Disturbed glutamate homeostasis may contribute to the pathological processes involved in Alzheimer’s disease (AD). Once glutamate is released from synapses or from other intracellular sources, it is rapidly cleared by glutamate transporters. EAAC1 (also called EAAT3 or SLC1A1) is the primary glutamate transporter in forebrain neurons. In addition to transporting glutamate, EAAC1 plays other roles in regulating GABA synthesis, reducing oxidative stress in neurons, and is important in supporting neuron viability. Currently, little is known about EAAC1 in AD. To address whether EAAC1 is disturbed in AD, immunohistochemistry was performed on tissue from hippocampus and frontal cortex of AD and normal control subjects matched for age and gender. While EAAC1 immunostaining in cortex appeared comparable to controls, in the hippocampus, EAAC1 aberrantly accumulated in the cell bodies and proximal neuritic processes of CA2–CA3 pyramidal neurons in AD patients. Biochemical analyses showed that Triton X-100-insoluble EAAC1 was significantly increased in the hippocampus of AD patients compared to both controls and Parkinson’s disease patients. These findings suggest that aberrant glutamate transporter expression is associated with AD-related neuropathology and that intracellular accumulation of detergent-insoluble EAAC1 is a feature of the complex biochemical lesions in AD that include altered protein solubility.
Glutamate Uptake; Glutamate toxicity; Synaptic dysfunction; protein aggregation; neurodegeneration; memantine; excitotoxicity
Ethanol has actions on cerebellar Purkinje neurons that can result either in a net excitation or in inhibition of neuronal activity. The present study examines the interplay of presynaptic and postsynaptic mechanisms to determine the net effect of ethanol on the neuronal firing rate of cerebellar Purkinje neurons.
Whole-cell voltage-clamp recording of miniature inhibitory postsynaptic currents (mIPSCs) from Purkinje neurons in cerebellar slices was used to examine the effect of ethanol on presynapticsynaptic release of γ-aminobutyric acid (GABA) and glutamate. Extracellular recording was used to examine the net action of both presynaptic and postsynaptic effects of ethanol on the firing rate of Purkinje neurons.
Under whole-cell voltage clamp, the frequency of bicuculline-sensitive miniature post-synaptic currents (mIPSCs) was increased dose-dependently by 25, 50, and 100 mM ethanol without any change in amplitude or decay time. Despite this evidence of increased release of GABA by ethanol, application of 50 mM ethanol caused an increase in firing in some neurons and a decrease in firing in others with a nonrandom distribution. When both glutamatergic and GABAergic influences were removed by simultaneous application of 6-cyano-7-nitroquinoxaline-2,3-dione and picrotoxin, respectively, ethanol caused only an increase in firing rate.
These data are consistent with a dual action of ethanol on cerebellar Purkinje neuron activity. Specifically, ethanol acts presynaptically to increase inhibition by release of GABA, while simultaneously acting postsynaptically to increase intrinsic excitatory drive.
Ethanol; GABA; Glutamate; mIPSC; Presynaptic; Postsynaptic; Intrinsic Drive
The neuronal Na+-dependent glutamate transporter, excitatory amino acid carrier 1 (EAAC1, also called EAAT3), has been implicated in the control of synaptic spillover of glutamate, synaptic plasticity, and the import of cysteine for neuronal synthesis of glutathione. EAAC1 protein is observed in both perisynaptic regions of the synapse and in neuronal cell bodies. Although amino acid residues in the carboxyl terminal tail have been implicated in the dendritic targeting of EAAC1 protein, it is not known if mRNA for EAAC1 may also be targeted to dendrites. Sorting of mRNA to specific cellular domains provides a mechanism by which signals can rapidly increase translation in a local environment; this form of regulated translation has been linked to diverse biological phenomena ranging from establishment of polarity during embryogenesis to synapse development and synaptic plasticity. In the present study, EAAC1 mRNA sequences were amplified from dendritic samples that were mechanically harvested from low-density hippocampal neuronal cultures. In parallel analyses, mRNA for histone deacetylase 2 (HDAC-2) and glial fibrillary acidic protein (GFAP) was not detected, suggesting that these samples are not contaminated with cell body or glial mRNAs. EAAC1 mRNA also co-localized with Map2a (a marker of dendrites) but not Tau1 (a marker of axons) in hippocampal neuronal cultures by in situ hybridization. In control rats, EAAC1 mRNA was observed in soma and proximal dendrites of hippocampal pyramidal neurons. Following pilocarpine- or kainate-induced seizures, EAAC1 mRNA was present in CA1 pyramidal cell dendrites up to 200 μm from the soma. These studies provide the first evidence that EAAC1 mRNA localizes to dendrites and suggest that dendritic targeting of EAAC1 mRNA is increased by seizure activity and may be regulated by neuronal activity/depolarization.
