) is hydrolyzed by monoacylglycerol lipase (MGL), which is shown to be localized at axon terminals. In the present study, we investigated how MGL regulates endocannabinoid signaling and influences synaptic transmission in the hippocampus. We found that MGL inhibitors, methyl arachidonoyl fluorophosphonate and arachidonoyl trifluoromethylketone, caused a gradual suppression of cannabinoid-sensitive IPSCs in cultured hippocampal neurons. This suppression was reversed by blocking CB1 receptors and was attenuated by inhibiting 2-AG synthesis, indicating that MGL scavenges constitutively released 2-AG. We also found that the MGL inhibitors significantly prolonged the suppression of both IPSCs and EPSCs induced by exogenous 2-AG and depolarization-induced suppression of inhibition/excitation, a phenomenon known to be mediated by retrograde endocannabinoid signaling. In contrast, inhibitors of other endocannabinoid hydrolyzing enzymes, fatty acid amide hydrolase and cyclooxygenase-2, had no effect on the 2-AG-induced IPSC suppression. These results strongly suggest that presynaptic MGL not only hydrolyzes 2-AG released from activated postsynaptic neurons but also contributes to degradation of constitutively produced 2-AG and prevention of its accumulation around presynaptic terminals. Thus, the MGL activity determines basal endocannabinoid tone and terminates retrograde endocannabinoid signaling in the hippocampus.

Pharmacological evidence for the involvement of diacylglycerol lipase in depolarization-induced endocanabinoid release.

Depolarization-induced suppression of inhibition (DSI) or excitation (DSE) is a well-known form of endocannabinoid-mediated short-term plasticity that is induced by postsynaptic depolarization. It is generally accepted that DSI/DSE is triggered by Ca(2+) influx through voltage-gated Ca(2+) channels. It is also demonstrated that DSI/DSE is mediated by 2-arachidonoylglycerol (2-AG). However, how Ca(2+) induces 2-AG production is still unclear. In the present study, we investigated molecular mechanisms underlying the Ca(2+)-driven 2-AG production. Using cannabinoid-sensitive inhibitory synapses of cultured hippocampal neurons, we tested several inhibitors for enzymes that are supposed to be involved in 2-AG metabolism. The chemicals we tested include inhibitors for phospholipase C (U73122 and ET-18), diacylglycerol kinase (DGK inhibitor 1), phosphatidic acid phosphohydrolase (propranolol), and diacylglycerol lipase (DGL; RHC-80267 and tetrahydrolipstatin (THL)). However, unfavorable side effects were observed with these inhibitors, except for THL. Furthermore, we found that RHC-80267 hardly inhibited the endocannabinoid release driven by G(q/11)-coupled receptors, which is thought to be DGL-dependent. By contrast, THL exhibited no side effects as long as we tested, and was confirmed to inhibit the DGL-dependent process. Using THL as a DGL inhibitor, we demonstrated that DGL is involved in both hippocampal DSI and cerebellar DSE. To test a possible involvement of PLCdelta in DSI, we examined hippocampal DSI in PLCdelta1, delta3 and delta4-knockout mice. However, there was no significant difference in the DSI magnitude between these knockout mice and wild-type mice. The present study clearly shows that DGL is a prerequisite for DSI/DSE. The enzymes yielding DG remain to be determined.

The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum.

Endocannabinoids work as retrograde messengers and contribute to short-term and long-term modulation of synaptic transmission via presynaptic cannabinoid receptors. It is generally accepted that the CB1 cannabinoid receptor (CB1) mediates the effects of endocannabinoid in inhibitory synapses. For excitatory synapses, however, contributions of CB1, "CB3," and some other unidentified receptors have been suggested. In the present study we used electrophysiological and immunohistochemical techniques and examined the type(s) of cannabinoid receptor functioning at hippocampal and cerebellar excitatory synapses. Our electrophysiological data clearly demonstrate the predominant contribution of CB1. At hippocampal excitatory synapses on pyramidal neurons the cannabinoid-induced synaptic suppression was reversed by a CB1-specific antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), and was absent in CB1 knock-out mice. At climbing fiber (CF) and parallel fiber (PF) synapses on cerebellar Purkinje cells the cannabinoid-dependent suppression was absent in CB1 knock-out mice. The presence of CB1 at presynaptic terminals was confirmed by immunohistochemical experiments with specific antibodies against CB1. In immunoelectron microscopy the densities of CB1-positive signals in hippocampal excitatory terminals and cerebellar PF terminals were much lower than in inhibitory terminals but were clearly higher than the background. Along the long axis of PFs, the CB1 was localized at a much higher density on the perisynaptic membrane than on the extrasynaptic and synaptic regions. In contrast, CB1 density was low in CF terminals and was not significantly higher than the background. Despite the discrepancy between the electrophysiological and morphological data for CB1 expression on CFs, these results collectively indicate that CB1 is responsible for cannabinoid-dependent suppression of excitatory transmission in the hippocampus and cerebellum.

