21st century excitatory amino acid research: A Q & A with Jeff Watkins and Dick Evans.

In 1981 Jeff Watkins and Dick Evans wrote what was to become a seminal review on excitatory amino acids (EAAs) and their receptors (Watkins and Evans, 1981). Bringing together various lines of evidence dating back over several decades on: the distribution in the nervous system of putative amino acid neurotransmitters; enzymes involved in their production and metabolism; the uptake and release of amino acids; binding of EAAs to membranes; the pharmacological action of endogenous excitatory amino acids and their synthetic analogues, and notably the actions of antagonists for the excitations caused by both nerve stimulation and exogenous agonists, often using pharmacological tools developed by Jeff and his colleagues, they provided a compelling account for EAAs, especially l-glutamate, as a bona fide neurotransmitter in the nervous system. The rest, as they say, is history, but far from being consigned to history, EAA research is in rude health well into the 21st Century as this series of Special Issues of Neuropharmacology exemplifies. With EAAs and their receptors flourishing across a wide range of disciplines and clinical conditions, we enter into a dialogue with two of the most prominent and influential figures in the early days of EAA research: Jeff Watkins and Dick Evans.

Enantiomeric N-substituted phthalimides with excitatory amino acids protect zebrafish larvae against PTZ-induced seizures.

Epilepsy is a chronic neurological disease with high prevalence and adverse impacts on the quality of life of patients and caregivers. Up to one-third of individuals with epilepsy do not respond to current pharmacotherapy, underscoring the importance of identifying new molecules for epilepsy control. Thalidomide, the first synthetized phthalimide, is a neuroactive molecule with anti-seizure drug properties. The phthalimide group has been studied in some N-phthaloyl amino acids due to its pharmacological properties. Here we examine enantiomers of phthaloyl aspartate (R and S) and phthaloyl glutamate (R and S) for anti-seizure effects using zebrafish as a model. The zebrafish model is rapidly growing in use as a preclinical screening tool for drug discovery in epilepsy. Pentylenetetrazol (PTZ) exposure was used to produce convulsive behavior in 7- and 10-days post-fertilization (dpf) zebrafish larvae; these ages correspond to before and after the blood-brain-barrier (BBB) is fully developed. Larvae were pre-treated for 60 min with: control, valproic acid sodium salt (SVP) 3 mM, or one of two concentrations of N-phthaloyl-R-glutamic acid (R-TGLU; 100, 316 µM) prior to PTZ addition. R-TGLU modified the locomotor phenotype and protected against PTZ in 7 and 10 dpf larvae at 316 µM, suggesting it crossed the BBB. We next tested the per se and anticonvulsant effect of the glutamate and aspartate phthalimides were tested at 237.1 and 316 µM concentration in 10dpf zebrafish. The four tested molecules produced an anticonvulsant effect at 237.1 µM concentration, however the behavioral changes that they induce suggest that they might act by different mechanisms.

The α2ß2 isoform combination dominates the astrocytic Na+ /K+ -ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation.

Synaptic activity results in transient elevations in extracellular K+ , clearance of which is critical for sustained function of the nervous system. The K+ clearance is, in part, accomplished by the neighboring astrocytes by mechanisms involving the Na+ /K+ -ATPase. The Na+ /K+ -ATPase consists of an α and a ß subunit, each with several isoforms present in the central nervous system, of which the α2ß2 and α2ß1 isoform combinations are kinetically geared for astrocytic K+ clearance. While transcript analysis data designate α2ß2 as predominantly astrocytic, the relative quantitative protein distribution and isoform pairing remain unknown. As cultured astrocytes altered their isoform expression in vitro, we isolated a pure astrocytic fraction from rat brain by a novel immunomagnetic separation approach in order to determine the expression levels of α and ß isoforms by immunoblotting. In order to compare the abundance of isoforms in astrocytic samples, semi-quantification was carried out with polyhistidine-tagged Na+ /K+ -ATPase subunit isoforms expressed in Xenopus laevis oocytes as standards to obtain an efficiency factor for each antibody. Proximity ligation assay illustrated that α2 paired efficiently with both ß1 and ß2 and the semi-quantification of the astrocytic fraction indicated that the astrocytic Na+ /K+ -ATPase is dominated by α2, paired with ß1 or ß2 (in a 1:9 ratio). We demonstrate that while the familial hemiplegic migraine-associated α2.G301R mutant was not functionally expressed at the plasma membrane in a heterologous expression system, α2+/G301R mice displayed normal protein levels of α2 and glutamate transporters and that the one functional allele suffices to manage the general K+ dynamics.