glutamate transport; EAAC1; EAAT3; epilepsy; pilocarpine; seizure; mRNA targeting
Granule and periglomerular cells in the main olfactory bulb express group I metabotropic glutamate receptors (mGluRs). The group I mGluR agonist 3,4-dihydroxyphenylglycine (DHPG) increases GABAergic spontaneous IPSCs (sIPSCs) in mitral cells, yet the presynaptic mechanism(s) involved and source(s) of the IPSCs are unknown. We investigated the actions of DHPG on sIPSCs and TTX-insensitive miniature IPSCs (mIPSCs) recorded in mitral and external tufted cells in rat olfactory bulb slices. DHPG, acting at mGluR1 and mGluR5, increased the rate but not amplitude of sIPSCs and mIPSCs in both cell types. The increase in mIPSCs depended on voltage-gated Ca2+ channels but persisted when ionotropic glutamate receptors and sodium spikes were blocked. Focal DHPG puffs onto granule cells or bath application after glomerular layer (GL) excision failed to increase mIPSCs in mitral cells. Additionally, GL excision reduced sIPSCs in mitral cells by 50%, suggesting that periglomerular cells exert strong tonic GABAergic inhibition of mitral cells. In contrast, GL DHPG puffs readily increased mIPSCs. These findings indicate that DHPG-evoked GABA release from granule cells requires spikes, whereas in the GL, DHPG facilitates periglomerular cell GABA release via both spike-dependent and spike-independent presynaptic mechanisms. We speculate that mGluRs amplify spike-driven lateral inhibition through the mitral-to-granule cell circuit, whereas GL mGluRs may play a more important role in amplifying intraglomerular inhibition after subthreshold input.
GABA; glutamate; periglomerular cells; external tufted cells; olfaction; rat
Recently, we demonstrated that mRNA for the neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), is found in dendrites of hippocampal neurons in culture and in dendrites of hippocampal pyramidal cells after pilocarpine-induced status epilepticus (SE). We also showed that SE increased the levels of EAAC1 mRNA ~15-fold in synaptoneurosomes. In the present study, the effects of SE on the distribution EAAC1 protein in hippocampus were examined. In addition, the effects of Group 1 mGluR receptor activation on the levels of EAAC1 protein were examined in synaptoneurosomes prepared from sham control animals and from animals that experience pilocarpine-induced SE. We find that EAAC1 immunoreactivity increases in pyramidal cells of the hippocampus after 3 h of SE. In addition, the group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), caused an increase in EAAC1 protein levels in hippocampal synaptoneurosomes; this effect of DHPG was much larger (~3- to 5-fold) after 3 h of SE. The DHPG-induced increases in EAAC1 protein were blocked by two different inhibitors of translation but not by inhibitors of transcription. mGluR1 or mGluR5 antagonists completely blocked the DHPG-induced increases in EAAC1 protein. DHPG also increased the levels of GluR2/3 protein, but this effect was not altered by SE. The DHPG-induced increase in EAAC1 protein was blocked by an inhibitor of the mammalian target of rapamycin (mTOR) or an inhibitor of extracellular signal-regulated kinase (ERK). These studies provide the first evidence EAAC1 translation can be regulated, and they show that regulated translation of EAAC1 is up-regulated after SE.
glutamate transport; EAAC1; epilepsy; pilocarpine; seizure; mGluR; dendritic targeting
Decreased GABAergic synaptic strength (“disinhibition”) in the spinal dorsal horn is a crucial mechanism contributing to the development and maintenance of pathological pain. However, mechanisms leading to disinhibition in the spinal dorsal horn remain elusive. We investigated the role of glial glutamate transporters (GLT-1 and GLAST) and glutamine synthetase in maintaining GABAergic synaptic activity in the spinal dorsal horn. Electrically evoked GABAergic inhibitory postsynaptic currents (eIPSCs), spontaneous IPSCs (sIPSCs) and miniature IPSCs (mIPSCs) were recorded in superficial spinal dorsal horn neurons of spinal slices from young adult rats. We used (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), to block both GLT-1 and GLAST and dihydrokainic acid (DHK) to block only GLT-1. We found that blockade of both GLAST and GLT-1 and blockade of only GLT-1 in the spinal dorsal horn decreased the amplitude of GABAergic eIPSCs, as well as both the amplitude and frequency of GABAergic sIPSCs or mIPSCs. Pharmacological inhibition of glial glutamine synthetase had similar effects on both GABAergic eIPSCs and sIPSCs. We provided evidence demonstrating that the reduction in GABAergic strength induced by the inhibition of glial glutamate transporters is due to insufficient GABA synthesis through the glutamate-glutamine cycle between astrocytes and neurons. Thus, our results indicate that deficient glial glutamate transporters and glutamine synthetase significantly attenuate GABAergic synaptic strength in the spinal dorsal horn, which may be a crucial synaptic mechanism underlying glial-neuronal interactions caused by dysfunctional astrocytes in pathological pain conditions.
