Glutamate-mediated Excitatory Postsynaptic Currents Research Articles

Glutamate, T cells and multiple sclerosis.

Glutamate is the major excitatory neurotransmitter in the nervous system, where it induces multiple beneficial and essential effects. Yet, excess glutamate, evident in a kaleidoscope of acute and chronic pathologies, is absolutely catastrophic, since it induces excitotoxicity and massive loss of brain function. Both the beneficial and the detrimental effects of glutamate are mediated by a large family of glutamate receptors (GluRs): the ionotropic glutamate receptors (iGluRs) and the metabotropic glutamate receptors (mGluRs), expressed by most/all cells of the nervous system, and also by many non-neural cells in various peripheral organs and tissues. T cells express on their cell surface several types of functional GluRs, and so do few other immune cells. Furthermore, glutamate by itself activates resting normal human T cells, and induces/elevates key T cell functions, among them: T cell adhesion, chemotactic migration, cytokine secretion, gene expression and more. Glutamate has also potent effects on antigen/mitogen/cytokine-activated T cells. Furthermore, T cells can even produce and release glutamate, and affect other cells and themselves via their own glutamate. Multiple sclerosis (MS) and its animal model Experimental Autoimmune Encephalomyelitis (EAE) are mediated by autoimmune T cells. In MS and EAE, there are excess glutamate levels, and multiple abnormalities in glutamate degrading enzymes, glutamate transporters, glutamate receptors and glutamate signaling. Some GluR antagonists block EAE. Enhancer of mGluR4 protects from EAE viaregulatory T cells (Tregs), while mGluR4 deficiency exacerbates EAE. The protective effect of mGluR4 on EAE calls for testing GluR4 enhancers in MS patients. Oral MS therapeutics, namely Fingolimod, dimethyl fumarate and their respective metabolites Fingolimod-phosphate and monomethyl fumarate, can protect neurons against acute glutamatergic excitotoxic damage. Furthermore, Fingolimod reduce glutamate-mediated intracortical excitability in relapsing-remitting MS. Glatiramer acetate -COPAXONE®, an immunomodulator drug for MS, reverses TNF-α-induced alterations of striatal glutamate-mediated excitatory postsynaptic currents in EAE-afflicted mice. With regard to T cells of MS patients: (1) The cell surface expression of a specific GluR: the AMPA GluR3 is elevated in T cells of MS patients during relapse and with active disease, (2) Glutamate and AMPA (a selective agonist for glutamate/AMPA iGluRs) augment chemotactic migration of T cells of MS patients, (3) Glutamate augments proliferation of T cells of MS patients in response to myelin-derived proteins: MBP and MOG, (4) T cells of MS patients respond abnormally to glutamate, (5) Significantly higher proliferation values in response to glutamate were found in MS patients assessed during relapse, and in those with gadolinium (Gd)+ enhancing lesions on MRI. Furthermore, glutamate released from autoreactive T cells induces excitotoxic cell death of neurons. Taken together, the evidences accumulated thus far indicate that abnormal glutamate levels and signaling in the nervous system, direct activation of T cells by glutamate, and glutamate release by T cells, can all contribute to MS. This may be true also to other neurological diseases. It is postulated herein that the detrimental activation of autoimmune T cells by glutamate in MS could lead to: (1) Cytotoxicityin the CNS: T cell-mediated killing of neurons and glia cells, which would subsequently increase the extracellular glutamate levels, and by doing so increase the excitotoxicitymediated by excess glutamate, (2) Release of proinflammatory cytokines, e.g., TNFα and IFNγ that increase neuroinflammation. Finally, if excess glutamate, abnormal neuronal signaling, glutamate-induced activation of T cells, and glutamate release by T cells are indeed all playing a key detrimental role in MS, then optional therapeutic tolls include GluR antagonists, although these may have various side effects. In addition, an especially attractive therapeutic strategy is the novel and entirely different therapeutic approach to minimize excess glutamateand excitotoxicity, titled: 'brain to blood glutamate scavenging', designed to lower excess glutamate levels in the CNS by 'pumping it out' from the brain to the blood. The glutamate scavanging is achieved by lowering glutamate levels in the blood by intravenous injection of the blood enzyme glutamate oxaloacetate transaminase (GOT). The glutamate-scavenging technology, which is still experimental, validated so far for other brain pathologies,but not tested on MS or EAE yet, may be beneficial for MS too, since it could decrease both the deleterious effects of excess glutamate on neural cells, and the activation of autoimmune T cells by glutamate in the brain. The topic of glutamate scavenging, and also its potential benefit for MS, are discussed towards the end of the review, and call for research in this direction.

