The Mood Stabilizer Lithium Slows Down Synaptic Vesicle Cycling at Glutamatergic Synapses.

Lithium is a mood stabilizer broadly used to prevent and treat symptoms of mania and depression in people with bipolar disorder (BD). Little is known, however, about its mode of action. Here, we analyzed the impact of lithium on synaptic vesicle (SV) cycling at presynaptic terminals releasing glutamate, a neurotransmitter previously implicated in BD and other neuropsychiatric conditions. We used the pHluorin-based synaptic tracer vGpH and a fully automated image processing pipeline to quantify the effect of lithium on both SV exocytosis and endocytosis in hippocampal neurons. We found that lithium selectively reduces SV exocytic rates during electrical stimulation, and markedly slows down SV recycling post-stimulation. Analysis of single-bouton responses revealed the existence of functionally distinct excitatory synapses with varying sensitivity to lithium-some terminals show responses similar to untreated cells, while others are markedly impaired in their ability to recycle SVs. While the cause of this heterogeneity is unclear, these data indicate that lithium interacts with the SV machinery and influences glutamate release in a large fraction of excitatory synapses. Together, our findings show that lithium down modulates SV cycling, an effect consistent with clinical reports indicating hyperactivation of glutamate neurotransmission in BD.

VEGF is an essential retrograde trophic factor for motoneurons.

VEGF was initially discovered due to its angiogenic activity and therefore named "vascular endothelial growth factor." However, its more recently discovered neurotrophic activity may be evolutionarily more ancient. Our previous work showed that all the changes produced by axotomy on the firing activity and synaptic inputs of abducens motoneurons were completely restored after VEGF administration. Therefore, we hypothesized that the lack of VEGF delivered by retrograde transport from the periphery should also affect the physiology of otherwise intact abducens motoneurons. For VEGF retrograde blockade, we chronically applied a neutralizing VEGF antibody to the lateral rectus muscle. Recordings of extracellular single-unit activity and eye movements were made in alert cats before and after the application of the neutralizing antibody. Our data revealed that intact, noninjured abducens motoneurons retrogradely deprived of VEGF exhibited noticeable changes in their firing pattern. There is a general decrease in firing rate and a significant reduction in eye position and eye velocity sensitivity (i.e., a decrease in the tonic and phasic components of their discharge, respectively). Moreover, by means of confocal immunocytochemistry, motoneurons under VEGF blockade showed a marked reduction in the density of afferent synaptic terminals contacting with their cell bodies. Altogether, the present findings demonstrate that the lack of retrogradely delivered VEGF renders abducens motoneurons into an axotomy-like state. This indicates that VEGF is an essential retrograde factor for motoneuronal synaptic drive and discharge activity.

Neuroprotective Role of the B Vitamins in the Modulation of the Central Glutamatergic Neurotransmission.

BACKGROUND: Regulation of glutamate release is crucial for maintaining normal brain function, but excess glutamate release is implicated in many neuropathological conditions. Therefore, the minimum glutamate release from presynaptic nerve terminals is an important neuroprotective mechanism. OBJECTIVE: In this mini-review, we analyze the three B vitamins, namely vitamin B2 (riboflavin), vitamin B6 (pyridoxine), and vitamin B12 (cyanocobalamin), that affect the 4-aminopyridine (4- AP)-evoked glutamate release from presynaptic nerve terminal in rat and discuss their neuroprotective role. METHODS: In this study, the measurements include glutamate release, DiSC3(5), and Fura-2. RESULTS: The riboflavin, pyridoxine, and cyanocobalamin produced significant inhibitory effects on 4-aminopyridine-evoked glutamate release from rat cerebrocortical nerve terminals (synaptosomes) in a dose-dependent relationship. These presynaptic inhibitory actions of glutamate release are attributed to inhibition of physiologic Ca2+-dependent vesicular exocytosis but not Ca2+-independent nonvesicular release. These effects also did not affect membrane excitability, while diminished cytosolic (Ca2+)c through a reduction of direct Ca2+ influx via Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ channels, rather than through indirect Ca2+induced Ca2+ release from ryanodine-sensitive intracellular stores. Furthermore, their effects were attenuated by GF109203X and Ro318220, two protein kinase C (PKC) inhibitors, suggesting suppression of PKC activity. Taken together, these results suggest that riboflavin, pyridoxine, and cyanocobalamin inhibit presynaptic vesicular glutamate release from rat cerebrocortical synaptosomes, through the depression Ca2+ influx via voltage- dependent Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ channels, and PKC signaling cascade. CONCLUSION: Therefore, these B vitamins may reduce the strength of glutamatergic synaptic transmission and is of considerable importance as potential targets for therapeutic agents in glutamate- induced excitation-related diseases.