Whereas ethanol has behavioral actions consistent with increased GABAergic function, attempts to demonstrate a direct enhancement of GABA-gated currents by ethanol have produced mixed results. Recent work has suggested that a part of the GABAergic profile of ethanol may result from enhanced GABA release from presynaptic terminals. The present study examines the effect of ethanol on GABA release in several brain regions to assess the regional nature of ethanol-induced GABA release. Whole-cell voltage clamp recording of spontaneous inhibitory postsynaptic currents (sIPSCs) from mechanically dissociated neurons and miniature inhibitory postsynaptic currents (mIPSCs) and paired-pulse ratio (PPR) from a slice preparation were used to quantify GABA release. Ethanol produced a concentration-dependent increase in the frequency of sIPSCs recorded from mechanically dissociated cerebellar Purkinje neurons and mIPSCs from substantia nigra neurons without having an effect on sIPSCs recorded from lateral septal or cerebrocortical neurons. This regional difference in the effect of ethanol on GABA release was confirmed with PPR recording from brain slices. These data indicate that ethanol can act on presynaptic terminals to increase GABA release in some brain regions while having little or no effect on GABA release in others. This regional difference is consistent with earlier in vivo studies in which ethanol affected neural activity and sensitivity to GABA in some, but not all, brain sites.
DYT1 dystonia is caused by a deletion in a glutamic acid residue in the C-terminus of the protein torsinA, whose function is still largely unknown. Alterations in GABAergic signaling have been involved in the pathogenesis of dystonia. We recorded GABA- and glutamate-mediated synaptic currents from a striatal slice preparation obtained from a mouse model of DYT1 dystonia. In medium spiny neurons (MSNs) from mice expressing human mutant torsinA (hMT), we observed a significantly higher frequency, but not amplitude, of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature currents (mIPSCs), whereas glutamate-dependent spontaneous excitatory synaptic currents (sEPSCs) were normal. No alterations were found in mice overexpressing normal human torsinA (hWT). To identify the possible sources of the increased GABAergic tone, we recorded GABAergic Fast-Spiking (FS) interneurons that exert a feed-forward inhibition on MSNs. However, both sEPSC and sIPSC recorded from hMT FS interneurons were comparable to hWT and non-transgenic (NT) mice. In physiological conditions, dopamine (DA) D2 receptor act presynaptically to reduce striatal GABA release. Of note, application of the D2-like receptor agonist quinpirole failed to reduce the frequency of sIPSCs in MSNs from hMT as compared to hWT and NT mice. Likewise, the inhibitory effect of quinpirole was lost on evoked IPSCs both in MSNs and FS interneurons from hMT mice. Our findings demonstrate a disinhibition of striatal GABAergic synaptic activity, that can be at least partially attributed to a D2 DA receptor dysfunction.
Electrophysiology; Dystonia; D2 dopamine receptor; Medium Spiny neurons; Fast-spiking interneuron
To define the changes in gene and protein expression of the neuronal glutamate transporter (EAAT3/EAAC1) in a rat model of temporal lobe epilepsy as well as in human hippocampal and neocortical epilepsy.
The expression of EAAT3/EAAC1 mRNA was measured by reverse Northern blotting in single dissociated hippocampal dentate granule cells from rats with pilocarpine-induced temporal lobe epilepsy (TLE) and age-matched controls, in dentate granule cells from hippocampal surgical specimens from patients with TLE, and in dysplastic neurons microdissected from human focal cortical dysplasia specimens. Immunolabeling of rat and human hippocampi and cortical dysplasia tissue with EAAT3/EAAC1 antibodies served to corroborate the mRNA expression analysis.
The expression of EAAT3/EAAC1 mRNA was increased by nearly threefold in dentate granule cells from rats with spontaneous seizures compared with dentate granule cells from control rats. EAAT3/EAAC1 mRNA levels also were high in human dentate granule cells from patients with TLE and were significantly elevated in dysplastic neurons in cortical dysplasia compared with nondysplastic neurons from postmortem control tissue. No difference in expression of another glutamate transporter, EAAT2/GLT-1, was observed. Immunolabeling demonstrated that EAAT3/EAAC1 protein expression was enhanced in dentate granule cells from both rats and humans with TLE as well as in dysplastic neurons from human cortical dysplasia tissue.
Elevations of EAAT3/EAAC1 mRNA and protein levels are present in neurons from hippocampus and neocortex in both rats and humans with epilepsy. Upregulation of EAAT3/EAAC1 in hippocampal and neocortical epilepsy may be an important modulator of extracellular glutamate concentrations and may occur as a response to recurrent seizures in these cell types.
