Glutamate Release In Rats Research Articles

Annexin A7 induction of neuronal apoptosis via effect on glutamate release in a rat model of subarachnoid hemorrhage.

Glutamate excitotoxicity and neuronal apoptosis are suggested to contribute to early brain injury after subarachnoid hemorrhage (SAH). Annexin A7 (ANXA7) has been shown to regulate glutamate release. However, the role of ANXA7 in early brain injury after SAH has not been illustrated. In this study, we aimed to investigate the effect of ANXA7 knockdown in reducing the severity of early brain injury after SAH, and determine the underlying mechanisms. Endovascular perforation was performed to induce SAH in male Sprague-Dawley rats. ANXA7-siRNA was administered via intraventricular injection 5 days before SAH induction. Neurological test, evaluation of SAH grade, assessment of blood-brain barrier (BBB) permeability, measurement of brain water content, Western blot, double immunofluorescence staining, TUNEL staining, and enzyme-linked immunosorbent assay (ELISA) were performed at 24 hours of SAH induction. ANXA7 protein expression increased significantly after SAH induction and was seen mainly in neurons. High expression of ANXA7 was associated with poor neurological status. ANXA7 knockdown dramatically ameliorated early brain injury through alleviating BBB disruption and brain edema. Further investigation of the mechanism showed that inhibiting ANXA7 expression can rescue neuronal apoptosis. In addition, ANXA7 knockdown also significantly reduced glutamate release, which was consistent with a significant increase of Bcl-2 expression and decreases of Bax and cleaved caspase-3 expression. ANXA7 can induce neuronal apoptosis by affecting glutamate release in rats with SAH. Downregulating the expression of ANXA7 can significantly attenuate early brain injury after SAH. Future therapy targeting ANXA7 may be a promising new choice.

Involvement of the Hypothalamic Glutamatergic System in the Development of Radiation-Induced Pica in Rats.

Since the peripheral serotoninergic pathway is involved in the development of radiation-induced nausea and vomiting, referred to as radiation sickness, serotonin 5-HT3 receptor antagonists are used as a preventive measure, although patients still suffer from these symptoms. Glutamate is known as the excitatory neurotransmitter and is involved in various autonomic symptoms. We investigated the effect of radiation on glutamate release in rats, as measured by in vivo brain microdialysis, and the effects of glutamate receptor antagonists on radiation-induced pica, which can be used as a behavioral assessment of radiation sickness in rats. A microdialysis probe was inserted into the hypothalamus of rats that received 4 Gy total-body irradiation (TBI) with or without pretreatment of 5-HT3 receptor antagonist (granisetron, 0.1 mg/kg, i.p.), and dialysates were collected for 3 h after TBI and subjected to HPLC assay of glutamate. In addition, rats were intracerebroventricularly injected with NMDA receptor antagonist (MK-801: 3 μg/rat) or AMPA receptor antagonist (CNQX: 1 μg/rat) before TBI, and radiation-induced pica was determined. An increase in glutamate release was observed within 1 h postirradiation. The increased glutamate release was suppressed by granisetron. We also found that CNQX, but not MK-801, effectively inhibited radiation-induced pica. These results indicate that the hypothalamic glutamatergic system contributes to radiation-induced pica through the AMPA receptors.

Fentanyl and naloxone effects on glutamate and GABA release rates from anterior hypothalamus in freely moving rats

Fentanyl, a μ-opioid receptor agonist, has been studied for its neuro/psycho-pharmacological effects since its first clinical use; however, its effect on the release rate of the Central Nervous System (CNS) neurotransmitters has not been yet elucidated. In the present study the influence of fentanyl on the release rates of glutamate and GABA is investigated. Specifically, we examined the effects of intravenous (10 μg/kg) as well as intrahypothalamic (0.1nmol/min) fentanyl administration on the release rates of GABA and glutamate in the superfusate of anterior hypothalamus, under tail pinch manipulation. The release rate of the neurotransmitters was monitored by the push–pull superfusion technique. To investigate the role of fentanyl the opioid antagonist, naloxone 0.1 mg/kg was administered intravenously, or 50nmol/min intrahypothalamicaly. The amino acids were determined by High Performance Liquid Chromatography (HPLC) and fluorimetric detection after NBD-Cl derivatisation. After intravenous fentanyl administration a significant decrease of glutamate and increase of GABA release rates were observed. However during the pain manipulations, the release rate of glutamate was increased. Intravenous naloxone did not affect significantly the release rates of both amino acids, while intrahypothalamic antagonist administration reversed the alterations in both neurotransmitters release rates. Our results demonstrate that there is an opioid-glutamatergic transmission pathway, located in hypothalamus and that opioids can activate NMDA receptors, thus reducing the nociceptive threshold and the opioid analgesic effect.