A dual role for the RhoGEF Ephexin5 in regulation of dendritic spine outgrowth.

The outgrowth of new dendritic spines is closely linked to the formation of new synapses, and is thought to be a vital component of the experience-dependent circuit plasticity that supports learning. Here, we examined the role of the RhoGEF Ephexin5 in driving activity-dependent spine outgrowth. We found that reducing Ephexin5 levels increased spine outgrowth, and increasing Ephexin5 levels decreased spine outgrowth in a GEF-dependent manner, suggesting that Ephexin5 acts as an inhibitor of spine outgrowth. Notably, we found that increased neural activity led to a proteasome-dependent reduction in the levels of Ephexin5 in neuronal dendrites, which could facilitate the enhanced spine outgrowth observed following increased neural activity. Surprisingly, we also found that Ephexin5-GFP levels were elevated on the dendrite at sites of future new spines, prior to new spine outgrowth. Moreover, lowering neuronal Ephexin5 levels inhibited new spine outgrowth in response to both global increases in neural activity and local glutamatergic stimulation of the dendrite, suggesting that Ephexin5 is necessary for activity-dependent spine outgrowth. Our data support a model in which Ephexin5 serves a dual role in spinogenesis, acting both as a brake on overall spine outgrowth and as a necessary component in the site-specific formation of new spines.

Activity-Dependent Degradation of Synaptic Vesicle Proteins Requires Rab35 and the ESCRT Pathway.

UNLABELLED: Synaptic vesicle (SV) pools must maintain a functional repertoire of proteins to efficiently release neurotransmitter. The accumulation of old or damaged proteins on SV membranes is linked to synaptic dysfunction and neurodegeneration. However, despite the importance of SV protein turnover for neuronal health, the molecular mechanisms underlying this process are largely unknown. Here, we have used dissociated rat hippocampal neurons to investigate the pathway for SV protein degradation. We find that neuronal activity drives the degradation of a subset of SV proteins and that the endosomal sorting complex required for transport (ESCRT) machinery and SV-associated GTPase Rab35 are key elements of this use-dependent degradative pathway. Specifically, neuronal activity induces Rab35 activation and binding to the ESCRT-0 protein Hrs, which we have identified as a novel Rab35 effector. These actions recruit the downstream ESCRT machinery to SV pools, thereby initiating SV protein degradation via the ESCRT pathway. Our findings show that the Rab35/ESCRT pathway facilitates the activity-dependent removal of specific proteins from SV pools, thereby maintaining presynaptic protein homeostasis. SIGNIFICANCE STATEMENT: Synaptic transmission is mediated by the release of chemical neurotransmitters from synaptic vesicles (SVs). This tightly regulated process requires a functional pool of SVs, necessitating cellular mechanisms for removing old or damaged proteins that could impair SV cycling. Here, we show that a subset of SV proteins is degraded in an activity-dependent manner and that key steps in this degradative pathway are the activation of the small GTPase Rab35 and the subsequent recruitment of the endosomal sorting complex required for transport (ESCRT) machinery to SV pools. Further, we demonstrate that ESCRT-0 component Hrs is an effector of Rab35, thus providing novel mechanistic insight into the coupling of neuronal activity with SV protein degradation and the maintenance of functional SV pools.

Short-Term Synaptic Plasticity at Interneuronal Synapses Could Sculpt Rhythmic Motor Patterns.