Topographical distance between presynaptic Ca2+ channels and exocytotic Ca2+ sensors contributes to differential facilitatory actions of roscovitine on neurotransmitter release at cerebellar glutamatergic and GABAergic synapses.

Calcium influx into presynaptic terminals through voltage-gated Ca2+ channels triggers univesicular or multivesicular release of neurotransmitters depending on the characteristics of the release machinery. However, the mechanisms underlying multivesicular release (MVR) and its regulation remain unclear. Previous studies showed that in rat cerebellum, the cyclin-dependent kinase inhibitor roscovitine profoundly increases excitatory postsynaptic current (EPSC) amplitudes at granule cell (GC)-Purkinje cell (PC) synapses by enhancing the MVR of glutamate. This compound can also moderately augment the amplitude and prolong the decay time of inhibitory postsynaptic currents (IPSCs) at molecular layer interneuron (MLI)-PC synapses via MVR enhancement and GABA spillover, thus allowing for persistent activation of perisynaptic GABA receptors. The enhanced MVR may depend on the driving force for Cav 2.1 channel-mediated Ca2+ influx. To determine whether the distinct spatiotemporal dynamics of presynaptic Ca2+ influence MVR, we compared the effects of slow and fast Ca2+ chelators, that is, EGTA and BAPTA, respectively, on roscovitine-induced actions at GC-PC and MLI-PC synapses. Membrane-permeable EGTA-AM decreased GC-PC EPSC and MLI-PC IPSC amplitudes to a similar extent but suppressed the roscovitine-induced enhancement of EPSCs. In contrast, BAPTA-AM attenuated the effects of roscovitine on IPSCs. These results suggest that roscovitine augmented glutamate release by activating the release machinery located distally from the Cav 2.1 channel clusters, while it enhanced GABA release in a manner less dependent on those at distal sites. Therefore, the spatial relationships among Ca2+ channels, buffers, and sensors are critical determinants of the differential facilitatory actions of roscovitine on glutamatergic and GABAergic synapses in the cerebellar cortex.

Anxiolytic effect of GABAergic neurons in the anterior cingulate cortex in a rat model of chronic inflammatory pain.

Chronic pain easily leads to concomitant mood disorders, and the excitability of anterior cingulate cortex (ACC) pyramidal neurons (PNs) is involved in chronic pain-related anxiety. However, the mechanism by which PNs regulate pain-related anxiety is still unknown. The GABAergic system plays an important role in modulating neuronal activity. In this paper, we aimed to study how the GABAergic system participates in regulating the excitability of ACC PNs, consequently affecting chronic inflammatory pain-related anxiety. A rat model of CFA-induced chronic inflammatory pain displayed anxiety-like behaviors, increased the excitability of ACC PNs, and reduced inhibitory presynaptic transmission; however, the number of GAD65/67 was not altered. Interestingly, intra-ACC injection of the GABAAR agonist muscimol relieved anxiety-like behaviors but had no effect on chronic inflammatory pain. Intra-ACC injection of the GABAAR antagonist picrotoxin induced anxiety-like behaviors but had no effect on pain in normal rats. Notably, chemogenetic activation of GABAergic neurons in the ACC alleviated chronic inflammatory pain and pain-induced anxiety-like behaviors, enhanced inhibitory presynaptic transmission, and reduced the excitability of ACC PNs. Chemogenetic inhibition of GABAergic neurons in the ACC led to pain-induced anxiety-like behaviors, reduced inhibitory presynaptic transmission, and enhanced the excitability of ACC PNs but had no effect on pain in normal rats. We demonstrate that the GABAergic system mediates a reduction in inhibitory presynaptic transmission in the ACC, which leads to enhanced excitability of pyramidal neurons in the ACC and is associated with chronic inflammatory pain-related anxiety.

