Collagen VI: Role in synaptic transmission and seizure-related excitability.

Collagen VI (Col-VI) is an extracellular matrix protein primarily known for its bridging role in connective tissues that has been suggested to play a neuroprotective role. In the present study we report increased mRNA and protein expression of Col-VI in the hippocampus and cortex at a late stage of epileptogenesis in a post-status epilepticus (SE) model of epilepsy and in brain tissue from patients with epilepsy. We further present a novel finding that exposure of mouse hippocampal slices to Col-VI augments paired-pulse facilitation in Schaffer collateral-CA1 excitatory synapses indicating decreased release probability of glutamate. In line with this finding, lack of Col-VI expression in the knock-out mice show paired-pulse depression in these synapses, suggesting increased release probability of glutamate. In addition, we observed dynamic changes in Col-VI blood plasma levels in rats after Kainate-induced SE, and increased levels of Col-VI mRNA and protein in autopsy or postmortem brain of humans suffering from epilepsy. Thus, our data indicate that elevated levels of ColVI following seizures leads to attenuated glutamatergic transmission, ultimately resulting in less overall network excitability. Presumably, increased Col-VI may act as part of endogenous compensatory mechanism against enhanced excitability during epileptogenic processes in the hippocampus, and could be further investigated as a potential functional biomarker of epileptogenesis, and/or a novel target for therapeutic intervention.

Adaptive Changes in Group 2 Metabotropic Glutamate Receptors Underlie the Deficit in Recognition Memory Induced by Methamphetamine in Mice.

Cognitive dysfunction is associated with methamphetamine use disorder (MUD). Here, we used genetic and pharmacological approaches to examine the involvement of either Group 2 metabotropic glutamate (mGlu2) or mGlu3 receptors in memory deficit induced by methamphetamine in mice. Methamphetamine treatment (1 mg/kg, i.p., once a day for 5 d followed by 7 d of withdrawal) caused an impaired performance in the novel object recognition test in wild-type mice, but not in mGlu2-/- or mGlu3-/- mice. Memory deficit in wild-type mice challenged with methamphetamine was corrected by systemic treatment with selectively negative allosteric modulators of mGlu2 or mGlu3 receptors (compounds VU6001966 and VU0650786, respectively). Methamphetamine treatment in wild-type mice caused large increases in levels of mGlu2/3 receptors, the Type 3 activator of G-protein signaling (AGS3), Rab3A, and the vesicular glutamate transporter, vGlut1, in the prefrontal cortex (PFC). Methamphetamine did not alter mGlu2/3-mediated inhibition of cAMP formation but abolished the ability of postsynaptic mGlu3 receptors to boost mGlu5 receptor-mediated inositol phospholipid hydrolysis in PFC slices. Remarkably, activation of presynaptic mGlu2/3 receptors did not inhibit but rather amplified depolarization-induced [3H]-D-aspartate release in synaptosomes prepared from the PFC of methamphetamine-treated mice. These findings demonstrate that exposure to methamphetamine causes changes in the expression and function of mGlu2 and mGlu3 receptors, which might alter excitatory synaptic transmission in the PFC and raise the attractive possibility that selective inhibitors of mGlu2 or mGlu3 receptors (or both) may be used to improve cognitive dysfunction in individuals affected by MUD.

Gypenoside XVII Reduces Synaptic Glutamate Release and Protects against Excitotoxic Injury in Rats.