Age-dependent oxidation of extracellular cysteine/cystine redox state (Eh(Cys/CySS)) in mouse lung fibroblasts is mediated by a decline in Slc7a11 expression.

Aging is associated with progressive oxidation of the extracellular environment. The redox state of human plasma, defined by the concentrations of cysteine (Cys) and cystine (CySS), becomes more oxidized as we age. Recently, we showed that fibroblasts isolated from the lungs of young and old mice retain this differential phenotype; old cells produce and maintain a more oxidizing extracellular redox potential (Eh(Cys/CySS)) than young cells. Microarray analysis identified down-regulation of Slc7a11, the light subunit of the CySS/glutamate transporter, as a potential mediator of age-related oxidation in these cells. The purpose of the present study was to investigate the mechanistic link between Slc7a11 expression and extracellular Eh(Cys/CySS). Sulforaphane treatment or overexpression of Slc7a11 was used to increase Slc7a11 in lung fibroblasts from old mice, and sulfasalazine treatment or siRNA-mediated knock down was used to decrease Slc7a11 in young fibroblasts. Slc7a11 mRNA levels were measured by real-time PCR, Slc7a11 activity was determined by measuring the rate of glutamate release, Cys, CySS, glutathione (GSH) and its disulfide (GSSG) were measured by HPLC, and Eh(Cys/CySS) was calculated from the Nernst equation. The results showed that both Eh(Cys/CySS) and Eh(GSH/GSSG) were more oxidized in the conditioned media of old cells than in young cells. Up-regulation of Slc7a11 via overexpression or sulforaphane treatment restored extracellular Eh(Cys/CySS) in cultures of old cells, whereas down-regulation reproduced the oxidizing Eh(Cys/CySS) in young cells. Only sulforaphane treatment was able to increase total GSH and restore Eh(GSH/GSSG), whereas overexpression, knock down and sulfasalazine had no effect on these parameters. In addition, inhibition of GSH synthesis with buthionine sulfoximine had no effect on the ability of cells to restore their extracellular redox potential in response to an oxidative challenge. In conclusion, our study reveals Slc7a11 is the key regulator of age-dependent changes in extracellular Eh(Cys/CySS) in primary mouse lung fibroblasts, and its effects are not dependent on GSH synthesis.

Berberine attenuates convulsing behavior and extracellular glutamate and aspartate changes in 4-aminopyridine treated rats.

Objective(s):K+ channel blocker 4-aminopyridine (4-AP) stimulates the release of glutamate from nerve terminals and induces seizures. Berberine as a potential herbal drug exerts several pharmacological actions on the central nervous system including anxiolytic, anticonvulsant, and neuroprotective properties. The present study aimed to investigate the effect of berberine on seizure onset and time course of the extracellular levels of excitatory amino acids (EAA), glutamate and aspartate, changes produced by 4-AP in rat hippocampus.Materials and Methods:The rats were given either saline or berberine (50, 100 and 200 mg/kg, IP) 40 min before administration of 4-AP (15 mg/kg, IP) and the onset of seizure was recorded. A group of rats also received diazepam (DZP, 15 mg/kg, IP) 20 min prior to 4-AP administration. Hippocampal extracellular levels of EAA were also measured using microdialysis assay. Analysis of the dialysate samples was performed by reversed-phase high performance liquid chromatography (HPLC) with precolumn derivatization with o-phthaldialdehyde and fluorescence detection.Results:Our findings suggest that berberine significantly delayed the seizure onset following 4-AP injection. There was a considerable increase in the extracellular glutamate and aspartate levels in 4-AP treated rats and 4-AP-evoked release of EAA was sharply reduced (about 4-5 fold especially at 20 min after 4-AP administration) in berberine treatment groups.Conclusion:The results of present study show that berberine attenuates 4-AP induced seizures by decreasing hippocampal aspartate and glutamate release in rats.