Enhanced ability of TRPV1 channels in regulating glutamatergic transmission after repeated morphine exposure in the nucleus accumbens of rat

Glutamatergic projections to nucleus accumbens (NAc) drive drug-seeking behaviors during opioids withdrawal. Modulating glutamatergic neurotransmission provides a novel pharmacotherapeutic avenue for treatment of opioids dependence. Great deals of researches have verified that transient receptor potential vanilloid 1 (TRPV1) channels alters synaptic transmitter release and regulate neural plasticity. In the present study, whole-cell patch clamp recordings were adopted to examine the activity of TRPV1 Channels in regulating glutamate-mediated excitatory postsynaptic currents (EPSCs) in NAc of rat during morphine withdrawal for 3days and 3weeks. The data showed that the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and the amplitudes of evoked excitatory postsynaptic currents (eEPSCs) were increased during morphine withdrawal after applied with capsaicin (TRPV1 agonist). Capsaicin decreased the paired pulse ratio (PPR) and increased sEPSCs frequency but not their amplitudes suggesting a presynaptic locus of action during morphine withdrawal. All these effects were fully blocked by the TRPV1 antagonist Capsazepine. Additionally, In the presence of AM251 (CB1 receptor antagonist), depolarization-induced release of endogenous cannabinoids activated TRPV1 channels to enhance glutamatergic neurotransmission during morphine withdrawal. The functional enhancement of TRPV1 Channels in facilitating glutamatergic transmission was not recorded in dorsal striatum. Our findings demonstrate the ability of TRPV1 in regulating excitatory glutamatergic transmission is enhanced during morphine withdrawal in NAc, which would deepen our understanding of glutamatergic modulation during opioids withdrawal.

Fumarates modulate microglia activation through a novel HCAR2 signaling pathway and rescue synaptic dysregulation in inflamed CNS.

Dimethyl fumarate (DMF), recently approved as an oral immunomodulatory treatment for relapsing-remitting multiple sclerosis (MS), metabolizes to monomethyl fumarate (MMF) which crosses the blood–brain barrier and has demonstrated neuroprotective effects in experimental studies. We postulated that MMF exerts neuroprotective effects through modulation of microglia activation, a critical component of the neuroinflammatory cascade that occurs in neurodegenerative diseases such as MS. To ascertain our hypothesis and define the mechanistic pathways involved in the modulating effect of fumarates, we used real-time PCR and biochemical assays to assess changes in the molecular and functional phenotype of microglia, quantitative Western blotting to monitor activation of postulated pathway components, and ex vivo whole-cell patch clamp recording of excitatory post-synaptic currents in corticostriatal slices from mice with experimental autoimmune encephalomyelitis (EAE), a model for MS, to study synaptic transmission. We show that exposure to MMF switches the molecular and functional phenotype of activated microglia from classically activated, pro-inflammatory type to alternatively activated, neuroprotective one, through activation of the hydroxycarboxylic acid receptor 2 (HCAR2). We validate a downstream pathway mediated through the AMPK–Sirt1 axis resulting in deacetylation, and thereby inhibition, of NF-κB and, consequently, of secretion of pro-inflammatory molecules. We demonstrate through ex vivo monitoring of spontaneous glutamate-mediated excitatory post-synaptic currents of single neurons in corticostriatal slices from EAE mice that the neuroprotective effect of DMF was exerted on neurons at pre-synaptic terminals by modulating glutamate release. By exposing control slices to untreated and MMF-treated activated microglia, we confirm the modulating effect of MMF on microglia function and, thereby, its indirect neuroprotective effect at post-synaptic level. These findings, whereby DMF-induced activation of a new HCAR2-dependent pathway on microglia leads to the modulation of neuroinflammation and restores synaptic alterations occurring in EAE, represent a possible novel mechanism of action for DMF in MS.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1422-3) contains supplementary material, which is available to authorized users.

Interleukin-1β causes excitotoxic neurodegeneration and multiple sclerosis disease progression by activating the apoptotic protein p53.