Excitotoxicity is a common pathological process in neurological diseases caused by excess glutamate. The purpose of this study was to evaluate the effect of gypenoside XVII (GP-17), a gypenoside monomer, on the glutamatergic system. In vitro, in rat cortical nerve terminals (synaptosomes), GP-17 dose-dependently decreased glutamate release with an IC50 value of 16 µM. The removal of extracellular Ca2+ or blockade of N-and P/Q-type Ca2+ channels and protein kinase A (PKA) abolished the inhibitory effect of GP-17 on glutamate release from cortical synaptosomes. GP-17 also significantly reduced the phosphorylation of PKA, SNAP-25, and synapsin I in cortical synaptosomes. In an in vivo rat model of glutamate excitotoxicity induced by kainic acid (KA), GP-17 pretreatment significantly prevented seizures and rescued neuronal cell injury and glutamate elevation in the cortex. GP-17 pretreatment decreased the expression levels of sodium-coupled neutral amino acid transporter 1, glutamate synthesis enzyme glutaminase and vesicular glutamate transporter 1 but increased the expression level of glutamate metabolism enzyme glutamate dehydrogenase in the cortex of KA-treated rats. In addition, the KA-induced alterations in the N-methyl-D-aspartate receptor subunits GluN2A and GluN2B in the cortex were prevented by GP-17 pretreatment. GP-17 also prevented the KA-induced decrease in cerebral blood flow and arginase II expression. These results suggest that (i) GP-17, through the suppression of N- and P/Q-type Ca2+ channels and consequent PKA-mediated SNAP-25 and synapsin I phosphorylation, reduces glutamate exocytosis from cortical synaptosomes; and (ii) GP-17 has a neuroprotective effect on KA-induced glutamate excitotoxicity in rats through regulating synaptic glutamate release and cerebral blood flow.

Astrocyte ryanodine receptors facilitate gliotransmission and astroglial modulation of synaptic plasticity.

Intracellular Ca2+-signaling in astrocytes is instrumental for their brain "housekeeping" role and astroglial control of synaptic plasticity. An important source for elevating the cytosolic Ca2+ level in astrocytes is a release from endoplasmic reticulum which can be triggered via two fundamental pathways: IP3 receptors and calcium-induced calcium release (CICR) mediated by Ca2+-sensitive ryanodine receptors (RyRs). While the physiological role for glial IP3 became a focus of intensive research and debate, ryanodine receptors received much less attention. We explored the role for ryanodine receptors in the modulation of cytosolic Ca2+-signaling in the cortical and hippocampal astrocytes, astrocyte-neuron communication and astroglia modulation of synaptic plasticity. Our data show that RyR-mediated Ca2+-induced Ca2+-release from ER brings substantial contribution into signaling in the functional microdomains hippocampal and neocortical astrocytes. Furthermore, RyR-mediated CICR activated the release of ATP and glutamate from hippocampal and neocortical astrocytes which, in turn, elicited transient purinergic and tonic glutamatergic currents in the neighboring pyramidal neurons. The CICR-facilitated release of ATP and glutamate was inhibited after intracellular perfusion of astrocytes with ryanodine and BAPTA and in the transgenic dnSNARE mice with impaired astroglial exocytosis. We also found out that RyR-mediated amplification of astrocytic Ca2+-signaling enhanced the long-term synaptic potentiation in the hippocampus and neocortex of aged mice. Combined, our data demonstrate that ryanodine receptors are essential for astrocytic Ca2+-signaling and efficient astrocyte-neuron communications. The RyR-mediated CICR contributes to astrocytic control of synaptic plasticity and can underlie, at least partially, neuroprotective and cognitive effects of caffein.

Complement tunes glutamate release and supports synaptic impairments in an animal model of multiple sclerosis.

BACKGROUND AND PURPOSE: To deepen our knowledge of the role of complement in synaptic impairment in experimental autoimmune encephalomyelitis (EAE) mice, we investigated the distribution of C1q and C3 proteins and the role of complement as a promoter of glutamate release in purified nerve endings (synaptosomes) and astrocytic processes (gliosomes) isolated from the cortex of EAE mice at the acute stage of the disease (21 ± 1 day post-immunization). EXPERIMENTAL APPROACH: EAE cortical synaptosomes and gliosomes were analysed for glutamate release efficiency (measured as release of preloaded [3H]D-aspartate ([3H]D-ASP)), C1q and C3 protein density, and for viability and ongoing apoptosis. KEY RESULTS: In healthy mice, complement releases [3H]D-ASP from gliosomes more efficiently than from synaptosomes. The releasing activity occurs in a dilution-dependent manner and involves the reversal of the excitatory amino acid transporters (EAATs). In EAE mice, the complement-induced releasing activity is significantly reduced in cortical synaptosomes but amplified in cortical gliosomes. These adaptations are paralleled by decreased density of the EAAT2 protein in synaptosomes and increased EAAT1 staining in gliosomes. Concomitantly, PSD95, GFAP, and CD11b, but not SNAP25, proteins are overexpressed in the cortex of the EAE mice. Similarly, C1q and C3 protein immunostaining is increased in EAE cortical synaptosomes and gliosomes, although signs of ongoing apoptosis or altered viability are not detectable. CONCLUSION AND IMPLICATIONS: Our results unveil a new noncanonical role of complement in the CNS of EAE mice relevant to disease progression and central synaptopathy that suggests new therapeutic targets for the management of MS.