The output of a neuronal network depends on the organization and functional properties of its component cells and synapses. While the characterization of synaptic properties has lagged cellular analyses, a potentially important aspect in rhythmically active networks is how network synapses affect, and are in turn affected by, network activity. This could lead to a potential circular interaction where short-term activity-dependent synaptic plasticity is both influenced by and influences the network output. The analysis of synaptic plasticity in the lamprey locomotor network was extended here to characterize the short-term plasticity of connections between network interneurons and to try and address its potential network role. Paired recordings from identified interneurons in quiescent networks showed synapse-specific synaptic properties and plasticity that supported the presence of two hemisegmental groups that could influence bursting: depression in an excitatory interneuron group, and facilitation in an inhibitory feedback circuit. The influence of activity-dependent synaptic plasticity on network activity was investigated experimentally by changing Ringer Ca(2+) levels, and in a simple computer model. A potential caveat of the experimental analyses was that changes in Ringer Ca(2+) (and compensatory adjustments in Mg(2+) in some cases) could alter several other cellular and synaptic properties. Several of these properties were tested, and while there was some variability, these were not usually significantly affected by the Ringer changes. The experimental analyses suggested that depression of excitatory inputs had the strongest influence on the patterning of network activity. The simulation supported a role for this effect, and also suggested that the inhibitory facilitating group could modulate the influence of the excitatory synaptic depression. Short-term activity-dependent synaptic plasticity has not generally been considered in spinal cord models. These results provide further evidence for short-term plasticity between locomotor network interneurons. As this plasticity could influence the patterning of the network output it should be considered as a potential functional component of spinal cord networks.

Retinal development impairment and degenerative alterations in adult rats subjected to post-natal malnutrition.

BACKGROUND: The early stages of central nervous system (CNS) development are extremely important. Key events such as neurogenesis, gliogenesis, synaptogenesis, and ontogenesis occur. Malnutrition promotes alterations in CNS development, including the retinal development. During retinal development, malnutrition can induce a delay in some important events, such as neurotransmitter expression and neurogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Postpartum Wistar rats were fed either a commercial diet or a multideficient diet. Pups were breastfed by these rats, and from PND21 were kept with the same diet until PND45. We investigated the effects of malnutrition on adult retinal tissue with regard to (1) endogenous gamma-amino butyric acid (GABA) release induced by excitatory amino acids (EAAs) and (2) the expression of cellular markers related to degenerative events, such as reactive gliosis, microglial activation, cell proliferation and cell death. Endogenous GABA release induced by EAAs was higher in the retina of malnourished rats. The Müller cell population was reduced and displayed alterations in their phenotype profile compatible with reactive gliosis. The expression of glutamine synthetase and markers of cellular proliferation were higher in the retina of malnourished rats. Additionally, retinal dysplasia-like structures were present, indicating disturbance in the cell cycle machinery. CONCLUSION/SIGNIFICANCE: The current study provides evidence that the adult retina shows degenerative processes induced by long-term malnutrition during the postnatal development. These findings have high clinical significance with regard to the identification of possible targets for interventions in malnourished patients.

Electrophysiological characterization of methyleugenol: a novel agonist of GABA(A) receptors.

Methyleugenol (ME) is a natural constituent isolated from many plant essential oils having multiple biological effects including anticonvulsant and anesthetic activities, although the underlying mechanisms remain unclear. Here, we identify ME as a novel agonist of ionotropic γ-aminobutyric acid (GABA) receptors. At lower concentrations (∼30 µM), ME significantly sensitized GABA-induced, but not glutamate- or glycine-induced, currents in cultured hippocampal neurons, indicative of a preferentially modulatory role of this compound for A type GABA receptors (GABAARs). In addition, ME at higher concentrations (≥100 µM) induced a concentration-dependent, Cl(-)-permeable current in hippocampal neurons, which was inhibited by a GABAAR channel blocker, picrotoxin, and a competitive GABAAR antagonist, bicuculline, but not a specific glycine receptor inhibitor, strychnine. Moreover, ME activated a similar current mediated by recombinant α1-ß2-γ2 or α5-ß2-γ2 GABAARs in human embryonic kidney (HEK) cells. Consequently, ME produced a strong inhibition of synaptically driven neuronal excitation in hippocampal neurons. Together, these results suggest that ME represents a novel agonist of GABAARs, shedding additional light on future development of new therapeutics targeting GABAARs. The present study also adds GABAAR activation to the list of molecular targets of ME that probably account for its biological activities.

Short-term episodes of hypoxia induce posthypoxic hyperexcitability and selective death of GABAergic hippocampal neurons.