BackgroundUnderstanding how inflammation causes neuronal damage is of paramount importance in multiple sclerosis (MS) and in other neurodegenerative diseases. Here we addressed the role of the apoptotic cascade in the synaptic abnormalities and neuronal loss caused by the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor (TNF-α) in brain tissues, and disease progression caused by inflammation in relapsing-remitting MS (RRMS) patients.ResultsThe effect of IL-1β, but not of TNF-α, on glutamate-mediated excitatory postsynaptic currents was blocked by pifithrin-α (PFT), inhibitor of p53. The protein kinase C (PKC)/transient receptor potential vanilloid 1 (TRPV1) pathway was involved in IL-1β-p53 interaction at glutamatergic synapses, as pharmacological modulation of this inflammation-relevant molecular pathway affected PFT effects on the synaptic action of IL-1β. IL-1β-induced neuronal swelling was also blocked by PFT, and IL-1β increased the expression of p21, a canonical downstream target of activated p53.Consistent with these in vitro results, the Pro/Pro genotype of p53, associated with low efficiency of transcription of p53-regulated genes, abrogated the association between IL-1β cerebrospinal fluid (CSF) levels and disability progression in RRMS patients. The interaction between p53 and CSF IL-1β was also evaluated at the optical coherence tomography (OCT), showing that IL-1β-driven neurodegenerative damage, causing alterations of macular volume and of retinal nerve fibre layer thickness, was modulated by the p53 genotype.ConclusionsInflammatory synaptopathy and neurodegeneration caused by IL-1β in RRMS patients involve the apoptotic cascade. Targeting IL-1β-p53 interaction might result in significant neuroprotection in MS.

Glatiramer Acetate Protects Against Inflammatory Synaptopathy in Experimental Autoimmune Encephalomyelitis

Glutamate-mediated excitotoxicity is supposed to induce neurodegeneration in multiple sclerosis (MS). Glatiramer acetate (GA) is an immunomodulatory agent used in MS treatment with potential neuroprotective action. Aim of the present study was to investigate whether GA has effects on glutamate transmission alterations occurring in experimental autoimmune encephalomyelitis (EAE), to disclose a possible mechanism of GA-induced neuroprotection in this mouse model of MS. Single neuron electrophysiological recordings and immunofluorescence analysis of microglia activation were performed in the striatum of EAE mice, treated or not with GA, at different stages of the disease. GA treatment was able to reverse the tumor necrosis factor-α (TNF-α)-induced alterations of striatal glutamate-mediated excitatory postsynaptic currents (EPSCs) of EAE mice. Incubation of striatal slices of control animals with lymphocytes taken from EAE mice treated with GA failed to replicate such an anti-glutamatergic effect, while activated microglial cells stimulated with GA in vitro mimicked the effect of GA treatment of EAE mice. Consistently, EAE mice treated with GA had less microglial activation and less TNF-α expression than untreated EAE animals. Furthermore, direct application of GA to EAE slices replicated the in vivo protective activity of GA. Our results show that GA is neuroprotective against glutamate toxicity independently of its peripheral immunodulatory action, and through direct modulation of microglial activation and TNF-α release in the grey matter of EAE and possibly of MS brains.

Interleukin‐1β causes synaptic hyperexcitability in multiple sclerosis

The frequency of inflammatory episodes in the early stages of multiple sclerosis (MS) has been correlated with late neurodegeneration, but the mechanism by which inflammation gives rise to delayed neuronal damage is unknown. Increased activity of the neurotransmitter glutamate is thought to play a role in the inflammation-driven neurodegenerative process of MS, and therefore we tested whether inflammatory cytokines released during acute MS attacks have the property of enhancing glutamate-mediated transmission and excitotoxicity in central neurons. We compared the effect of cerebrospinal fluid (CSF) from active and quiescent MS patients on glutamate-mediated excitatory postsynaptic currents (EPSCs) and excitotoxic damage in rodent brain slices. We also measured CSF concentrations of tumor necrosis factor-α, of interleukin-1β (IL-1β), and of IL-1 receptor antagonist (IL-1ra), and correlated cytokine levels with cortical excitability assessed in MS patients by means of paired-pulse transcranial magnetic stimulation (TMS). CSF from MS patients with enhanced brain lesions at magnetic resonance imaging was able to increase spontaneous EPSC frequency and glutamate-mediated neuronal swelling in vitro, through a mechanism dependent on enhanced IL-1β signaling and increased glutamate α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor stimulation. Furthermore, IL-1β/IL-1ra ratio was significantly higher in the CSF of active MS subjects, and correlated with intracortical facilitation, an accredited TMS measure of glutamate transmission. Finally, we identified for the first time transient receptor potential vanilloid 1 channels as essential intermediates for the synaptic action of IL-1β on central glutamatergic synapses. Our results provide compelling evidence of the synaptic mechanism linking inflammation and excitotoxic neurodegeneration in MS.