Propylparaben applied after pilocarpine-induced status epilepticus modifies hippocampal excitability and glutamate release in rats

Propylparaben (PPB) induces cardioprotection after ischemia–reperfusion injury by inhibiting voltage-dependent Na+ channels. The present study focuses on investigating whether the i.p. application of 178mg/kg PPB after pilocarpine-induced status epilepticus (SE) reduces the acute and long-term consequences of seizure activity. Initially, we investigated the effects of a single administration of PPB after SE. Our results revealed that compared to rats receiving diazepam (DZP) plus vehicle after 2h of SE, animals receiving a single dose of PPB 1h after DZP injection presented 126% (p<0.001) lower extracellular levels of glutamate in the hippocampus. This effect was associated with an increased potency of low-frequency oscillations (0.1–13Hz bands, p<0.001), a reduced potency of 30–250Hz bands (p<0.001) and less neuronal damage in the hippocampus. The second experiment examined whether the subchronic administration of PPB during the post-SE period is able to prevent the long-term consequences of seizure activity. In comparison to animals that were treated subchronically with vehicle after SE, rats administered with PPB for 5 days presented lower hippocampal excitability and interictal glutamate release, astrogliosis, and neuroprotection in the dentate gyrus. Our data indicate that PPB, when applied after SE, can be used as a therapeutic strategy to reduce the consequences of seizure activity.

Pulsed radiofrequency attenuates diabetic neuropathic pain and suppresses formalin-evoked spinal glutamate release in rats.

BACKGROUND: Pulsed radiofrequency (PRF) has been used to treat chronic pain for years, but its effectiveness and mechanism in treating diabetic neuropathic pain are still unexplored. The aim of this study was to elucidate the modulation of diabetic neuropathic pain induced by streptozotocin and the release of spinal excitatory amino acids by PRF.METHODS: Diabetes was induced by intraperitoneal administration of streptozotocin. Pulsed radiofrequency was applied to L5 and L6 dorsal roots at 42 °C for 2 min. The responses of all of the groups to thermal, mechanical and cold stimuli were measured for a period of 6 d after this process. Seven days after PRF treatment, intrathecal microdialysis was used to examine the effect of pulsed radiofrequency on the formalin-evoked spinal release of excitatory amino acids and concurrent behaviour responses from diabetic rats.RESULTS: Three weeks after intraperitoneal streptozotocin treatment and before PRF application, mechanical, thermal and cold hypersensitivity occurred. Application of PRF significantly alleviated hyperglycaemia-induced mechanical, thermal and cold hypersensitivity and also attenuated the increase in formalin-evoked CSF glutamate concentration, compared with sham treated diabetic rats.CONCLUSION: It may be concluded that PRF has an analgesic effect on neuropathic pain by suppressing the nociception-induced release of excitatory neurotransmitters. PRF may provide a novel promising therapeutic approach for managing diabetic neuropathic pain.

High-Throughput Assay Development for Cystine-Glutamate Antiporter (xc-) Highlights Faster Cystine Uptake than Glutamate Release in Glioma Cells.

The cystine-glutamate antiporter (system xc -) is a Na+-independent amino acid transporter that exchanges extracellular cystine for intracellular glutamate. It is thought to play a critical role in cellular redox processes through regulation of intracellular glutathione synthesis via cystine uptake. In gliomas, system xc - expression is universally up-regulated while that of glutamate transporters down-regulated, leading to a progressive accumulation of extracellular glutamate and excitotoxic cell death of the surrounding non-tumorous tissue. Additionally, up-regulation of system xc - in activated microglia has been implicated in the pathogenesis of several neurodegenerative disorders mediated by excess glutamate. Consequently, system xc - is a new drug target for brain cancer and neuroinflammatory diseases associated with excess extracellular glutamate. Unfortunately no potent and selective small molecule system xc - inhibitors exist and to our knowledge, no high throughput screening (HTS) assay has been developed to identify new scaffolds for inhibitor design. To develop such an assay, various neuronal and non-neuronal human cells were evaluated as sources of system xc -. Human glioma cells were chosen based on their high system xc - activity. Using these cells, [14C]-cystine uptake and cystine-induced glutamate release assays were characterized and optimized with respect to cystine and protein concentrations and time of incubation. A pilot screen of the LOPAC/NINDS libraries using glutamate release demonstrated that the logistics of the assay were in place but unfortunately, did not yield meaningful pharmacophores. A larger, HTS campaign using the 384-well cystine-induced glutamate release as primary assay and the 96-well 14C-cystine uptake as confirmatory assay is currently underway. Unexpectedly, we observed that the rate of cystine uptake was significantly faster than the rate of glutamate release in human glioma cells. This was in contrast to the same rates of cystine uptake and glutamate release previously reported in normal human fibroblast cells.