Glutamate transporter; EAAT3/EAAC1; Epilepsy; Dentate; Dysplasia
Excitatory amino acid transporters (EAATs) are the primary regulators of extracellular glutamate concentrations in the central nervous system. Their dysfunction may contribute to several neurological diseases. To date, five distinct mammalian glutamate transporters have been cloned. In brain, EAAC1 (excitatory amino acid carrier 1) is the primary neuronal glutamate transporter, localized on the perisynaptic membranes that are near release sites. Despite its potential importance in synaptic actions, little is known concerning the regulation of EAAC1 trafficking from the endoplasmic reticulum (ER) to the cell surface. Previously, we identified an EAAC1-associated protein, GTRAP3-18, an ER protein that prevents ER exit of EAAC1 when induced. Here we show that RTN2B, a member of the reticulon protein family that mainly localizes in the ER and ER exit sites interacts with EAAC1 and GTRAP3-18. EAAC1 and GTRAP3-18 bind to different regions of RTN2B. Each protein can separately and independently form complexes with EAAC1. RTN2B enhances ER exit and the cell surface composition of EAAC1 in heterologous cells. Expression of short interfering RNA-mediated knockdown of RTN2B decreases the EAAC1 protein level in neurons. Overall, our results suggest that RTN2B functions as a positive regulator in the delivery of EAAC1 from the ER to the cell surface. These studies indicate that transporter exit from the ER controlled by the interaction with its ER binding partner represents a critical regulatory step in glutamate transporter trafficking to the cell surface.
Glucocorticoids influence vagal parasympathetic output to the viscera via mechanisms that include modulation of neural circuitry in the dorsal vagal complex, a principal autonomic regulatory center. Glucocorticoids can modulate synaptic neurotransmitter release elsewhere in the brain by inducing release of retrograde signalling molecules. We tested the hypothesis that the glucocorticoid agonist dexamethasone (DEX) modulates GABA release in the rat dorsal motor nucleus of the vagus (DMV). Whole-cell patch-clamp recordings revealed that DEX (1-10 µM) rapidly (i.e. within three minutes) increased the frequency of tetrodotoxin-resistant, miniature IPSCs (mIPSCs) in 67% of DMV neurons recorded in acutely prepared slices. Glutamate-mediated mEPSCs were also enhanced by DEX (10 µM), and blockade of ionotropic glutamate receptors reduced the DEX effect on mIPSC frequency. Antagonists of type I or II corticosteroid receptors blocked the effect of DEX on mIPSCs. The effect was mimicked by application of the membrane-impermeant BSA-conjugated DEX, and intracellular blockade of G protein function with GDP βS in the recorded cell prevented the effect of DEX. The enhancement of GABA release was blocked by the TRPV1 antagonists, 5’-iodoresiniferatoxin or capsazepine, but was not altered by the cannabinoid type 1 receptor antagonist AM251. The DEX effect was prevented by blocking fatty acid amide hydrolysis or by inhibiting anandamide transport, implicating involvement of the endocannabinoid system in the response. These findings indicate that DEX induces an enhancement of GABA release in the DMV, which is mediated by activation of TRPV1 receptors on afferent terminals. The effect is likely induced by anandamide or other ‘endovanilloid’, suggesting activation of a local retrograde signal originating from DMV neurons to enhance synaptic inhibition locally in response to glucocorticoids.
Excitatory amino acid carrier 1 (EAAC1) is a glutamate transporter found in neuronal tissues and is extensively expressed in the retina. EAAC1 plays a role in a variety of neural functions, but its biological functions in the retina has not been fully determined. The purpose of this study was to identify proteins regulated by EAAC1 in the retina of mice. To accomplish this, we used a proteomics-based approach to identify proteins that are up- or down-regulated in EAAC1-deficient (EAAC1-/-) mice.
Proteomic analyses and two-dimensional gel electorphoresis were performed on the retina of EAAC1-/- mice, and the results were compared to that of wild type mice. The protein spots showing significant differences were selected for identification by mass spectrometric analyses. Thirteen proteins were differentially expressed; nine proteins were up-regulated and five proteins were down-regulated in EAAC1-/- retina. Functional clustering showed that identified proteins are involved in various cellular process, e.g. cell cycle, cell death, transport and metabolism.
We identified thirteen proteins whose expression is changed in EAAC-/- mice retinas. These proteins are known to regulate cell proliferation, death, transport, metabolism, cell organization and extracellular matrix.