Oxytocin in the anterior cingulate cortex attenuates neuropathic pain and emotional anxiety by inhibiting presynaptic long-term potentiation.

Oxytocin is a well-known neurohypophysial hormone that plays an important role in behavioral anxiety and nociception. Two major forms of long-term potentiation, presynaptic LTP (pre-LTP) and postsynaptic LTP (post-LTP), have been characterized in the anterior cingulate cortex (ACC). Both pre-LTP and post-LTP contribute to chronic-pain-related anxiety and behavioral sensitization. The roles of oxytocin in the ACC have not been studied. Here, we find that microinjections of oxytocin into the ACC attenuate nociceptive responses and anxiety-like behavioral responses in animals with neuropathic pain. Application of oxytocin selectively blocks the maintenance of pre-LTP but not post-LTP. In addition, oxytocin enhances inhibitory transmission and excites ACC interneurons. Similar results are obtained by using selective optical stimulation of oxytocin-containing projecting terminals in the ACC in animals with neuropathic pain. Our results demonstrate that oxytocin acts on central synapses and reduces chronic-pain-induced anxiety by reducing pre-LTP.

Recruitment of release sites underlies chemical presynaptic potentiation at hippocampal mossy fiber boutons.

Synaptic plasticity is a cellular model for learning and memory. However, the expression mechanisms underlying presynaptic forms of plasticity are not well understood. Here, we investigate functional and structural correlates of presynaptic potentiation at large hippocampal mossy fiber boutons induced by the adenylyl cyclase activator forskolin. We performed 2-photon imaging of the genetically encoded glutamate sensor iGluu that revealed an increase in the surface area used for glutamate release at potentiated terminals. Time-gated stimulated emission depletion microscopy revealed no change in the coupling distance between P/Q-type calcium channels and release sites mapped by Munc13-1 cluster position. Finally, by high-pressure freezing and transmission electron microscopy analysis, we found a fast remodeling of synaptic ultrastructure at potentiated boutons: Synaptic vesicles dispersed in the terminal and accumulated at the active zones, while active zone density and synaptic complexity increased. We suggest that these rapid and early structural rearrangements might enable long-term increase in synaptic strength.

Ceftriaxone regulates glutamate production and vesicular assembly in presynaptic terminals through GLT-1 in APP/PS1 mice.

Perturbations in the glutamate-glutamine cycle and glutamate release from presynaptic terminals have been involved in the development of cognitive deficits in Alzheimer's disease (AD) patients and mouse models. Glutamate transporter-1 (GLT-1) removes glutamate from the synaptic cleft and transports it into astrocytes, where it is used as substrate for the glutamate-glutamine cycle. Ceftriaxone has been reported to improve cognitive deficits in AD mice by increasing GLT-1 expression, glutamate transformation to glutamine, and glutamine efflux from astrocytes. However, the impact of ceftriaxone on glutamine metabolism in neurons is unknown. The present study aimed to investigate whether ceftriaxone regulated the production and vesicular assembly of glutamate in the presynaptic terminals of neurons and to determine GLT-1 involvement in this process. We used the amyloid precursor protein (APP)/presenilin-1 (PS1) AD mouse model and GLT-1 knockdown APP/PS1 (GLT-1+/-/APP/PS1) mice. The expression levels of sodium-coupled neutral amino-acid transporter 1 (SNAT1) and vesicular glutamate transporters 1 and 2 (VGLUT1/2) were analyzed by immunofluorescence and immunohistochemistry staining as well as by Western blotting. Glutaminase activity was assayed by fluorometry. Ceftriaxone treatment significantly increased SNAT1 expression and glutaminase activity in neurons in APP/PS1 mice. Similarly, VGLUT1/2 levels were increased in the presynaptic terminals of APP/PS1 mice treated with ceftriaxone. The deletion of one GLT-1 allele in APP/PS1 mice prevented the ceftriaxone-induced upregulation of SNAT1 and VGLUT1/2 expression, indicating that GLT-1 played an important role in ceftriaxone effect. Based on the role of SNAT1, glutaminase, and VGLUT1/2 in the glutamate-glutamine cycle in neurons, the present results suggested that ceftriaxone improved the production and vesicular assembly of glutamate as a neurotransmitter in presynaptic terminals by acting on GLT-1 in APP/PS1 mice.