Hypotheses for Hair Cell Ribbon Synapses Probed by a Biomimetic System

The conversion of sound waves to electrical impulse in the auditory nerve of the inner ear takes place in the organ of Corti through the process of synaptic transmission in and around the ribbon synapse. Finding the relation between the functional and physiological characteristics of the ribbon synapse is one of the central issues in auditory science nowadays. There has been a hypothesis on inner hair cells (IHC) that complex broad band sounds can be encoded by multiple ribbon synapses in an IHC. We provide a hypothesis on the spectral analysis by multiple ribbon synapses, where heterogenous glutamate release rates in a IHC is the key to the spectral analysis. To demonstrate our hypothesis, we fabricate a neuromorphic device that models the hair cell bundle and its afferent neuron, and investigate its response to pure-tone and human voice sounds. Our biomimetic experimental results go beyond theoretical simulation While mechanical oscillators’ velocity dependence of relaxation and noise can not but be assumed theoretically, our biomimetic approach goes beyond this limitation of such assumption and is self-consistent by its own. We observe that for the neuronal spike rates to mimic the voice sound signal of a word, a distribution of the spontaneous neurotransmitter relaxation rates is necessary. We also discuss a physical microscopic model for the ribbon synapse which is based on charge transport theory.

Calmodulin enhances ribbon replenishment and shapes filtering of synaptic transmission by cone photoreceptors.

At the first synapse in the vertebrate visual pathway, light-evoked changes in photoreceptor membrane potential alter the rate of glutamate release onto second-order retinal neurons. This process depends on the synaptic ribbon, a specialized structure found at various sensory synapses, to provide a supply of primed vesicles for release. Calcium (Ca(2+)) accelerates the replenishment of vesicles at cone ribbon synapses, but the mechanisms underlying this acceleration and its functional implications for vision are unknown. We studied vesicle replenishment using paired whole-cell recordings of cones and postsynaptic neurons in tiger salamander retinas and found that it involves two kinetic mechanisms, the faster of which was diminished by calmodulin (CaM) inhibitors. We developed an analytical model that can be applied to both conventional and ribbon synapses and showed that vesicle resupply is limited by a simple time constant, τ = 1/(Dρδs), where D is the vesicle diffusion coefficient, δ is the vesicle diameter, ρ is the vesicle density, and s is the probability of vesicle attachment. The combination of electrophysiological measurements, modeling, and total internal reflection fluorescence microscopy of single synaptic vesicles suggested that CaM speeds replenishment by enhancing vesicle attachment to the ribbon. Using electroretinogram and whole-cell recordings of light responses, we found that enhanced replenishment improves the ability of cone synapses to signal darkness after brief flashes of light and enhances the amplitude of responses to higher-frequency stimuli. By accelerating the resupply of vesicles to the ribbon, CaM extends the temporal range of synaptic transmission, allowing cones to transmit higher-frequency visual information to downstream neurons. Thus, the ability of the visual system to encode time-varying stimuli is shaped by the dynamics of vesicle replenishment at photoreceptor synaptic ribbons.