The dorsal (DR) and median (MR) raphe nuclei contain 5-hydroxytryptamine (5-HT) cell bodies that give rise to the majority of the ascending 5-HT projections to the forebrain. The DR and MR have differential roles in mediating stress, anxiety and depression. Glutamate and GABA activity sculpt putative 5-HT neuronal firing and 5-HT release in a seemingly differential manner in the MR and DR, yet isolated glutamate and GABA activity within the DR and MR has not been systematically characterized. Visualized whole-cell voltage-clamp techniques were used to record excitatory and inhibitory postsynaptic currents (EPSC and IPSC) in 5-HT-containing neurons. There was a regional variation in action potential-dependent (spontaneous) and basal [miniature (m)] glutamate and GABAergic activity. mEPSC activity was greater than mIPSC activity in the DR, whereas in the MR the mIPSC activity was greater. These differences in EPSC and IPSC frequency indicate that glutamatergic and GABAergic input have distinct cytoarchitectures in the DR and MR. 5-HT1B receptor activation decreased mEPSC frequency in the DR and the MR, but selectively inhibited mIPSC activity only in the MR. This finding, in concert with its previously described function as an autoreceptor, suggests that 5-HT1B receptors influence the ascending 5-HT system through multiple mechanisms. The disparity in organization and integration of glutamatergic and GABAergic input to DR and MR neurons and their regulation by 5-HT1B receptors may contribute to the distinction in MR and DR regulation of forebrain regions and their differential function in the aetiology and pharmacological treatment of psychiatric disease states.
5-HT1B receptor; AMPA/kainate receptor; EPSC; GABAA receptor; IPSC; serotonin
We investigated possible alterations of pharmacologically-isolated, evoked GABAA inhibitory postsynaptic potentials (eIPSPs) and miniature GABAA inhibitory postsynaptic currents (mIPSCs) in the rat central amygdala (CeA) elicited by acute application of μ-opioid receptor (MOR) agonists (DAMGO and morphine; 1 μM) and by chronic morphine treatment with morphine pellets. The acute activation of MORs decreased the amplitudes of eIPSPs, increased paired-pulse facilitation (PPF) of eIPSPs and decreased the frequency (but not the amplitude) of mIPSCs in a majority of CeA neurons, suggesting that acute MOR-dependent modulation of this GABAergic transmission is mediated predominantly via presynaptic inhibition of GABA release. We observed no significant changes in the membrane properties, eIPSPs, PPF or mIPSCs of CeA neurons during chronic morphine treatment compared to CeA of naïve or sham rats. Superfusion of the MOR antagonist CTOP (1 μM) increased the mean amplitude of eIPSPs in a majority of CeA neurons to the same degree in both naïve/sham and morphine-treated rats, suggesting a tonic activation of MORs in both conditions. Superfusion of DAMGO decreased eIPSP amplitudes and the frequency of mIPSCs equally in both naïve/sham and morphine-treated rats but decreased the amplitude of mIPSCs only in morphine treated rats, an apparent postsynaptic action. Our combined findings suggest the development of tolerance of the CeA GABAergic system to inhibitory effects of acute activation of MORs on presynaptic GABA release and possible alteration of MOR-dependent postsynaptic mechanisms that may represent important neuroadaptations of the GABAergic and MOR systems during chronic morphine treatment.
addiction; drug abuse; electrophysiology; extended amygdala; opiate; tolerance
The formation and maturation of GABAergic synapses was studied in cultured hippocampal pyramidal neurons by both performing immunocytochemistry for GABAergic markers and recording miniature inhibitory postsynaptic currents (mIPSCs). Nascent GABAergic synapses appeared between 3-8 days in vitro (DIV), with GABAA receptor subunit clusters appearing first, followed by GAD-65 puncta, and then functional synapses. The number of GABAergic synapses increased from 7-14 DIV, with a corresponding increase in frequency of mIPSCs. Moreover, these new GABAergic synapses formed on neuronal processes farther away from the soma, contributing to decreased mIPSC amplitude and slowed mIPSC 19-90% rise-time. The mIPSC decay quickened from 7-14 DIV with a parallel change in the distribution of the α5 subunit from diffuse expression at 7 DIV to clustered expression at 14 DIV. These α5 clusters were mostly extrasynaptic. The α1 subunit was expressed as clusters in none of the neurons at 7 DIV, in 20% at 14 DIV, and in 80% at 21 DIV. Most of these α1 clusters were expressed at GABAergic synapses. In addition, puncta of GABA transporter 1 (GAT-1) were localized to GABAergic synapses at 14 DIV, but were not expressed at 7 DIV. These studies demonstrate that mIPSCs appear after pre- and postsynaptic elements are in place. Furthermore, the process of maturation of GABAergic synapses involves increased synapse formation at distal processes, expression of new GABAA receptor subunits, and GAT-1 expression at synapses; these changes are reflected in altered frequency, kinetics and drug sensitivity of mIPSCs.