Ilex kudingcha C.J. Tseng Mitigates Phenotypic Characteristics of Human Autism Spectrum Disorders in a Drosophila Melanogaster Rugose Mutant.

Autism spectrum disorders (ASD) have heterogeneous etiologies involving dysfunction of central nervous systems, for which no effective pan-specific treatments are available. Ilex kudingcha (IK) C.J. Tseng is a nootropic botanical used in Asia for neuroprotection and improvement of cognition. This study establishes that a chemically characterized extract from IK (IKE) mitigates behavioral traits in the Drosophila melanogaster rugose mutant, whose traits resemble human ASD, and examines possible mechanisms. IKE treatment significantly ameliorated deficits in social interaction, short-term memory, and locomotor activity in Drosophila rugose, and significantly increased synaptic bouton number of size more than 2 µm2 in the neuromuscular junctions (NMJs) of Drosophila rugose. To clarify mechanism(s) of IKE action, methylphenidate (MPH), a dopamine transporter inhibitor, was included as a reference drug in the behavioral assays: MPH significantly improved social interaction and short-term memory deficit in Drosophila rugose; administration of the dopamine D1 receptor antagonist SCH23390 and dopamine D2 receptor antagonist sulpiride reversed the ameliorative effects of both MPH and IKE on the social interaction deficits of Drosophila rugose. To extend analysis of IKE treatment to the vertebrate central nervous system, ASD-associated gene expression in mouse hippocampus was studied by RNA-seq: IKE treatment altered the expression of genes coding phosphoinositide 3-kinases/protein kinase B (PI3K-Akt), proteins in glutamatergic, dopaminergic, serotonergic, and GABAergic synapses, cAMP response element-binding protein (CREB), and RNA transporter proteins. These results provide a foundation for further analysis of IKE as a candidate for treatment of some forms of ASD.

Sigma 1 receptor agonist cutamesine promotes plasticity of serotonergic boutons in lumbar enlargement in spinal cord injured rats.

Cutamesine, a sigma-1 receptor agonist, functions in both neuroprotection and neurite outgrowth. We assessed the therapeutic effects of cutamesine in a rodent spinal cord injury (SCI) model to demonstrate pre-clinical proof-of-concept. First of all, in order to determine optimal cutamesine dose, cutamesine was administered to normal rats and BDNF protein levels in the lumbar spinal cord were assessed by Western blot. Next, for the SCI model, spinal cords of adult female Sprague-Dawley rats were contused using an Infinite Horizon Impactor. Two weeks post-injury, rats were randomly assigned to receive daily subcutaneous injections of either cutamesine (3.0 mg/kg/day) or saline (as a control) for another two weeks. Immunohistochemistry for BDNF and 5-HT was assessed at four and twelve weeks post-injury in the lumbar spinal cord. Locomotor function was assessed weekly using the BBB locomotor scale until twelve weeks after SCI and CatWalk XT 10.5 gait analysis was conducted at twelve weeks after SCI. In normal rats, cutamesine treatment (3.0 mg/kg/day) significantly up-regulated BDNF expression in the lumbar spinal cord. In SCI rats, cutamesine treatment (3.0 mg/kg/day) significantly increased the fluorescence intensity of neuronal BDNF and serotonin boutons in the injured spinal cord compared to saline. However, cutamesine treatment did not promote significant locomotor recovery. Recent work indicates that cutamesine treatment alone did not promote locomotor recovery in spite of immunohistological changes. Future work will explore the influence of combining cutamesine with other treatment promoting plasticity (e.g. rehabilitative training) in SCI rats.