Physiological temperatures drive glutamate release onto trigeminal superficial dorsal horn neurons

Trigeminal sensory afferent fibers terminating in nucleus caudalis (Vc) relay sensory information from craniofacial regions to the brain and are known to express transient receptor potential (TRP) ion channels. TRP channels are activated by H(+), thermal, and chemical stimuli. The present study investigated the relationships among the spontaneous release of glutamate, temperature, and TRPV1 localization at synapses in the Vc. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded from Vc neurons (n = 151) in horizontal brain-stem slices obtained from Sprague-Dawley rats. Neurons had basal sEPSC rates that fell into two distinct frequency categories: High (≥10 Hz) or Low (<10 Hz) at 35°C. Of all recorded neurons, those with High basal release rates (67%) at near-physiological temperatures greatly reduced their sEPSC rate when cooled to 30°C without amplitude changes. Such responses persisted during blockade of action potentials indicating that the High rate of glutamate release arises from presynaptic thermal mechanisms. Neurons with Low basal frequencies (33%) showed minor thermal changes in sEPSC rate that were abolished after addition of TTX, suggesting these responses were indirect and required local circuits. Activation of TRPV1 with capsaicin (100 nM) increased miniature EPSC (mEPSC) frequency in 70% of neurons, but half of these neurons had Low basal mEPSC rates and no temperature sensitivity. Our evidence indicates that normal temperatures (35-37°C) drive spontaneous excitatory synaptic activity within superficial Vc by a mechanism independent of presynaptic action potentials. Thus thermally sensitive inputs on superficial Vc neurons may tonically activate these neurons without afferent stimulation.

Excitotoxic neuronal cell death during an oligodendrocyte-directed CD8+ T cell attack in the CNS gray matter

BackgroundNeural-antigen reactive cytotoxic CD8+ T cells contribute to neuronal dysfunction and degeneration in a variety of inflammatory CNS disorders. Facing excess numbers of target cells, CNS-invading CD8+ T cells cause neuronal cell death either via confined release of cytotoxic effector molecules towards neurons, or via spillover of cytotoxic effector molecules from 'leaky’ immunological synapses and non-confined release by CD8+ T cells themselves during serial and simultaneous killing of oligodendrocytes or astrocytes.MethodsWild-type and T cell receptor transgenic CD8+ T cells were stimulated in vitro, their activation status was assessed by flow cytometry, and supernatant glutamate levels were determined using an enzymatic assay. Expression regulation of molecules involved in vesicular glutamate release was examined by quantitative real-time PCR, and mechanisms of non-vesicular glutamate release were studied by pharmacological blocking experiments. The impact of CD8+ T cell-mediated glutamate liberation on neuronal viability was studied in acute brain slice preparations.ResultsFollowing T cell receptor stimulation, CD8+ T cells acquire the molecular repertoire for vesicular glutamate release: (i) they upregulate expression of glutaminase required to generate glutamate via deamination of glutamine and (ii) they upregulate expression of vesicular proton-ATPase and vesicular glutamate transporters required for filling of vesicles with glutamate. Subsequently, CD8+ T cells release glutamate in a strictly stimulus-dependent manner. Upon repetitive T cell receptor stimulation, CD25high CD8+ T effector cells exhibit higher estimated single cell glutamate release rates than CD25low CD8+ T memory cells. Moreover, glutamate liberation by oligodendrocyte-reactive CD25high CD8+ T effector cells is capable of eliciting collateral excitotoxic cell death of neurons (despite glutamate re-uptake by glia cells and neurons) in intact CNS gray matter.ConclusionGlutamate release may represent a crucial effector pathway of neural-antigen reactive CD8+ T cells, contributing to excitotoxicity in CNS inflammation.

Facilitation of short‐term memory by histaminergic neurons in the nucleus accumbens is independent of cholinergic and glutamatergic transmission

Here, we have investigated whether learning and/or short-term memory was associated with release of ACh and glutamate in the rat nucleus accumbens (NAc). Additionally, neurotransmitter release in the NAc was assessed during facilitation of cognitive processes by antagonists of inhibitory histamine autoreceptors. The olfactory, social memory test was used in combination with push-pull superfusion of the NAc. A male, juvenile rat was exposed twice to an adult male rat at intervals of 60 or 90 min, and release of ACh and glutamate was determined in the NAc of the conscious adult rat. Histamine receptor antagonists were applied i.c.v. First exposure of a juvenile rat to an adult rat increased ACh and glutamate release in the NAc of the adult rat. Repetition of exposure after 60 min did not change release of ACh and glutamate, while contact time to recognition (CTR) was shortened. Repetition of exposure after an interval of 90 min prolonged CTR and enhanced accumbal ACh and glutamate release rates. Injection (i.c.v.) of thioperamide (histamine H3 receptor antagonist) together with famotidine (H₂ receptor antagonist), 80 min prior to second exposure, diminished CTR and abolished ACh and glutamate release when second exposure was carried out 90 min after the first one. Histaminergic neurons per se facilitated short-term memory, without activation of cholinergic and/or glutamatergic neurons in the NAc of rats. Cholinergic and glutamatergic neurons within the NAc contributed to learning but not to recall of memory.