Transient receptor potential vanilloid 1 channels modulate the synaptic effects of TNF-α and of IL-1β in experimental autoimmune encephalomyelitis

Transient receptor potential vanilloid 1 (TRPV1) channels are involved in several inflammatory diseases. However, their action is still controversial, and both pro-inflammatory and anti-inflammatory roles have been described. We used a strain of TRPV1-KO mice to characterize the role of these channels in experimental autoimmune encephalomyelitis (EAE), which models multiple sclerosis (MS) in mice.EAE mice showed higher lethality in the peak phase of the disease and a better recovery of the surviving animals in the chronic stages, compared to their wild-type (WT) counterparts. By means of whole-cell patch clamp experiments in corticostriatal brain slices, we found that the absence of TRPV1 channels exacerbated the defect of glutamate transmission occurring in the peak phase of EAE, and attenuated the alterations of GABA synapses in the chronic phase of EAE, thus paralleling the dual effects of TRPV1-KO on the motor deficits of EAE mice. Furthermore, in slices from non-EAE mice, we found that genetic or pharmacological blockade of TRPV1 channels enhanced the synaptic effects of tumor necrosis factor α (TNF-α) on glutamate-mediated excitatory postsynaptic currents, and prevented the action of interleukin 1β (IL-1β) on GABAergic inhibitory postsynaptic currents.Together, our results suggest that TRPV1 channels contrast TNF-α-mediated synaptic deficits in the peak phase of EAE and, in the chronic stages, enhance IL-1β-induced GABAergic defects. The opposing interplay with the synaptic actions of the two major pro-inflammatory cytokines might explain the bimodal effects of TRPV1 ablation on the motor deficits of EAE, and suggests that the inflammatory milieu determines whether TRPV1 channels exert preferentially aversive or protective effects on neurons during neuroinflammatory diseases.

Cannabinoid CB1 receptors regulate neuronal TNF-α effects in experimental autoimmune encephalomyelitis

Cannabinoid CB1 receptors (CB1Rs) regulate the neurodegenerative damage of experimental autoimmune encephalomyelitis (EAE) and of multiple sclerosis (MS). The mechanism by which CB1R stimulation exerts protective effects is still unclear. Here we show that pharmacological activation of CB1Rs dampens the tumor necrosis factor α (TNFα)-mediated potentiation of striatal spontaneous glutamate-mediated excitatory postsynaptic currents (EPSCs), which is believed to cogently contribute to the inflammation-induced neurodegenerative damage observed in EAE mice. Furthermore, mice lacking CB1Rs showed a more severe clinical course and, in parallel, exacerbated alterations of sEPSC duration after induction of EAE, indicating that endogenous cannabinoids activate CB1Rs and mitigate the synaptotoxic action of TNFα in EAE. Consistently, we found that mice lacking the fatty acid amide hydrolase (FAAH), and thus expressing abnormally high brain levels of the endocannabinoid anandamide, developed a less severe EAE associated with preserved TNFα-induced sEPSC alterations.CB1Rs are important modulators of EAE pathophysiology, and might play a mechanistic role in the neurodegenerative damage of MS patients.

Before or after does it matter? Different protocols of environmental enrichment differently influence motor, synaptic and structural deficits of cerebellar origin

Cerebellar compensation is a reliable model of lesion-induced plasticity occurring through profound synaptic and neurochemical modifications in cortical and sub-cortical regions. As the recovery from cerebellar deficits progresses, the firstly enhanced glutamate striatal transmission is then normalized. The time course of cerebellar compensation and the concomitant striatal modifications might be influenced by protocols of environmental enrichment (EE) differently timed in respect to cerebellar lesion. In the present study, we analyzed the effects of different EE protocols on postural and locomotor behaviors (by means of a neurological rating scale), and on striatal synaptic activity (by means of recordings of spontaneous glutamate-mediated excitatory postsynaptic currents (sEPSCs)) and on morphological correlates (by means of density and dendritic length of Fast Spiking (FS) interneurons) following hemicerebellectomy (HCb) in rats. Cerebellar motor deficits were reduced faster in the enriched animals in comparison to standard housed HCbed rats. The beneficial influence of EE was higher in the animals enriched before the HCb than in rats enriched only after the lesion. In parallel, the HCb-induced increase in striatal sEPSCs was not observed in rats enriched before HCb and attenuated in rats enriched after HCb. Furthermore, the EE prevented the shrinkage of dendritic arborization of FS striatal interneurons. Also this effect was more marked in animals enriched before than after the HCb. The exposure to EE exerted either neuro-protective or therapeutic actions on the cerebellar deficits. The experience-dependent changes of the synaptic and neuronal connectivity observed in the striatal neurons may represent one of the mechanisms through which the enrichment facilitates functional compensation following the cerebellar damage.