synaptogenesis; GABAA receptor; GABA transporter; mIPSC
Since inhibitory synaptic transmission is a major mechanism of general anesthesia, we examined the effects of isoflurane on properties of GABAergic inhibitory currents in the reticular thalamic nucleus (nRT) in brain slices. The evoked inhibitory postsynaptic currents (eIPSCs) and spontaneous miniature synaptic currents (mIPSCs) of visualized nRT cells in young and adult rats were recorded. Consistent with postsynaptic effects on GABAA receptors, isoflurane prolonged the decay-time constants of both eIPSCs and mIPCSs. Surprisingly, isoflurane completely inhibited the amplitude of eIPSCs at clinically relevant concentrations (IC50 of 240 ± 20 μM), increased the paired-pulse ratio, and decreased the frequency of mIPSCs, indicating that presynaptic mechanisms may also contribute to the effects of isoflurane on IPSCs. The overall effect of isoflurane on eIPSCs in nRT cells was a decrease of net charge-transfer across the postsynaptic membrane. The application of 100 μM nickel (Ni2+) and the more specific R-type Ca2+ channel blocker SNX-482 (0.5 μM) decreased eIPSC amplitudes, increased the paired-pulse ratio, and attenuated isoflurane-induced inhibition of eIPSCs. In addition, isoflurane potently blocked currents in recombinant human CaV2.3 (α1E) channels with an IC50 of 206 ± 22 μM. Importantly, in vivo electroencephalographic (EEG) recordings in adult CaV2.3 knockout mice demonstrated alterations in isoflurane-induced burst-suppression activity. Because the thalamus has a key function in processing sensory information, sleep, and cognition, modulation of its GABAergic tone by presynaptic R-type Ca2+ channels may contribute to the clinical effects of general anesthesia.
anesthesia; calcium channels; calcium current; GABAA receptor; GABAergic neuron; thalamus
Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na+-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.
Pyramidal neurons in hippocampal CA1 regions are highly sensitive to cerebral ischemia. Alterations of excitatory and inhibitory synaptic transmission may contribute to the ischemia-induced neuronal degeneration. However, little is known about the changes of GABAergic synaptic transmission in the hippocampus following reperfusion. We examined the GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons 12 hours and 24 hours after transient forebrain ischemia. The amplitudes of evoked IPSCs (eIPSCs) were increased significantly 12 hours after ischemia and returned to control levels 24 hours following reperfusion. The potentiation of eIPSCs was accompanied by an increase of miniature IPSCs (mIPSCs) amplitude, and an enhanced response to exogenous application of GABA, indicating the involvement of postsynaptic mechanisms. Furthermore, there was no obvious change of the paired-pulse ratio (PPR) of eIPSCs and the frequency of mIPSCs, suggesting that the potentiation of eIPSCs might not be due to the increased presynaptic release. Blockade of adenosine A1 receptors led to a decrease of eIPSCs amplitude in post-ischemic neurons but not in control neurons, without affecting the frequency of mIPSCs and the PPR of eIPSCs. Thus, tonic activation of adenosine A1 receptors might, at least in part, contribute to the enhancement of inhibitory synaptic transmission in CA1 neurons after forebrain ischemia. The transient enhancement of inhibitory neurotransmission might temporarily protect CA1 pyramidal neurons, and delay the process of neuronal death after cerebral ischemia.
GABAA receptor; Cell death; Stroke; Excitotoxicity
The kinetics of inhibitory postsynaptic currents (IPSCs) influence many neuronal processes, such as the frequencies of oscillations or the duration of shunting inhibition. The subunit composition of recombinant GABAA receptors (GABAARs) strongly affects the deactivation kinetics of GABA-evoked currents. However, for GABAergic synapses, the relationship between the subunit composition and IPSC decay is less clear. Here we addressed this by combining whole-cell recordings of miniature IPSCs (mIPSCs) and quantitative immunolocalization of synaptic GABAAR subunits. In cerebellar stellate, thalamic relay and main olfactory bulb (MOB) deep short-axon cells of Wistar rats the only synaptic α subunit was the α1, and zolpidem-sensitive mIPSCs had weighted decay time constants (τw) of 4-6 ms. Nucleus reticularis thalami neurons expressed only the α3 as synaptic α subunit and exhibited slow (τw = 28 ms), zolpidem-insensitive, mIPSCs. By contrast, MOB external tufted cells contained two α subunit types (α1 and α3) at their synapses. Quantitative analysis of multiple immunolabeled images revealed small within-cell, but large between-cell variability in synaptic α1:α3 ratios. This corresponded to large cell-to-cell variability in the decay (τw = 3-30 ms) and zolpidem sensitivity of mIPSCs. Currents evoked by rapid application of GABA to patches excised from HEK cells expressing different mixtures of α1 and α3 subunits displayed highly variable deactivation times that correlated with the α1:α3 cDNA ratio. Our results demonstrate that diversity in the decay of IPSCs can be generated by varying the expression of different GABAAR subunits that alone confer different decay kinetics, allowing the time course of inhibition to be tuned to individual cellular requirements.