Zinc modulation of calcium activity at the photoreceptor terminal: A calcium imaging study

There is abundant experimental evidence that zinc ions (Zn2+) are present in the synaptic vesicles of vertebrate photoreceptors, and that they are co-released with glutamate. Here we show that increasing the concentration of extracellular zinc (2 μM–2 mM) suppresses the entry of calcium into the synaptic terminals of isolated salamander double cones. The resultant dose-dependent curve was fit by an inverse Hill equation having an IC50 of 38 μM, and Hill coefficient of 1.1. Because there is currently no reliable way to measure the concentration of extracellular zinc, it is not known whether the zinc released under normal circumstances is of physiological significance. In an attempt to circumvent this problem we used zinc chelators to reduce the available pool of endogenous zinc. This enabled us to determine how the absence of zinc affected calcium entry. We found that when intra- or extra-cellular zinc was chelated by 250 μM of membrane-permeable TPEN or 500 μM of membrane-impermeable histidine, there was a significant rise in the depolarization-induced intracellular calcium level within photoreceptor terminals. This increase in internal [Ca2+] will undoubtedly lead to a concomitant increase in glutamate release. In addition, we found that blocking the L-type calcium channels that are expressed on the synaptic terminals of photoreceptors with 50 μM nicardipine or 100 μM verapamil abolished the effects of zinc chelation. These findings are a good indication that, when released in vivo, the zinc concentration is sufficient to suppress voltage-gated calcium channels, and reduce the rate of glutamate release from photoreceptor terminals.

Nonlinear Decoding and Asymmetric Representation of Neuronal Input Information by CaMKIIα and Calcineurin

How information encoded in glutamate release rates at individual synapses is converted into biochemical activation patterns of postsynaptic enzymes remains unexplored. To address this, we developed a dual fluorescence resonance energy transfer (FRET) imaging platform and recorded CaMKIIα and calcineurin activities in hippocampal neurons while varying glutamate uncaging frequencies. With little spine morphological change, 5 Hz spine glutamate uncaging strongly stimulated calcineurin, but not CaMKIIα. In contrast, 20 Hz spine glutamate uncaging, which induced spine growth, activated both CaMKIIα and calcineurin with distinct spatiotemporal kinetics. Higher temporal resolution recording in the soma revealed that CaMKIIα activity summed supralinearly and sensed both higher frequency and input number, thus acting as an input frequency/number decoder. In contrast, calcineurin activity summated sublinearly with increasing input number and showed little frequency dependence, thus functioning as an input number counter. These results provide evidence that CaMKIIα and calcineurin are fine-tuned to unique bandwidths and compute input variables in an asymmetric manner.

Glutamate Spillover between Mammalian Cone Photoreceptors

Cone photoreceptors transmit signals at high temporal frequencies and mediate fine spatial vision. High-frequency transmission requires a high rate of glutamate release, which could promote spillover to neighboring cells, whereas spatial vision requires that cones within a tightly packed array signal light to postsynaptic bipolar cells with minimal crosstalk. Glutamate spread from the cone terminal is thought to be limited by presynaptic transporters and nearby glial processes. In addition, there is no ultrastructural evidence for chemical synapses between mammalian cones, although such synapses have been described in lower vertebrate retinas. We tested for cone-cone glutamate diffusion by recording from adjacent cone pairs in the ground squirrel retina, and instead found that the glutamate released by one cone during electrical stimulation activates glutamate transporter Cl(-) conductances on neighboring cones. Unlike in other systems, where crosstalk is diminished by increasing the temperature and by moving to a more intact preparation, glutamate spread persisted at physiological temperatures (37°C) and in retinal flat mounts. The glutamate-gated anion conductance in cones has a reversal potential of ∼-30 mV compared with a cone resting potential of ∼-50 mV; thus, crosstalk should have a depolarizing effect on the cone network. Cone-cone glutamate spread is regulated by the physiological stimulus, light, and under physiological conditions can produce a response of ∼2 mV, equivalent to 13-20% of a cone's light response. We conclude that in the absence of discrete chemical synapses, glutamate flows between cones during a light response and may mediate a spatially distributed positive feedback.