The link between inflammation, synaptic transmission and neurodegeneration in multiple sclerosis

Multiple sclerosis (MS) has been classically regarded as a disorder of the white matter of the central nervous system (CNS). However, early alterations of the neuronal compartment occurring in this disorder are partially independent of demyelination. Soluble inflammatory cytokines and glutamate have been proposed as major determinants of neurodegeneration in MS as well as in its experimental animal model, namely experimental autoimmune encephalomyelitis (EAE). The relationship between these two major determinants has been largely elusive. In recent years, unexpected connections have emerged between immune cells and soluble cytokines on the one hand, and synaptic transmission and neurodegeneration on the other. Neurophysiological recordings have recently shown that glutamate-mediated excitatory postsynaptic currents are enhanced during the early phase of EAE, because of altered expression and phosphorylation of AMPA receptors and the downregulation of the immediate early gene Arc/Arg3.1. The synaptic alterations occurring during neuroinflammatory diseases are largely mediated by inflammatory cytokines released from infiltrating T cells and from activated microglia, and are responsible, at least in part, for irreversible dendritic pathology. Collectively, the data covered in this review article suggest that CNS-confined inflammation in MS is associated with the release of soluble molecules, which are capable of altering excitatory synaptic transmission and, finally, of stimulating secondary neurodegenerative gray matter pathology.

Enhanced Pre-Synaptic Glutamate Release in Deep-Dorsal Horn Contributes to Calcium Channel Alpha-2-Delta-1 Protein-Mediated Spinal Sensitization and Behavioral Hypersensitivity

Nerve injury-induced expression of the spinal calcium channel alpha-2-delta-1 subunit (Cavα2δ1) has been shown to mediate behavioral hypersensitivity through a yet identified mechanism. We examined if this neuroplasticity modulates behavioral hypersensitivity by regulating spinal glutamatergic neurotransmission in injury-free transgenic mice overexpressing the Cavα2δ1 proteins in neuronal tissues. The transgenic mice exhibited hypersensitivity to mechanical stimulation (allodynia) similar to the spinal nerve ligation injury model. Intrathecally delivered antagonists for N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptors, but not for the metabotropic glutamate receptors, caused a dose-dependent allodynia reversal in the transgenic mice without changing the behavioral sensitivity in wild-type mice. This suggests that elevated spinal Cavα2δ1 mediates allodynia through a pathway involving activation of selective glutamate receptors. To determine if this is mediated by enhanced spinal neuronal excitability or pre-synaptic glutamate release in deep-dorsal horn, we examined wide-dynamic-range (WDR) neuron excitability with extracellular recording and glutamate-mediated excitatory postsynaptic currents with whole-cell patch recording in deep-dorsal horn of the Cavα2δ1 transgenic mice. Our data indicated that overexpression of Cavα2δ1 in neuronal tissues led to increased frequency, but not amplitude, of miniature excitatory post synaptic currents mediated mainly by AMPA/kainate receptors at physiological membrane potentials, and also by NMDA receptors upon depolarization, without changing the excitability of WDR neurons to high intensity stimulation. Together, these findings support a mechanism of Cavα2δ1-mediated spinal sensitization in which elevated Cavα2δ1 causes increased pre-synaptic glutamate release that leads to reduced excitation thresholds of post-synaptic dorsal horn neurons to innocuous stimuli. This spinal sensitization mechanism may mediate at least partially the neuropathic pain states derived from increased pre-synaptic Cavα2δ1 expression.

Autoinactivation of Neuronal AMPA Receptors via Glutamate-Regulated TARP Interaction

Neuronal AMPA receptors autoinactivate at high concentrations of glutamate, i.e., the current declines at glutamate concentrations above 10-100 microM. The mechanisms underlying this phenomenon are unclear. Stargazin-like TARPs are AMPA receptor auxiliary subunits that modulate receptor trafficking and channel properties. Here, we found that neuronal AMPA receptors and recombinant AMPA receptors coexpressed with stargazin autoinactivate at high concentrations of glutamate, whereas recombinant AMPA receptors expressed alone do not. The reduction of currents at high glutamate concentrations is not associated with a reduction of AMPA receptor number, but rather with the loss of stargazin-associated allosteric modulation of channel gating. We show that receptor desensitization promotes the dissociation of TARP-AMPA receptor complexes in a few milliseconds. This dissociation mechanism contributes to synaptic short-term modulation. The results demonstrate a mechanism for dynamic regulation of AMPA receptor activity to tune synaptic strength.