inhibition; immunohistochemistry; patch-clamp; GABA; zolpidem
Zolpidem is a widely prescribed sleep aid with relative selectivity for GABAA receptors containing α1–3 subunits. We examined the effects of zolpidem on the inhibitory currents mediated by GABAA receptors using whole-cell patch-clamp recordings from DMV neurons in transverse brainstem slices from rat. Zolpidem prolonged the decay time of mIPSCs and of muscimol-evoked whole-cell GABAergic currents, and it occasionally enhanced the amplitude of mIPSCs. The effects were blocked by flumazenil, a benzodiazepine antagonist. Zolpidem also hyperpolarized the resting membrane potential, with a concomitant decrease in input resistance and action potential firing activity in a subset of cells. Zolpidem did not clearly alter the GABAA receptor-mediated tonic current (Itonic) under baseline conditions, but after elevating extracellular GABA concentration with nipecotic acid, a non-selective GABA transporter blocker, zolpidem consistently and significantly increased the tonic GABA current. This increase was suppressed by flumazenil and gabazine. These results suggest that α1–3 subunits are expressed in synaptic GABAA receptors on DMV neurons. The baseline tonic GABA current is likely not mediated by these same low affinity, zolpidem-sensitive GABAA receptors. However, when the extracellular GABA concentration is increased, zolpidem-sensitive extrasynaptic GABAA receptors containing α1–3 subunits contribute to the Itonic.
Benzodiazepine; Parasympathetic; GABA receptor; IPSC; Patch-clamp
Alterations in network activity trigger compensatory changes in excitation and inhibition that restore neuronal firing rate to an optimal range. One example of such synaptic homeostasis is the downregulation of inhibitory transmission by chronic inactivity, in part, through the reduction of vesicular transmitter content. The enzyme glutamic acid decarboxylase 67 (GAD67) is critical for GABA synthesis, but its involvement in homeostatic plasticity is unclear. We explored the role of GAD67 in activity-dependent synaptic plasticity using a mouse line (Gad1−/−) in which GAD67 expression is disrupted by genomic insertion of the green fluorescent protein (GFP). Homozygous deletion of Gad1 significantly reduced miniature inhibitory postsynaptic current (mIPSC) amplitudes and GABA levels in cultured hippocampal neurons. The fractional block of mIPSC amplitude by a low affinity, competitive GABAA receptor antagonist was higher in GAD67-lacking neurons, suggesting that GABA concentration in the synaptic cleft is lower in knockout animals. Chronic suppression of activity by the application of tetrodotoxin (TTX) reduced mIPSC amplitudes and the levels of GAD67 and GABA. Moreover, TTX reduced GFP levels in interneurons, suggesting that GAD67 gene expression is a key regulatory target of activity. These in vitro experiments were corroborated by in vivo studies in which olfactory deprivation reduced mIPSC amplitudes and GFP levels in glomerular neurons in the olfactory bulb. Importantly, TTX-induced downregulation of mIPSC was attenuated in Gad1−/− neurons. Altogether, these findings indicate that activity-driven expression of GAD67 critically controls GABA synthesis and, thus, vesicular filling of the transmitter.
Activation of the dopaminergic (DA) neurons of the ventral tegmental area (VTA) by ethanol has been implicated in its rewarding and reinforcing effects. We previously demonstrated that ethanol enhances GABA release onto VTA-DA neurons via activation of 5-HT2C receptors and subsequent release of calcium from intracellular stores. Here we demonstrate that excitation of VTA-DA neurons by ethanol is limited by an ethanol-enhancement in GABA release.
In this study, we performed whole-cell voltage clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs) and cell-attached recordings of action potential firing from VTA-DA neurons in midbrain slices from young Long Evans rats. Acute exposure to ethanol (75 mM) transiently enhanced the firing rate of VTA-DA neurons as well as the frequency of mIPSCs. Simultaneous blockade of both GABAA and GABAB receptors (Picrotoxin (75 μM) and SCH50911 (20 μM)) disinhibited VTA-DA firing rate whereas a GABAA agonist (muscimol, 1 μM) strongly inhibited firing rate. In the presence of picrotoxin, ethanol enhanced VTA-DA firing rate more than in the absence of picrotoxin. Additionally, a sub-maximal concentration of muscimol together with ethanol inhibited VTA-DA firing rate more than muscimol alone. DAMGO (3 μM) inhibited mIPSC frequency but did not block the ethanol-enhancement in mIPSC frequency. DAMGO (1 and 3 μM) had no effect on VTA-DA firing rate. Naltrexone (60 μM) had no effect on basal or ethanol-enhancement of mIPSC frequency. Additionally, naltrexone (20 and 60 μM) did not block the ethanol-enhancement in VTA-DA firing rate. Overall, the present results indicate that the ethanol enhancement in GABA release onto VTA-DA neurons limits the stimulatory effect of ethanol on VTA-DA neuron activity and may have implications for the rewarding properties of ethanol.