Thermally Active TRPV1 Tonically Drives Central Spontaneous Glutamate Release

Central synapses spontaneously release neurotransmitter at low rates. In the brainstem, cranial visceral afferent terminals in caudal solitary tract nucleus (NTS) display pronounced, activity-dependent, asynchronous release of glutamate and this extra release depends on TRPV1 receptors (TRPV1+). Asynchronous release is absent for afferents lacking TRPV1 (TRPV1-) and resting EPSC frequency was greater in TRPV1+. Here, we studied this basal activity difference by assessing thermal sensitivity of spontaneous and miniature synaptic events in TRPV1+ and TRPV1- second-order NTS neurons. The spontaneous EPSC rate decreased when temperature was decreased, increased steeply between 30 and 42°C only in TRPV1+ neurons, and was calcium-dependent. TRPV1-specific antagonist SB366791, but not TTX, strongly attenuated thermal responses. Temperature changes failed to alter EPSC frequency in TRPV1- neurons. EPSC amplitudes and decay kinetics changed little with temperature. IPSCs in these second-order NTS neurons were unaltered by temperature. Such results suggest that activated, presynaptic TRPV1+ receptors trigger continuous resting release of glutamate vesicles at physiological temperatures only in capsaicin-responsive terminals. In mechanically isolated individual neurons harvested from medial NTS, increases in temperature increased the rate of glutamate release only in TRPV1+ neurons, whereas IPSC rates were unaffected. Cadmium failed to block thermal increases in glutamate release, suggesting that calcium entry through TRPV1 channels may trigger glutamate release independently of voltage-activated calcium channels. Together, our findings indicate a new form of afferent signaling in which TRPV1 channels within central terminals of peripheral afferents tonically generate glutamate release in NTS at 37°C in the absence of afferent action potentials.

TRPM1 is required for the depolarizing light response in retinal ON-bipolar cells

The ON pathway of the visual system, which detects increases in light intensity, is established at the first retinal synapse between photoreceptors and ON-bipolar cells. Photoreceptors hyperpolarize in response to light and reduce the rate of glutamate release, which in turn causes the depolarization of ON-bipolar cells. This ON-bipolar cell response is mediated by the metabotropic glutamate receptor, mGluR6, which controls the activity of a depolarizing current. Despite intensive research over the past two decades, the molecular identity of the channel that generates this depolarizing current has remained elusive. Here, we present evidence indicating that TRPM1 is necessary for the depolarizing light response of ON-bipolar cells, and further that TRPM1 is a component of the channel that generates this light response. Gene expression profiling revealed that TRPM1 is highly enriched in ON-bipolar cells. In situ hybridization experiments confirmed that TRPM1 mRNA is found in cells of the retinal inner nuclear layer, and immunofluorescent confocal microscopy showed that TRPM1 is localized in the dendrites of ON-bipolar cells in both mouse and macaque retina. The electroretinogram (ERG) of TRPM1-deficient (TRPM1(-/-)) mice had a normal a-wave, but no b-wave, indicating a loss of bipolar cell response. Finally, whole-cell patch-clamp recording from ON-bipolar cells in mouse retinal slices demonstrated that genetic deletion of TRPM1 abolished chemically simulated light responses from rod bipolar cells and dramatically altered the responses of cone ON-bipolar cells. Identification of TRPM1 as a mGluR6-coupled cation channel reveals a key step in vision, expands the role of the TRP channel family in sensory perception, and presents insights into the evolution of vertebrate vision.