GABAA presynaptic inhibition regulates the gain and kinetics of retinal output neurons.

Output signals of neural circuits, including the retina, are shaped by a combination of excitatory and inhibitory signals. Inhibitory signals can act presynaptically on axon terminals to control neurotransmitter release and regulate circuit function. However, it has been difficult to study the role of presynaptic inhibition in most neural circuits due to lack of cell type-specific and receptor type-specific perturbations. In this study, we used a transgenic approach to selectively eliminate GABAA inhibitory receptors from select types of second-order neurons - bipolar cells - in mouse retina and examined how this affects the light response properties of the well-characterized ON alpha ganglion cell retinal circuit. Selective loss of GABAA receptor-mediated presynaptic inhibition causes an enhanced sensitivity and slower kinetics of light-evoked responses from ON alpha ganglion cells thus highlighting the role of presynaptic inhibition in gain control and temporal filtering of sensory signals in a key neural circuit in the mammalian retina.

Absence of Thyroid Hormone Induced Delayed Dendritic Arborization in Mouse Primary Hippocampal Neurons Through Insufficient Expression of Brain-Derived Neurotrophic Factor.

Thyroid hormone (TH) plays important roles in the developing brain. TH deficiency in early life leads to severe developmental impairment in the hippocampus. However, the mechanisms of TH action in the developing hippocampus are still largely unknown. In this study, we generated 3,5,3'-tri-iodo-l-thyronine (T3)-free neuronal supplement, based on the composition of neuronal supplement 21 (NS21), to examine the effect of TH in the developing hippocampus using primary cultured neurons. Effects of TH on neurons were compared between cultures in this T3-free culture medium (-T3 group) and a medium in which T3 was added (+T3 group). Morphometric analysis and RT-qPCR were performed on 7, 10, and 14 days in vitro (DIV). On 10 DIV, a decreased dendrite arborization in -T3 group was observed. Such difference was not observed on 7 and 14 DIV. Brain-derived neurotrophic factor (Bdnf) mRNA levels also decreased significantly in -T3 group on 10 DIV. We then confirmed protein levels of phosphorylated neurotrophic tyrosine kinase type 2 (NTRK2, TRKB), which is a receptor for BDNF, on 10 DIV by immunocytochemistry and Western blot analysis. Phosphorylated NTRK2 levels significantly decreased in -T3 group compared to +T3 group on 10 DIV. Considering the role of BDNF on neurodevelopment, we examined its involvement by adding BDNF on 8 and 9 DIV. Addition of 10 ng/ml BDNF recovered the suppressed dendrite arborization induced by T3 deficiency on 10 DIV. We show that the lack of TH induces a developmental delay in primary hippocampal neurons, likely caused through a decreased Bdnf expression. Thus, BDNF may play a role in TH-regulated dendritogenesis.

SCAMP5 mediates activity-dependent enhancement of NHE6 recruitment to synaptic vesicles during synaptic plasticity.