reward; mesolimbic; alcohol dependence; electrophysiology; inhibitory synaptic transmission
Vesicular GABA and intraterminal glutamate concentrations are in equilibrium, suggesting inhibitory efficacy may depend on glutamate availability. Two main intraterminal glutamate sources are uptake by neuronal glutamate transporters and glutamine synthesized through the astrocytic glutamate-glutamine cycle. We examined the involvement of the glutamate-glutamine cycle in modulating GABAergic synaptic efficacy. In the absence of neuronal activity, disruption of the glutamate-glutamine cycle by blockade of neuronal glutamine transport with α-(methylamino) isobutyric acid (MeAIB; 5 mm) or inhibition of glutamine synthesis in astrocytes with methionine sulfoximine (MSO; 1.5 mm) had no effect on miniature IPSCs recorded in hippocampal area CA1 pyramidal neurons. However, after a period of moderate synaptic activity, application of MeAIB, MSO, or dihydrokainate (250 μm; an astrocytic glutamate transporter inhibitor) significantly reduced evoked IPSC (eIPSC) amplitudes. The MSO effect could be reversed by exogenous application of glutamine (5 mm), whereas glutamine could not rescue the eIPSC decreases induced by the neuronal glutamine transporter inhibitor MeAIB. The activity-dependent reduction in eIPSCs by glutamate-glutamine cycle blockers was accompanied by an enhanced blocking effect of the low-affinity GABAA receptor antagonist, TPMPA [1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid], consistent with diminished GABA release. We further corroborated this hypothesis by examining MeAIB effects on minimal stimulation-evoked quantal IPSCs (meIPSCs). We found that, in MeAIB-containing medium, moderate stimulation induced depression in potency of meIPSCs but no change in release probability, consistent with reduced vesicular GABA content. We conclude that the glutamate-glutamine cycle is a major contributor to synaptic GABA release under physiological conditions, which dynamically regulates inhibitory synaptic strength.
GABAergic modulation; hippocampus; neurotransmitter; interneuron; astrocyte; astroglia; glutamate transporters; synaptic transmission; GABA; glutamine; patch clamp; unitary
Glutathione (GSH) is a tripeptide consisting of glutamate, cysteine, and glycine; it has a variety of functions in the central nervous system. Brain GSH depletion is considered a preclinical sign in age-related neurodegenerative diseases, and it promotes the subsequent processes toward neurotoxicity. A neuroprotective mechanism accomplished by increasing GSH synthesis could be a promising approach in the treatment of neurodegenerative diseases. In neurons, cysteine is the rate-limiting substrate for GSH synthesis. Excitatory amino acid carrier 1 (EAAC1) is a neuronal cysteine/glutamate transporter in the brain. EAAC1 translocation to the plasma membrane promotes cysteine uptake, leading to GSH synthesis, while being negatively regulated by glutamate transport associated protein 3-18 (GTRAP3-18). Our recent studies have suggested GTRAP3-18 as an inhibitory factor for neuronal GSH synthesis. Inhibiting GTRAP3-18 function is an endogenous mechanism to increase neuron-specific GSH synthesis in the brain. This review gives an overview of EAAC1-mediated GSH synthesis, and its regulatory mechanisms by GTRAP3-18 in the brain, and a potential approach against neurodegeneration.
glutathione; cysteine uptake; GTRAP3-18; EAAC1; neurodegeneration
Neuropathic pain is a chronic and intractable symptom associated with nerve injury. The periaqueductal gray (PAG) is important in the endogenous pain control system and is the main site of the opioidergic analgesia. To investigate whether neuropathic pain affects the endogenous pain control system, we examined the effect of neuropathic pain induced by sacral nerve transection on presynaptic GABA release, the kinetics of postsynaptic GABA-activated Cl- currents, and the modulatory effect of μ-opioid receptor (MOR) activation in mechanically isolated PAG neurons with functioning synaptic boutons.
In normal rats, MOR activation inhibited the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) to 81.3% of the control without any alteration in their amplitude. In neuropathic rats, the inhibition of mIPSC frequency by MOR activation was 82.4%. The frequency of GABAergic mIPSCs in neuropathic rats was 151.8% of normal rats without any difference in the mIPSC amplitude. Analysis of mIPSC kinetics showed that the fast decay time constant and synaptic charge transfer of mIPSCs in neuropathic rats were 76.0% and 73.2% of normal rats, respectively.
These results indicate that although the inhibitory effect of MOR activation on presynaptic GABA release is similar in both neuropathic and normal rats, neuropathic pain may inhibit endogenous analgesia in the PAG through an increase in presynaptic GABA release.
Neuropathic pain; Endogenous pain control system; Opioid analgesia; GABAergic synaptic transmission; Periaqueductal gray