Maternal deprivation causes CaMKII downregulation and modulates glutamate, norepinephrine and serotonin in limbic brain areas in a rat model of single prolonged stress.

BACKGROUND: Early life stress is a major risk factor for later development of psychiatric disorders, including post-traumatic stress disorder (PTSD). An intricate relationship exists between various neurotransmitters (such as glutamate, norepinephrine or serotonin), calcium/calmodulin-dependent protein kinase II (CaMKII), as an important regulator of glutamatergic synaptic function, and PTSD. Here, we developed a double-hit model to investigate the interaction of maternal deprivation (MD) as an early life stress model and single prolonged stress (SPS) as a PTSD model at the behavioral and molecular levels. METHODS: Male Wistar rats exposed to these stress paradigms were subjected to a comprehensive behavioral analysis. In hippocampal synaptosomes we investigated neurotransmitter release and glutamate concentration. The expression of CaMKII and the content of monoamines were determined in selected brain regions. Brain-derived neurotrophic factor (BDNF) mRNA was quantified by radioactive in situ hybridization. RESULTS: We report a distinct behavioral phenotype in the double-hit group. Double-hit and SPS groups had decreased hippocampal presynaptic glutamatergic function. In hippocampus, double-hit stress caused a decrease in autophosphorylation of CaMKII. In prefrontal cortex, both SPS and double-hit stress had a similar effect on CaMKII autophosphorylation. Double-hit stress, rather than SPS, affected the norepinephrine and serotonin levels in prefrontal cortex, and suppressed BDNF gene expression in prefrontal cortex and hippocampus. LIMITATIONS: The study was conducted in male rats only. The affected brain regions cannot be restricted to hippocampus, prefrontal cortex and amygdala. CONCLUSION: Double-hit stress caused more pronounced and distinct behavioral, molecular and functional changes, compared to MD or SPS alone.

Interactions between Glycine and Glutamate through Activation of Their Transporters in Hippocampal Nerve Terminals.

Evidence supports the pathophysiological relevance of crosstalk between the neurotransmitters Glycine and Glutamate and their close interactions; some reports even support the possibility of Glycine-Glutamate cotransmission in central nervous system (CNS) areas, including the hippocampus. Functional studies with isolated nerve terminals (synaptosomes) permit us to study transporter-mediated interactions between neurotransmitters that lead to the regulation of transmitter release. Our main aims here were: (i) to investigate release-regulating, transporter-mediated interactions between Glycine and Glutamate in hippocampal nerve terminals and (ii) to determine the coexistence of transporters for Glycine and Glutamate in these terminals. Purified synaptosomes, analyzed at the ultrastructural level via electron microscopy, were used as the experimental model. Mouse hippocampal synaptosomes were prelabeled with [3H]D-Aspartate or [3H]Glycine; the release of radiolabeled tracers was monitored with the superfusion technique. The main findings were that (i) exogenous Glycine stimulated [3H]D-Aspartate release, partly by activation of GlyT1 and in part, unusually, through GlyT2 transporters and that (ii) D-Aspartate stimulated [3H]glycine release by a process that was sensitive to Glutamate transporter blockers. Based on the features of the experimental model used, it is suggested that functional transporters for Glutamate and Glycine coexist in a small subset of hippocampal nerve terminals, a condition that may also be compatible with cotransmission; glycinergic and glutamatergic transporters exhibit different functions and mediate interactions between the neurotransmitters. It is hoped that increased information on Glutamate-Glycine interactions in different areas, including the hippocampus, will contribute to a better knowledge of drugs acting at "glycinergic" targets, currently under study in relation with different CNS pathologies.

Reduction of BDNF Levels and Biphasic Changes in Glutamate Release in the Prefrontal Cortex Correlate with Susceptibility to Chronic Stress-Induced Anhedonia.