Gabapentin produces PKA‐dependent pre‐synaptic inhibition of GABAergic synaptic transmission in LC neurons following partial nerve injury in mice

We have previously demonstrated that gabapentin supraspinally activates the descending noradrenergic system to ameliorate pain hypersensitivity in mice with partial nerve ligation. To clarify the supraspinal mechanism of action of gabapentin, whole-cell patch-clamp recordings were performed on locus coeruleus (LC) neurons in brainstem slices prepared from mice after peripheral nerve injury or mice subjected to a sham-operation, and the effects of gabapentin in the modulation of synaptic transmission were studied. Bath application of gabapentin (10, 30 and 100 muM) in a concentration-dependent manner reduced the GABA(A) receptor-mediated inhibitory post-synaptic currents (IPSCs) in slices prepared from partially nerve-ligated mice, whereas glutamate-mediated excitatory post-synaptic currents were hardly affected. By contrast, gabapentin did not reduce IPSCs in slices taken from mice given a sham operation. Although gabapentin altered neither the amplitude nor the frequency of miniature IPSCs, it reduced IPSCs together with an increase in the paired-pulse ratio, suggesting that gabapentin acts on the pre-synaptic GABAergic nerve terminals in the LC. As the protein kinase A (PKA) inhibitor H-89 but not the protein kinase C inhibitor chelerythrine abolished the inhibitory action of gabapentin on IPSCs, PKA-mediated phosphorylation seems to be important for supraspinal gabapentin responsiveness in neuropathic conditions. Together, gabapentin generates PKA-dependent pre-synaptic inhibition of GABAergic synaptic transmission, and thereby removes the inhibitory influence on LC neurons only under neuropathic pain states. These findings provide crucial evidence of how supraspinally acting gabapentin recruits the descending noradrenergic system.

5-HT inhibits synaptic transmission in rat subthalamic nucleus neurons in vitro

The subthalamic nucleus (STN) plays a pivotal role in normal and abnormal motor function. We used patch pipettes to study effects of 5-HT on synaptic currents evoked in STN neurons by focal electrical stimulation of rat brain slices. 5-HT (10 μM) reduced glutamate-mediated excitatory postsynaptic currents (EPSCs) by 35±4%. However, a much higher concentration of 5-HT (100 μM) was required to inhibit GABA-mediated inhibitory postsynaptic currents (IPSCs) to a comparable extent. Concentration–response curves showed that the 5-HT inhibitory concentration 50% (IC 50) for inhibition of IPSCs (20.2 μM) was more than fivefold greater than the IC 50 for inhibition of EPSCs (3.4 μM). The 5-HT-induced reductions in EPSCs and IPSCs were accompanied by increases in paired-pulse ratios, indicating that 5-HT acts presynaptically to inhibit synaptic transmission. The 5-HT 1B receptor antagonist NAS-181 significantly antagonized 5-HT-induced inhibitions of EPSCs and IPSCs. These studies show that 5-HT inhibits synaptic transmission in the STN by activating presynaptic 5-HT 1B receptors.

Two Families of TARP Isoforms that Have Distinct Effects on the Kinetic Properties of AMPA Receptors and Synaptic Currents

Transmembrane AMPA receptor regulatory proteins (TARPs) are auxiliary AMPA receptor subunits that regulate both the trafficking and gating properties of AMPA receptors, and different TARP isoforms display distinct expression patterns in brain. Here, we compared the effects of four TARP isoforms on the kinetics of AMPA receptor currents. Each isoform slowed the deactivation of GluR1 currents, but the slowing was greatest with gamma-4 and gamma-8. Isoform-specific differences in desensitization were also observed that correlated with effects on deactivation. TARP isoforms also differentially modulated responses to trains of glutamate applications designed to mimic high-frequency presynaptic firing. Importantly, whereas both stargazin and gamma-4 rescued excitatory synaptic transmission in cerebellar granule cells from stargazer mice, the decay of miniature EPSCs was 2-fold slower in neurons expressing gamma-4. The results show that heterogeneity in the composition of AMPA receptor/TARP complexes contributes to synapse-specific differences in EPSC decays and frequency-dependent modulation of neurotransmission.

Long latency of evoked quantal transmitter release from somata of locus coeruleus neurons in rat pontine slices

The locus coeruleus (LC) harbors a compact group of noradrenergic cell bodies projecting to virtually all parts of the central nervous system. By using combined measurements of amperometry and patch-clamp, quantal vesicle release of noradrenaline (NA) was detected as amperometric spikes, after depolarization of the LC neurons. After a pulse depolarization, the average latency of amperometric spikes was 1,870 ms, whereas the latency of glutamate-mediated excitatory postsynaptic currents was 1.6 ms. A substantial fraction of the depolarization-induced amperometric spikes originated from the somata. In contrast to glutamate-mediated excitatory postsynaptic currents, NA secretion was strongly modulated by the action potential frequency (0.5-50 Hz). Somatodendritic NA release from LC upon enhanced cell activity produced autoinhibition of firing and of NA release. We conclude that, in contrast to classic synaptic transmission, quantal NA release from LC somata is characterized by a number of distinct properties, including long latency and high sensitivity to action potential frequency.