We have previously developed a rat hippocampal neuronal cell model for the registration of the preconditioning effect and posthypoxic hyperexcitability (Turovskaya et al., 2011). Repeated episodes of short-term hypoxia are reported to suppress the amplitude of Ca(2+) response to NMDA in majority of neurons, reflecting the effect of preconditioning in the culture. In addition, exposure to hypoxia causes posthypoxic hyperexcitability: this is characterized by the onset of spontaneous synchronous Ca(2+) transients in a population of neurons in a neural network during the period of reoxygenation after each hypoxic episode. The nature of this phenomenon is unknown, although it has been observed that there always exists a minority of neurons in which there is no effect of hypoxic preconditioning. In this small population of neurons, the amplitude of Ca(2+) response to NMDA is not suppressed, but rather increases after each episode of hypoxia. Here we report the type of these neurons and their role in the generation of posthypoxic hyperexcitability. We compared the effect of short-term hypoxia on the amplitude of the Ca(2+) response to NMDA and the Ca(2+) transient generation in two populations of neurons - inhibitory GABAergic and excitatory glutamatergic. We have demonstrated that the neurons in which the preconditioning effect was not observed are GABAergic. Moreover at the instant moment of the posthypoxic synchronous Ca(2+)-transient generation (during reoxygenation) there is a global increase of [Ca(2+)]i and subsequent apoptosis in some GABAergic neurons. Anti-inflammatory cytokine interleukin-10 prevents the development of posthypoxic hyperexcitability, inhibiting the spontaneous synchronous Ca(2+) transients. At the same time, interleukin-10 protects GABAergic neurons from death, by restoring the effect of hypoxic preconditioning in them. Activation of one of the signaling pathways initiated by interleukin-10 appears to be necessary for the development of hypoxic preconditioning in GABAergic neurons. Overall our results indicate that short-term episodes of hypoxia can damage GABAergic neurons and weaken the inhibitory action of GABAergic neurons in a neural network. Activation of PI3K-dependent survival signaling pathways in neurons of this type is a possible strategy to protect these cells against hypoxia.

Polysaccharide glucomannan isolated from Heterodermia obscurata attenuates acute and chronic pain in mice.

Glucomannan (GM) is a polysaccharide obtained from Heterodermia obscurata lichens. The present study was conducted to elucidate the antinociceptive effect of GM in behavioural models of acute and chronic pain in mice. GM reduced mechanical allodynia and the levels of interleukin 1-ß (IL-1ß) in spinal cord and nerve in the partial sciatic nerve ligation (PSNL) model. Systemic treatment with GM inhibited the nociception induced by intraplantar injection of glutamate and by intrathecal injection of N-methyl-d-aspartic acid (NMDA), (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), tumour necrosis factor α (TNF-α) and IL-1ß. Taken together, our data demonstrate that GM has significant antinociceptive effect in acute and chronic pain, suggesting a potential interest in the development of new clinically relevant drugs for the management of pain.

[Inhibitory regulation of glutamate receptors in the frog motoneuron].

The interaction of exogenously applied excitatory (glutamate and their agonists NMDA, AMPA, kainate) and inhibitory (glycine and GABA) amino acid effects was studied intracellularly in the motoneurones of the isolated frog spinal cord. During simultaneous glycine or GABA bath applications GLU-, AMPA-, KA- and NMDA-evoked responses were, respectively, decreased up to 45.8 +/- 2.9% (n = 12) and 67.8 +/- 3.9% (n = 16), 13.9 +/- 4.3% (n = 9) and 32.1 +/- 8.3% (n = 12), 36.8 +/- 8.2% (n = 7) and 48.0 +/- 11.8% (n = 6), 7.7 +/- 3.5% (n = 9) and 18.1 +/- 3.8% (n = 14) from the control. Sequential applications of EAA after glycine or GABA as well as the applications of EAA-agonist and glycine (GABA) mixture demonstrated similar results. The decrease of EAA-responses by glycine and GABA was abolished by selective GlyR antagonist strychnine (1 microM) and the selective GABAR antagonist SR95531 (gabazine, 20 MM), respectively. The data revealed differences in inhibitory effect of glycine and GABA on the excitation responses mediated by different types of glutamate receptors in the frog motoneurones: the predominant inhibitory effect of glycine and GABA on NMDA-responses and weak inhibitory effect on KA- and GLU-responses. Inhibitory effect of glycine was twice as much as that of GABA at the same concentration.