Chronic stress has been considered to induce depressive symptoms, such as anhedonia, particularly in susceptible individuals. Synaptic plasticity in the prefrontal cortex (PFC) is closely associated with susceptibility or resilience to chronic stress-induced anhedonia. However, effects of chronic stress with different durations on the neurobiological mechanisms that underlie susceptibility to anhedonia remain unclear. The present study investigated effects of chronic mild stress (CMS) for 14, 21, and 35 d on anhedonia-like behavior and glutamate synapses in the PFC. We found that brain-derived neurotrophic factor (BDNF) levels in the PFC significantly decreased only in anhedonia-susceptible rats that were exposed to CMS for 14, 21, and 35 d. Additionally, 14 d of CMS increased prefrontal glutamate release, and 35 d of CMS decreased glutamate release, in addition to reducing synaptic proteins and spine density in the PFC. Moreover, we found that anhedonia-like behavior in a subset of rats spontaneously decreased, accompanied by the restoration of BDNF levels and glutamate release, on day 21 of CMS. Ketamine treatment restored the reduction of BDNF levels and biphasic changes in glutamate release that were induced by CMS. Our findings revealed a progressive reduction of synaptic plasticity and biphasic changes in glutamate release in the PFC during CMS. Reductions of BDNF levels may be key neurobiological markers of susceptibility to stress-induced anhedonia.

The Key Role of Astrocytes in Amyotrophic Lateral Sclerosis and Their Commitment to Glutamate Excitotoxicity.

In the last two decades, there has been increasing evidence supporting non-neuronal cells as active contributors to neurodegenerative disorders. Among glial cells, astrocytes play a pivotal role in driving amyotrophic lateral sclerosis (ALS) progression, leading the scientific community to focus on the "astrocytic signature" in ALS. Here, we summarized the main pathological mechanisms characterizing astrocyte contribution to MN damage and ALS progression, such as neuroinflammation, mitochondrial dysfunction, oxidative stress, energy metabolism impairment, miRNAs and extracellular vesicles contribution, autophagy dysfunction, protein misfolding, and altered neurotrophic factor release. Since glutamate excitotoxicity is one of the most relevant ALS features, we focused on the specific contribution of ALS astrocytes in this aspect, highlighting the known or potential molecular mechanisms by which astrocytes participate in increasing the extracellular glutamate level in ALS and, conversely, undergo the toxic effect of the excessive glutamate. In this scenario, astrocytes can behave as "producers" and "targets" of the high extracellular glutamate levels, going through changes that can affect themselves and, in turn, the neuronal and non-neuronal surrounding cells, thus actively impacting the ALS course. Moreover, this review aims to point out knowledge gaps that deserve further investigation.

Pregabalin-Tolperisone Combination to Treat Neuropathic Pain: Improved Analgesia and Reduced Side Effects in Rats.

The current treatment of neuropathic pain (NP) is unsatisfactory; therefore, effective novel agents or combination-based analgesic therapies are needed. Herein, oral tolperisone, pregabalin, and duloxetine were tested for their antinociceptive effect against rat partial sciatic nerve ligation (pSNL)-induced tactile allodynia described by a decrease in the paw withdrawal threshold (PWT) measured by a dynamic plantar aesthesiometer. On day 7 after the operation, PWTs were assessed at 60, 120, and 180 min post-treatment. Chronic treatment was continued for 2 weeks, and again, PWTs were measured on day 14 and 21. None of the test compounds produced an acute antiallodynic effect. In contrast, after chronic treatment, tolperisone and pregabalin alleviated allodynia. In other experiments, on day 14, the acute antiallodynic effect of the tolperisone/pregabalin or duloxetine combination was measured. As a novel finding, a single dose of the tolperisone/pregabalin combination could remarkably alleviate allodynia acutely. It also restored the neuropathy-induced elevated CSF glutamate content. Furthermore, the combination is devoid of adverse effects related to motor and gastrointestinal transit functions. Tolperisone and pregabalin target voltage-gated sodium and calcium channels, respectively. The dual blockade effect of the combination might explain its advantageous acute analgesic effect in the present work.

Caveolin-1 is essential for the increased release of glutamate in the anterior cingulate cortex in neuropathic pain mice.