Interaction between glutamate and GABA systems in the integration of sympathetic outflow by the paraventricular nucleus of the hypothalamus

The paraventricular nucleus (PVN) of the hypothalamus is a central site known to modulate sympathetic outflow. Excitatory and inhibitory neurotransmitters within the PVN dictate final outflow. The goal of the present study was to examine the role of the interaction between the excitatory neurotransmitter glutamate and the inhibitory neurotransmitter GABA in the regulation of sympathetic activity. In alpha-chloralose- and urethane-anesthetized rats, microinjection of glutamate and N-methyl-D-aspartate (NMDA; 50, 100, and 200 pmol) into the PVN produced dose-dependent increases in renal sympathetic nerve activity, blood pressure, and heart rate. These responses were blocked by the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP-5). Microinjection of bicuculline, a GABA(A) receptor antagonist, into the PVN (50, 100, and 200 pmol) also produced significant, dose-dependent increases in renal sympathetic nerve activity, blood pressure, and heart rate; AP-5 also blocked these responses. Using microdialysis and HPLC/electrochemical detection techniques, we observed that bicuculline infusion into the PVN increased glutamate release. Using an in vitro hypothalamic slice preparation, we found that bicuculline increased the frequency of glutamate-mediated excitatory postsynaptic currents in PVN-rostral ventrolateral medullary projecting neurons, supporting a GABA(A)-mediated tonic inhibition of this excitatory input into these neurons. Together, these data indicate that 1) glutamate, via NMDA receptors, excites the presympathetic neurons within the PVN and increases sympathetic outflow and 2) this glutamate excitatory input is tonically inhibited by a GABA(A)-mediated mechanism.

Are Altered Excitatory Synapses Found in Neuronal Migration Disorders?

Physiological and Morphological Characterization of Dentate Granule Cells in the p35 Knock-out Mouse Hippocampus: Evidence for an Epileptic Circuit Patel LS, Wenzel HJ, Schwartzkroin PA J Neurosci 2004;24:9005–9014 There is a high correlation between pediatric epilepsies and neuronal migration disorders. What remains unclear is whether intrinsic features of the individual dysplastic cells give rise to heightened seizure susceptibility, or whether these dysplastic cells contribute to seizure activity by establishing abnormal circuits that alter the balance of inhibition and excitation. Mice lacking a functional p35 gene provide an ideal model in which to address these questions, because these knockout animals not only exhibit aberrant neuronal migration but also demonstrate spontaneous seizures. Extracellular field recordings from hippocampal slices, characterizing the input–output relation in the dentate, revealed little difference between wild-type and knockout mice under both normal and elevated extracellular potassium conditions. However, in the presence of the GABAA antagonist bicuculline, p35 knockout slices, but not wild-type slices, exhibited prolonged depolarizations in response to stimulation of the perforant path. No significant differences were found in the intrinsic properties of dentate granule cells (i.e., input resistance, time constant, action-potential generation) from wild-type versus knockout mice. However, antidromic activation (mossy fiber stimulation) evoked an excitatory synaptic response in more than 65% of granule cells from p35 knockout slices that was never observed in wild-type slices. Ultrastructural analyses identified morphological substrates for this aberrant excitation: recurrent axon collaterals, abnormal basal dendrites, and mossy fiber terminals forming synapses onto the spines of neighboring granule cells. These studies suggest that granule cells in p35 knockout mice contribute to seizure activity by forming an abnormal excitatory feedback circuit. Prolonged NMDA-mediated Synaptic Response, Altered Ifenprodil Sensitivity, and Generation of Epileptiform-like Events in a Malformed Hippocampus of Rats Exposed to Methylazoxymethanol in Utero Calcagnotto ME, Baraban SC J Neurophysiol 2005 [Epub ahead of print] Cortical malformations are often associated with refractory epilepsy and cognitive deficit. Clinical and experimental studies have demonstrated an important role for glutamate-mediated synaptic transmission in these conditions. With whole-cell voltage-clamp techniques, we examined evoked glutamate-mediated excitatory postsynaptic currents (eEPSCs) and responses to exogenously applied glutamate on hippocampal heterotopic cells in an animal model of malformation (i.e., rats exposed to methylazoxymethanol [MAM] in utero). Analysis of eEPSCs revealed that the late N-methyl-D-aspartate (NMDA) receptor–mediated eEPSC component was significantly increased on heterotopic cells compared with age-matched normotopic pyramidal cells. At a holding potential of +40 mV, heterotopic cells also exhibited eEPSCs with a slower decay-time constant. No differences in the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) component of eEPSCs were detected. In 23% of heterotopic pyramidal cells, electrical stimulation evoked prolonged burstlike responses. Focal application of glutamate (10 m M) targeted to different sites near the heterotopia also evoked epileptiform-like bursts on heterotopic cells. Ifenprodil (10 μM), an NR2B subunit antagonist, only slightly reduced the NMDA receptor–mediated component and amplitude of eEPSCs on heterotopic cells (MAM) but significantly decreased the late component and peak amplitude of eEPSCs in normotopic cells (Control). Our data demonstrate a functional alteration in the NMDA-mediated component of excitatory synaptic transmission in heterotopic cells and suggest that this alteration may be attributable, at least in part, to changes in composition and function of the NMDAR subunit. Changes in NMDA-receptor function may directly contribute to the hyperexcitability and cognitive deficits reported in animal models and patients with brain malformations.