Effects of monomethylarsonic and monomethylarsonous acid on evoked synaptic potentials in hippocampal slices of adult and young rats

Arsenite and its metabolites, dimethylarsinic or dimethylarsinous acid, have previously been shown to disturb synaptic transmission in hippocampal slices of rats (Krüger, K., Gruner, J., Madeja, M., Hartmann, L.M., Hirner, A.V., Binding, N., Muβhoff, U., 2006a. Blockade and enhancement of glutamate receptor responses in Xenopus oocytes by methylated arsenicals. Arch. Toxicol. 80, 492–501, Krüger, K., Straub, H., Binding, N., Muβhoff, U., 2006b. Effects of arsenite on long-term potentiation in hippocampal slices from adult and young rats. Toxicol. Lett. 165, 167–173, Krüger, K., Repges, H., Hippler, J., Hartmann, L.M., Hirner, A.V., Straub, H., Binding, N., Muβhoff, U., 2007. Effects of dimethylarsinic and dimethylarsinous acid on evoked synaptic potentials in hippocampal slices of young and adult rats. Toxicol. Appl. Pharmacol. 225, 40–46). The present experiments investigate, whether the important arsenic metabolites monomethylarsonic acid (MMA V) and monomethylarsonous acid (MMA III) also influence the synaptic functions of the hippocampus. In hippocampal slices of young (14–21 days-old) and adult (2–4 months-old) rats, evoked synaptic field potentials from the Schaffer collateral-CA1 synapse were measured under control conditions and during and after 30 and 60 min of application of the arsenic compounds. MMA V had no effect on the synapse functions neither in slices of adult nor in those from young rats. However, MMA III strongly influenced the synaptic transmission: it totally depressed the amplitudes of fEPSPs at concentrations of 50 μmol/l (adult rats) and 25 μmol/l (young rats) and LTP amplitudes at concentrations of 25 μmol/l (adult rats) and 10 μmol/l (young rats), respectively. In contrast, application of 1 μmol/l MMA III led to an enhancement of the LTP amplitude in young rats, which is interpretable by an enhancing effect on NMDA receptors and a lack of the blocking effect on AMPA receptors at this concentration (Krüger, K., Gruner, J., Madeja, M., Hartmann, L.M., Hirner, A.V., Binding, N., Muβhoff, U., 2006a. Blockade and enhancement of glutamate receptor responses in Xenopus oocytes by methylated arsenicals. Arch. Toxicol. 80, 492–501). These effects are probably not mediated by changes in cell excitability or in presynaptic glutamate release rates, since antidromically induced population spikes and paired-pulse facilitation failed to show any MMA III effect. The impairment of the excitatory CA1 synapse is more likely caused by the action of MMA III on postsynaptic glutamatergic receptors and may be jointly responsible for dysfunctions of cognitive effects in arsenic toxicity.

Cocaine serves as a peripheral interoceptive conditioned stimulus for central glutamate and dopamine release.

Intravenous injections of cocaine HCl are habit-forming because, among their many actions, they elevate extracellular dopamine levels in the terminal fields of the mesocorticolimbic dopamine system. This action, thought to be very important for cocaine's strong addiction liability, is believed to have very short latency and is assumed to reflect rapid brain entry and pharmacokinetics of the drug. However, while intravenous cocaine HCl has almost immediate effects on behavior and extracellular dopamine levels, recent evidence suggests that its central pharmacological effects are not evident until 10 or more seconds after IV injection. Thus the immediate effects of a given intravenous cocaine injection on extracellular dopamine concentration and behavior appear to occur before there is sufficient time for cocaine to act centrally as a dopamine uptake inhibitor. To explore the contribution of peripheral effects of cocaine to the early activation of the dopamine system, we used brain microdialysis to measure the effects of cocaine methiodide (MI)—a cocaine analogue that does not cross the blood brain barrier—on glutamate (excitatory) input to the dopamine cells. IP injections of cocaine MI were ineffective in cocaine-naïve animals but stimulated ventral tegmental glutamate release in rats previously trained to lever-press for cocaine HCl. This peripherally triggered glutamate input was sufficient to reinstate cocaine-seeking in previously trained animals that had undergone extinction of the habit. These findings offer an explanation for short-latency behavioral responses and immediate dopamine elevations seen following cocaine injections in cocaine-experienced but not cocaine-naïve animals.