Neuropathic pain has a complex pathogenesis. Here, we examined the role of caveolin-1 (Cav-1) in the anterior cingulate cortex (ACC) in a chronic constriction injury (CCI) mouse model for the enhancement of presynaptic glutamate release in chronic neuropathic pain. Cav-1 was localized in glutamatergic neurons and showed higher expression in the ACC of CCI versus sham mice. Moreover, the release of glutamate from the ACC of the CCI mice was greater than that of the sham mice. Inhibition of Cav-1 by siRNAs greatly reduced the release of glutamate of ACC, while its overexpression (induced by injecting Lenti-Cav-1) reversed this process. The chemogenetics method was then used to activate or inhibit glutamatergic neurons in the ACC area. After 21 days of injection of AAV-hM3Dq in the sham mice, the release of glutamate was increased, the paw withdrawal latency was shortened, and expression of Cav-1 in the ACC was upregulated after intraperitoneal injection of 2 mg/kg clozapine N-oxide. Injection of AAV-hM4Di in the ACC of CCI mice led to the opposite effects. Furthermore, decreasing Cav-1 in the ACC in sham mice injected with rAAV-hM3DGq did not increase glutamate release. These findings suggest that Cav-1 in the ACC is essential for enhancing glutamate release in neuropathic pain.

Glutamate-releasing BEST1 channel is a new target for neuroprotection against ischemic stroke with wide time window.

Many efforts have been made to understand excitotoxicity and develop neuroprotectants for the therapy of ischemic stroke. The narrow treatment time window is still to be solved. Given that the ischemic core expanded over days, treatment with an extended time window is anticipated. Bestrophin 1 (BEST1) belongs to a bestrophin family of calcium-activated chloride channels. We revealed an increase in neuronal BEST1 expression and function within the peri-infarct from 8 to 48 h after ischemic stroke in mice. Interfering the protein expression or inhibiting the channel function of BEST1 by genetic manipulation displayed neuroprotective effects and improved motor functional deficits. Using electrophysiological recordings, we demonstrated that extrasynaptic glutamate release through BEST1 channel resulted in delayed excitotoxicity. Finally, we confirmed the therapeutic efficacy of pharmacological inhibition of BEST1 during 6-72 h post-ischemia in rodents. This delayed treatment prevented the expansion of infarct volume and the exacerbation of neurological functions. Our study identifies the glutamate-releasing BEST1 channel as a potential therapeutic target against ischemic stroke with a wide time window.

Photons Induce Vesicular Exocytotic Release of Glutamate in a Power-Dependent Way.

Increasing evidence indicates that photobiomodulation, based on tissue irradiation with photons in the red to near-infrared spectrum, may be an effective therapeutic approach to central nervous system disorders. Although nervous system functionality has been shown to be affected by photons in animal models, as well as in preliminary evidence in healthy subjects or in patients with neuropsychiatric disorders, the mechanisms involved in the photobiomodulation effects have not yet been clarified. We previously observed that photobiomodulation could stimulate glutamate release. Here, we investigate mechanisms potentially involved in the glutamate-releasing effect of photons from adult mouse cerebrocortical nerve terminals. We report evidence of photon ability to induce an exocytotic vesicular release of glutamate from the terminals of glutamatergic neurons in a power-dependent way. It can be hypothesized that photobiomodulation, depending on the potency, can release glutamate in a potentially neurotoxic or physiological range.

Cognitive dysfunction and impaired neuroplasticity following repeated exposure to the synthetic cannabinoid JWH-018 in male mice.