Endocannabinoids and synaptic function in the CNS.

Marijuana affects neural functions through the binding of its active component (Delta(9)-THC) to cannabinoid receptors in the CNS. Recent studies have elucidated that endogenous ligands for cannabinoid receptors, endocannabinoids, serve as retrograde messengers at central synapses. Endocannabinoids are produced on demand in activity-dependent manners and released from postsynaptic neurons. The released endocannabinoids travel backward across the synapse, activate presynaptic CB1 cannabinoid receptors, and modulate presynaptic functions. Retrograde endocannabinoid signaling is crucial for certain forms of short-term and long-term synaptic plasticity at excitatory or inhibitory synapses in many brain regions, and thereby contributes to various aspects of brain function including learning and memory. Molecular identities of the CB1 receptor and enzymes involved in production and degradation of endocannabinoids have been elucidated. Anatomical studies have demonstrated unique distributions of these molecules around synapses, which provide morphological bases for the roles of endocannabinoids as retrograde messengers. CB1-knockout mice exhibit various behavioral abnormalities and multiple defects in synaptic plasticity, supporting the notion that endocannabinoid signaling is involved in various aspects of neural function. In this review article, the authors describe molecular mechanisms of the endocannabinoid-mediated synaptic modulation and its possible physiological significance.

Hypoxia-induced inhibition of the endocannabinoid system in glioblastoma cells.

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 up-regulates cerebral brain-derived neurotrophic factor and facilitates motor learning in mice.

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.

Synaptically driven endocannabinoid release requires Ca2+-assisted metabotropic glutamate receptor subtype 1 to phospholipase Cbeta4 signaling cascade in the cerebellum.

Endocannabinoids mediate retrograde signaling and modulate synaptic transmission in various regions of the CNS. Depolarization-induced elevation of intracellular Ca2+ concentration causes endocannabinoid-mediated suppression of excitatory/inhibitory synaptic transmission. Activation of G(q/11)-coupled receptors including group I metabotropic glutamate receptors (mGluRs) also causes endocannabinoid-mediated suppression of synaptic transmission. However, precise mechanisms of endocannabinoid production initiated by physiologically relevant synaptic activity remain to be determined. To address this problem, we made whole-cell recordings from Purkinje cells (PCs) in mouse cerebellar slices and examined their excitatory synapses arising from climbing fibers (CFs) and parallel fibers (PFs). We first characterized three distinct modes to induce endocannabinoid release by analyzing CF to PC synapses. The first mode is strong activation of mGluR subtype 1 (mGluR1)-phospholipase C (PLC) beta4 cascade without detectable Ca2+ elevation. The second mode is Ca2+ elevation to a micromolar range without activation of the mGluR1-PLCbeta4 cascade. The third mode is the Ca2+-assisted mGluR1-PLCbeta4 cascade that requires weak mGluR1 activation and Ca2+ elevation to a submicromolar range. By analyzing PF to PC synapses, we show that the third mode is essential for effective endocannabinoid release from PCs by excitatory synaptic activity. Furthermore, our biochemical analysis demonstrates that combined weak mGluR1 activation and mild depolarization in PCs effectively produces 2-arachidonoylglycerol (2-AG), a candidate of endocannabinoid, whereas either stimulus alone did not produce detectable 2-AG. Our results strongly suggest that under physiological conditions, excitatory synaptic inputs to PCs activate the Ca2+-assisted mGluR1-PLCbeta4 cascade, and thereby produce 2-AG, which retrogradely modulates synaptic transmission to PCs.

Electrophysiological evidence showing muscarinic agonist-antagonist activities of N-desmethylclozapine using hippocampal excitatory and inhibitory neurons.

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.