Synaptically Released GABA Activates Both Pre- and Postsynaptic GABAB Receptors in the Rat Globus Pallidus

The globus pallidus (GP) contains abundant GABAergic synapses and GABA(B) receptors. To investigate whether synaptically released GABA can activate pre- and postsynaptic GABA(B) receptors in the GP, physiological recordings were performed using rat brain slice preparations. Cell-attached recordings from GABA(A) antagonist-treated preparations revealed that repetitive local stimulation induced a GABA(B) antagonist-sensitive pause in spontaneous firings of GP neurons. Whole cell recordings revealed that the repetitive stimulation evoked fast excitatory postsynaptic potentials followed by a slow inhibitory postsynaptic potential (IPSP) in GP neurons. The slow IPSP was insensitive to a GABA(A) receptor antagonist, increased in amplitude with the application of ionotropic glutamate receptor antagonists, and was suppressed by the GABA(B) antagonist CGP55845. The reversal potential of the slow IPSP was close to the potassium equilibrium potential. These results suggest that synaptically released GABA activated postsynaptic GABA(B) receptors and induced the pause and the slow IPSP. On the other hand, in the neurons that were treated to block postsynaptic GABA(B) responses, CGP55845 increased the amplitudes of repetitive local stimulation-induced GABA(A)-mediated inhibitory postsynaptic currents (IPSCs) but not the ionotropic glutamate-mediated excitatory postsynaptic currents. Moreover, the GABA(B) receptor specific agonist baclofen reduced the frequency of miniature IPSCs without altering their amplitude distributions. These results suggest that synaptically released GABA also activated presynaptic GABA(B) autoreceptors, resulting in decreased GABA release in the GP. Together, we infer that both pre- and postsynaptic GABA(B) receptors may play crucial roles in the control of GP neuronal activity.

Prolonged NMDA-Mediated Responses, Altered Ifenprodil Sensitivity, and Epileptiform-Like Events in the Malformed Hippocampus of Methylazoxymethanol Exposed Rats

Cortical malformations are often associated with refractory epilepsy and cognitive deficit. Clinical and experimental studies have demonstrated an important role for glutamate-mediated synaptic transmission in these conditions. Using whole cell voltage-clamp techniques, we examined evoked glutamate-mediated excitatory postsynaptic currents (eEPSCs) and responses to exogenously applied glutamate on hippocampal heterotopic cells in an animal model of malformation i.e., rats exposed to methylazoxymethanol (MAM) in utero. Analysis revealed that the late N-methyl-D-aspartate (NMDA) receptor-mediated eEPSC component was significantly increased on heterotopic cells compared with age-matched normotopic pyramidal cells. At a holding potential of +40 mV, heterotopic cells also exhibited eEPSCs with a slower decay-time constant. No differences in the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) component of eEPSCs were detected. In 23% of heterotopic pyramidal cells, electrical stimulation evoked prolonged burst-like responses. Focal application of glutamate (10 mM) targeted to different sites near the heterotopia also evoked epileptiform-like bursts on heterotopic cells. Ifenprodil (10 microM), an NR2B subunit antagonist, only slightly reduced the NMDA receptor (NMDAR)-mediated component and amplitude of eEPSCs on heterotopic cells (MAM) but significantly decreased the late component and peak amplitude of eEPSCs in normotopic cells (control). Our data demonstrate a functional alteration in the NMDA-mediated component of excitatory synaptic transmission in heterotopic cells and suggest that this alteration may be attributable, at least in part, to changes in composition and function of the NMDAR subunit. Changes in NMDAR function may directly contribute to the hyperexcitability and cognitive deficits reported in animal models and patients with brain malformations.