Na+(K+)/H+ exchanger 6 (NHE6) on synaptic vesicle (SV) is critical for the presynaptic regulation of quantal size at the glutamatergic synapses by converting the chemical gradient (ΔpH) into membrane potential (Δψ) across the SV membrane. We recently found that NHE6 directly interacts with secretory carrier membrane protein 5 (SCAMP5), and SCAMP5-dependent recruitment of NHE6 to SVs controls the strength of synaptic transmission by modulation of quantal size of glutamate release at rest. It is, however, unknown whether NHE6 recruitment by SCAMP5 plays a role during synaptic plasticity. Here, we found that the number of NHE6-positive presynaptic boutons was significantly increased by the chemical long-term potentiation (cLTP). Since cLTP involves new synapse formation, our results indicated that NHE6 was recruited not only to the existing presynaptic boutons but also to the newly formed presynaptic boutons. Knock down of SCAMP5 completely abrogated the enhancement of NHE6 recruitment by cLTP. Interestingly, despite an increase in the number of NHE6-positive boutons by cLTP, the quantal size of glutamate release at the presynaptic terminals remained unaltered. Together with our recent results, our findings indicate that SCAMP5-dependent recruitment of NHE6 plays a critical role in manifesting presynaptic efficacy not only at rest but also during synaptic plasticity. Since both are autism candidate genes, reduced presynaptic efficacy by interfering with their interaction may underlie the molecular mechanism of synaptic dysfunction observed in autism.

NMDA GluN2C/2D receptors contribute to synaptic regulation and plasticity in the anterior cingulate cortex of adult mice.

INTRODUCTION: N-Methyl-D-aspartate receptors (NMDARs) play a critical role in different forms of plasticity in the central nervous system. NMDARs are always assembled in tetrameric form, in which two GluN1 subunits and two GluN2 and/or GluN3 subunits combine together. Previous studies focused mainly on the hippocampus. The anterior cingulate cortex (ACC) is a key cortical region for sensory and emotional functions. NMDAR GluN2A and GluN2B subunits have been previously investigated, however much less is known about the GluN2C/2D subunits. RESULTS: In the present study, we found that the GluN2C/2D subunits are expressed in the pyramidal cells of ACC of adult mice. Application of a selective antagonist of GluN2C/2D, (2R*,3S*)-1-(9-bromophenanthrene-3-carbonyl) piperazine-2,3-dicarboxylic acid (UBP145), significantly reduced NMDAR-mediated currents, while synaptically evoked EPSCs were not affected. UBP145 affected neither the postsynaptic long-term potentiation (post-LTP) nor the presynaptic LTP (pre-LTP). Furthermore, the long-term depression (LTD) was also not affected by UBP145. Finally, both UBP145 decreased the frequency of the miniature EPSCs (mEPSCs) while the amplitude remained intact, suggesting that the GluN2C/2D may be involved in presynaptic regulation of spontaneous glutamate release. CONCLUSIONS: Our results provide direct evidence that the GluN2C/2D contributes to evoked NMDAR mediated currents and mEPSCs in the ACC, which may have significant physiological implications.

Insulin Bidirectionally Alters NAc Glutamatergic Transmission: Interactions between Insulin Receptor Activation, Endogenous Opioids, and Glutamate Release.

Human fMRI studies show that insulin influences brain activity in regions that mediate reward and motivation, including the nucleus accumbens (NAc). Insulin receptors are expressed by NAc medium spiny neurons (MSNs), and studies of cultured cortical and hippocampal neurons suggest that insulin influences excitatory transmission via presynaptic and postsynaptic mechanisms. However, nothing is known about how insulin influences excitatory transmission in the NAc. Furthermore, insulin dysregulation accompanying obesity is linked to cognitive decline, depression, anxiety, and altered motivation that rely on NAc excitatory transmission. Using whole-cell patch-clamp and biochemical approaches, we determined how insulin affects NAc glutamatergic transmission in nonobese and obese male rats and the underlying mechanisms. We find that there are concentration-dependent, bidirectional effects of insulin on excitatory transmission, with insulin receptor activation increasing and IGF receptor activation decreasing NAc excitatory transmission. Increases in excitatory transmission were mediated by activation of postsynaptic insulin receptors located on MSNs. However, this effect was due to an increase in presynaptic glutamate release. This suggested feedback from MSNs to presynaptic terminals. In additional experiments, we found that insulin-induced increases in presynaptic glutamate release are mediated by opioid receptor-dependent disinhibition. Furthermore, obesity resulted in a loss of insulin receptor-mediated increases in excitatory transmission and a reduction in NAc insulin receptor surface expression, while preserving reductions in transmission mediated by IGF receptors. These results provide the first insights into how insulin influences excitatory transmission in the adult brain, and evidence for a previously unidentified form of opioid receptor-dependent disinhibition of NAc glutamatergic transmission.SIGNIFICANCE STATEMENT Data here provide the first insights into how insulin influences excitatory transmission in the adult brain, and identify previously unknown interactions between insulin receptor activation, opioids, and glutamatergic transmission. These data contribute to our fundamental understanding of insulin's influence on brain motivational systems and have implications for the use of insulin as a cognitive enhancer and for targeting of insulin receptors and IGF receptors to alter motivation.