BACKGROUND AND PURPOSE: Psychotic disorders have been reported in long-term users of synthetic cannabinoids. This study aims at investigating the long-lasting effects of repeated JWH-018 exposure. EXPERIMENTAL APPROACH: Male CD-1 mice were injected with vehicle, JWH-018 (6 mg·kg-1 ), the CB1 -antagonist NESS-0327 (1 mg·kg-1 ) or co-administration of NESS-0327 and JWH-018, every day for 7 days. After 15 or 16 days washout, we investigated the effects of JWH-018 on motor function, memory, social dominance and prepulse inhibition (PPI). We also evaluated glutamate levels in dialysates from dorsal striatum, striatal dopamine content and striatal/hippocampal neuroplasticity focusing on the NMDA receptor complex and the neurotrophin BDNF. These measurements were accompanied by in vitro electrophysiological evaluations in hippocampal preparations. Finally, we investigated the density of CB1 receptors and levels of the endocannabinoid anandamide (AEA) and 2-arachidonoylglycerol (2-AG) and their main synthetic and degrading enzymes in the striatum and hippocampus. KEY RESULTS: The repeated treatment with JWH-018 induced psychomotor agitation while reducing social dominance, recognition memory and PPI in mice. JWH-018 disrupted hippocampal LTP and decreased BDNF expression, reduced the synaptic levels of NMDA receptor subunits and decreased the expression of PSD95. Repeated exposure to JWH-018, reduced hippocampal CB1 receptor density and induced a long-term alteration in AEA and 2-AG levels and their degrading enzymes, FAAH and MAGL, in the striatum. CONCLUSION AND IMPLICATIONS: Our findings suggest that repeated administration of a high dose of JWH-018 leads to the manifestation of psychotic-like symptoms accompanied by alterations in neuroplasticity and change in the endocannabinoid system.

Striatal astrocytic A2A-D2 receptor-receptor interactions and their role in neuropsychiatric disorders.

It is now generally accepted that astrocytes are active players in synaptic transmission, so that a neurocentric perspective of the integrative signal communication in the central nervous system is shifting towards a neuro-astrocentric perspective. Astrocytes respond to synaptic activity, release chemical signals (gliotransmitters) and express neurotransmitter receptors (G protein-coupled and ionotropic receptors), thus behaving as co-actors with neurons in signal communication in the central nervous system. The ability of G protein-coupled receptors to physically interact through heteromerization, forming heteromers and receptor mosaics with new distinct signal recognition and transduction pathways, has been intensively studied at neuronal plasma membrane, and has changed the view of the integrative signal communication in the central nervous system. One of the best-known examples of receptor-receptor interaction through heteromerization, with relevant consequences for both the physiological and the pharmacological points of view, is given by adenosine A2A and dopamine D2 receptors on the plasma membrane of striatal neurons. Here we review evidence that native A2A and D2 receptors can interact through heteromerization at the plasma membrane of astrocytes as well. Astrocytic A2A-D2 heteromers were found able to control the release of glutamate from the striatal astrocyte processes. A2A-D2 heteromers on striatal astrocytes and astrocyte processes are discussed as far as their potential relevance in the control of glutamatergic transmission in striatum is concerned, including potential roles in glutamatergic transmission dysregulation in pathological conditions including schizophrenia or the Parkinson's disease. This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".

Mangiferin depresses vesicular glutamate release in synaptosomes from the rat cerebral cortex by decreasing synapsin I phosphorylation.

Mangiferin is a glucosyl xanthone that has been shown to be a neuroprotective agent against brain disorders involving excess glutamate. However, the effect of mangiferin on the function of the glutamatergic system has not been investigated. In this study, we used synaptosomes from the rat cerebral cortex to investigate the effect of mangiferin on glutamate release and identify the possible underlying mechanism. We observed that mangiferin produced a concentration-dependent reduction in the release of glutamate elicited by 4-aminopyridine with an IC50 value of 25 µM. Inhibition of glutamate release was blocked by removing extracellular calcium and by treatment with the vacuolar-type H+-ATPase inhibitor bafilomycin A1, which prevents the uptake and storage of glutamate in vesicles. Moreover, we showed that mangiferin decreased the 4-aminopyridine-elicited FM1-43 release and synaptotagmin 1 luminal domain antibody (syt1-L ab) uptake from synaptosomes, which correlated with decreased synaptic vesicle exocytosis. Transmission electron microscopy in synaptosomes also showed that mangiferin attenuated the 4-aminopyridine-elicited decrease in the number of synaptic vesicles. In addition, antagonism of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase A (PKA) counteracted mangiferin's effect on glutamate release. Mangiferin also decreased the phosphorylation of CaMKII, PKA, and synapsin I elicited by 4-aminopyridine treatment. Our data suggest that mangiferin reduces PKA and CaMKII activation and synapsin I phosphorylation, which could decrease synaptic vesicle availability and lead to a subsequent reduction in vesicular glutamate release from synaptosomes.