A high-affinity, partial antagonist effect of 3,4-diaminopyridine mediates action potential broadening and enhancement of transmitter release at NMJs.

3,4-Diaminopyridine (3,4-DAP) increases transmitter release from neuromuscular junctions (NMJs), and low doses of 3,4-DAP (estimated to reach ∼1 µM in serum) are the Food and Drug Administration (FDA)-approved treatment for neuromuscular weakness caused by Lambert-Eaton myasthenic syndrome. Canonically, 3,4-DAP is thought to block voltage-gated potassium (Kv) channels, resulting in prolongation of the presynaptic action potential (AP). However, recent reports have shown that low millimolar concentrations of 3,4-DAP have an off-target agonist effect on the Cav1 subtype ("L-type") of voltage-gated calcium (Cav) channels and have speculated that this agonist effect might contribute to 3,4-DAP effects on transmitter release at the NMJ. To address 3,4-DAP's mechanism(s) of action, we first used the patch-clamp electrophysiology to characterize the concentration-dependent block of 3,4-DAP on the predominant presynaptic Kv channel subtypes found at the mammalian NMJ (Kv3.3 and Kv3.4). We identified a previously unreported high-affinity (1-10 µM) partial antagonist effect of 3,4-DAP in addition to the well-known low-affinity (0.1-1 mM) antagonist activity. We also showed that 1.5-µM DAP had no effects on Cav1.2 or Cav2.1 current. Next, we used voltage imaging to show that 1.5- or 100-µM 3,4-DAP broadened the AP waveform in a dose-dependent manner, independent of Cav1 calcium channels. Finally, we demonstrated that 1.5- or 100-µM 3,4-DAP augmented transmitter release in a dose-dependent manner and this effect was also independent of Cav1 channels. From these results, we conclude that low micromolar concentrations of 3,4-DAP act solely on Kv channels to mediate AP broadening and enhance transmitter release at the NMJ.

Fingolimod inhibits glutamate release through activation of S1P1 receptors and the G protein ßγ subunit-dependent pathway in rat cerebrocortical nerve terminals.

Fingolimod, a sphingosine-1-phosphate (S1P) receptor modulator approved for treating multiple sclerosis, is reported to prevent excitotoxic insult. Because excessive glutamate release is a major cause of neuronal damage in various neurological disorders, the effect of fingolimod on glutamate release in rat cerebrocortical nerve terminals (synaptosomes) was investigated in the current study. Fingolimod decreased 4-aminopyridine (4-AP)-stimulated glutamate release and calcium concentration elevation. Fingolimod-mediated inhibition of 4-AP-induced glutamate release was dependent on extracellular calcium, persisted in the presence of the glutamate transporter inhibitor DL-TBOA or intracellular Ca2+-releasing inhibitors dantrolene and CGP37157, and was prevented by blocking vesicular transporters or N- and P/Q-type channels. Western blot and immunocytochemical analysis revealed the presence of S1P1 receptor proteins in presynaptic terminals. Fingolimod-mediated inhibition of 4-AP-induced glutamate release was also abolished by the sphingosine kinase inhibitor DMS, selective S1P1 receptor antagonist W146, Gi/o protein inhibitor pertussis toxin, and G protein ßγ subunit inhibitor gallein; however, it was unaffected by the adenylyl cyclase inhibitor SQ22536, protein kinase A inhibitor H89, and phospholipase C inhibitor U73122. These data indicate that fingolimod decreases glutamate release from rat cerebrocortical synaptosomes by suppressing N- and P/Q-type Ca2+ channel activity; additionally, the activation of presynaptic S1P1 receptors and the G protein ßγ subunit participates in achieving the effect.

Presynaptic vesicular accumulation is required for antipsychotic efficacy in psychotic-like rats.

BACKGROUND: The therapeutic effects of antipsychotic drugs (APDs) are mainly attributed to their postsynaptic inhibitory functions on the dopamine D2 receptor, which, however, cannot explain the delayed onset of full therapeutic efficacy. It was previously shown that APDs accumulate in presynaptic vesicles during chronic treatment and are released like neurotransmitters in an activity-dependent manner triggering an auto-inhibitory feedback mechanism. Although closely mirroring therapeutic action onset, the functional consequence of the APD accumulation process remained unclear. AIMS: Here we tested whether the accumulation of the APD haloperidol (HAL) is required for full therapeutic action in psychotic-like rats. METHODS: We designed a HAL analog compound (HAL-F), which lacks the accumulation property of HAL, but retains its postsynaptic inhibitory action on dopamine D2 receptors. RESULTS/OUTCOMES: By perfusing LysoTracker fluorophore-stained cultured hippocampal neurons, we confirmed the accumulation of HAL and the non-accumulation of HAL-F. In an amphetamine hypersensitization psychosis-like model in rats, we found that subchronic intracerebroventricularly delivered HAL (0.1 mg/kg/day), but not HAL-F (0.3-1.5 mg/kg/day), attenuates psychotic-like behavior in rats. CONCLUSIONS/INTERPRETATION: These findings suggest the presynaptic accumulation of HAL may serve as an essential prerequisite for its full antipsychotic action and may explain the time course of APD action. Targeting accumulation properties of APDs may, thus, become a new strategy to improve APD action.

Isoflurane decreases substantia gelatinosa neuron excitability and synaptic transmission from periphery in the rat spinal dorsal horn.

Isoflurane is an inhaled anesthetic, though its actions at the cellular level remain controversial. By using acute spinal cord slices from adult rats and the whole-cell recording technique, we found that aqueous isoflurane at the minimum alveolar concentration decreased postsynaptic neural excitability and enhanced membrane conductance, while suppressing glutamate release from presynaptic afferent onto substantia gelatinosa (lamina II) neurons in the dorsal horn. The data demonstrate that isoflurane modulates synaptic transmission from peripheral to the spinal cord via both pre- and postsynaptic effects and these actions may underlie its spinal anesthesia.

Rho-kinase inhibition by fasudil modulates pre-synaptic vesicle dynamics.

The Rho kinase (ROCK) signaling pathway is an attractive therapeutic target in neurodegeneration since it has been linked to the prevention of neuronal death and neurite regeneration. The isoquinoline derivative fasudil is a potent ROCK inhibitor, which is already approved for chronic clinical treatment in humans. However, the effects of chronic fasudil treatments on neuronal function are still unknown. We analyzed here chronic fasudil treatment in primary rat hippocampal cultures. Neurons were stimulated with 20 Hz field stimulation and we investigated pre-synaptic mechanisms and parameters regulating synaptic transmission after fasudil treatment by super resolution stimulated emission depletion (STED) microscopy, live-cell fluorescence imaging, and western blotting. Fasudil did not affect basic synaptic function or the amount of several synaptic proteins, but it altered the chronic dynamics of the synaptic vesicles. Fasudil reduced the proportion of the actively recycling vesicles, and shortened the vesicle lifetime, resulting overall in a reduction of the synaptic response upon stimulation. We conclude that fasudil does not alter synaptic structure, accelerates vesicle turnover, and decreases the number of released vesicles. This broadens the known spectrum of effects of this drug, and suggests new potential clinical uses.