Plantainoside D Reduces Depolarization-Evoked Glutamate Release from Rat Cerebral Cortical Synaptosomes.

Inhibiting the excessive release of glutamate in the brain is emerging as a promising therapeutic option and is efficient for treating neurodegenerative disorders. The aim of this study is to investigate the effect and mechanism of plantainoside D (PD), a phenylenthanoid glycoside isolated from Plantago asiatica L., on glutamate release in rat cerebral cortical nerve terminals (synaptosomes). We observed that PD inhibited the potassium channel blocker 4-aminopyridine (4-AP)-evoked release of glutamate and elevated concentration of cytosolic Ca2+. Using bafilomycin A1 to block glutamate uptake into synaptic vesicles and EDTA to chelate extracellular Ca2+, the inhibitory effect of PD on 4-AP-evoked glutamate release was prevented. In contrast, the action of PD on the 4-AP-evoked release of glutamate in the presence of dl-TBOA, a potent nontransportable inhibitor of glutamate transporters, was unaffected. PD does not alter the 4-AP-mediated depolarization of the synaptosomal membrane potential, suggesting that the inhibitory effect of PD on glutamate release is associated with voltage-dependent Ca2+ channels (VDCCs) but not the modulation of plasma membrane potential. Pretreatment with the Ca2+ channel blocker (N-type) ω-conotoxin GVIA abolished the inhibitory effect of PD on the evoked glutamate release, as did pretreatment with the protein kinase C inhibitor GF109203x. However, the PD-mediated inhibition of glutamate release was eliminated by applying the mitochondrial Na+/Ca2+ exchanger inhibitor CGP37157 or dantrolene, which inhibits Ca2+ release through ryanodine receptor channels. These data suggest that PD mediates the inhibition of evoked glutamate release from synaptosomes primarily by reducing the influx of Ca2+ through N-type Ca2+ channels, subsequently reducing the protein kinase C cascade.

Brainstem serotonin neurons selectively gate retinal information flow to thalamus.

Retinal ganglion cell (RGC) types relay parallel streams of visual feature information. We hypothesized that neuromodulators might efficiently control which visual information streams reach the cortex by selectively gating transmission from specific RGC axons in the thalamus. Using fiber photometry recordings, we found that optogenetic stimulation of serotonergic axons in primary visual thalamus of awake mice suppressed ongoing and visually evoked calcium activity and glutamate release from RGC boutons. Two-photon calcium imaging revealed that serotonin axon stimulation suppressed RGC boutons that responded strongly to global changes in luminance more than those responding only to local visual stimuli, while the converse was true for suppression induced by increases in arousal. Converging evidence suggests that differential expression of the 5-HT1B receptor on RGC presynaptic terminals, but not differential density of nearby serotonin axons, may contribute to the selective serotonergic gating of specific visual information streams before they can activate thalamocortical neurons.

Endogenous Modulators of NMDA Receptor Control Dendritic Field Expansion of Cortical Neurons.

Impairments of N-methyl-D-aspartate receptor (NMDAR) activity have been implicated in several neuropsychiatric disorders, with pharmacological inhibition of NMDAR-mediated currents and associated neurobehavioral changes considered as a model of schizophrenia. We analyzed the effects of brief and long-term exposure of rat cortical cultures to the most prevalent endogenous modulators of NMDAR (kynurenic acid, pregnenolone sulfate, spermidine, and zinc) on neuronal viability, stimulation-induced release of glutamate, and dendritic morphology with synaptic density. Both, glutamate release and neuronal viability studies revealed no difference between the test and control groups. No differences were also observed in the number of dendritic branching and length, or density of synaptic connections and neuronal soma size. Comparison of the extent of dendritic projections and branching patterns, however, revealed enhanced distal arborization with the expansion of the dendritic area under prolonged treatment of cultures with physiological concentrations of NMDAR modulators, with differences reaching significance in spermidine and pregnenolone sulfate tests. Measurements of the density of glutamatergic synapses showed consistency across all neuronal groups, except those treated with pregnenolone sulfate, which showed a reduction of PSD-95-positive elements. Overall, our data suggest that constitutive glutamatergic activity mediated by NMDAR controls the dendritic field expansion and can influence the integrative properties of cortical neurons.