Input to the lateral habenula from the basal ganglia is excitatory, aversive, and suppressed by serotonin.

The lateral habenula (LHb) has recently been identified as a key regulator of the reward system by driving inhibition onto dopaminergic neurons. However, the nature and potential modulation of the major input to the LHb originating from the basal ganglia are poorly understood. Although the output of the basal ganglia is thought to be primarily inhibitory, here we show that transmission from the basal ganglia to the LHb is excitatory, glutamatergic, and suppressed by serotonin. Behaviorally, activation of this pathway is aversive, consistent with its role as an "antireward" signal. Our demonstration of an excitatory projection from the basal ganglia to the LHb explains how LHb-projecting basal ganglia neurons can have similar encoding properties as LHb neurons themselves. Our results also provide a link between antireward excitatory synapses and serotonin, a neuromodulator implicated in depression.

Presynaptic inhibition of glutamate transmission by α2 receptors in the VTA.

The ventral tegmental area (VTA) forms part of the mesocorticolimbic system and plays a pivotal role in reward and reinforcing actions of drugs of abuse. Glutamate transmission within the VTA controls important aspects of goal-directed behavior and motivation. Noradrenergic receptors also present in the VTA have important functions in the modulation of neuronal activity. Here we studied the effects of α2 noradrenergic receptor activation in the alteration of glutamate neurotransmission in VTA dopaminergic neurons from male Sprague-Dawley rats. We used whole-cell patch-clamp recordings from putative VTA dopaminergic neurons and measured excitatory postsynaptic currents. Clonidine (40 µm) and UK 14,304 (40 µm), both α2 receptor agonists, reduced (approximately 40%) the amplitude of glutamate-induced excitatory postsynaptic currents. After clonidine administration, there was a dose-dependent reduction over the concentration range of 15-40 µm. Using yohimbine (20 µm) and two other α2 adrenergic receptor antagonists, idaxozan (40 µm) and atipemazole (20 µm), we demonstrated that the inhibitory action is specifically mediated by α2 receptors. Moreover, by inhibiting protein kinases with H-7 (75 µm), Rp-adenosine 3',5'-cyclic (11 µm) and chelerythrine (1 µm) it was shown that the clonidine-induced inhibition seems to involve a selective activation of the protein kinase C intracellular pathway. Increased paired-pulse ratios and changes in spontaneous and miniature excitatory postsynaptic current frequencies but not amplitudes indicated that the effect of the α2 agonist was presynaptically mediated. It is suggested that the suppression of glutamate excitatory inputs onto VTA dopaminergic neurons might be relevant in the regulation of reward and drug-seeking behaviors.

Role of pulmonary stretch receptors and sympathetic system in the inhibition of reflex bradycardia produced by chemical stimulation of the periaqueductal gray matter of the rat.

The present study examined the role of the sympathetic system and pulmonary afferent feedback in the baroreflex inhibition by chemical stimulation of the dorsal periaqueductal gray matter (DPAG) of the anesthetized rat. The baroreflex bradycardia was induced by phenylephrine infusions (PHE, 50 µg/ml/min, i.v.) given either alone or combined with glutamate microinjections (GLU, 10 nmol/100 nl) into the DPAG. GLU microinjections alone produced marked increases in respiratory amplitude (67±19%), but barely changed the respiratory frequency (15±3 cpm) and blood pressure (14±2 mm Hg), and did not affect the heart rate. In contrast, the same injections produced a 92% inhibition of PHE-induced bradycardia (from -62 to -5 bpm). Because GLU microinjections per se had little effects on blood pressure, the baroreflex inhibition should be credited to the deactivation of both the vagal and sympathetic reflex pathways at the medulla. Indeed, the baroreflex was inhibited in only 47% following the DPAG stimulation of atenolol-treated rats. The GLU-evoked inhibition of baroreflex was also correlated with concomitant increases in respiratory amplitude. The role of pulmonary feedback in baroreflex inhibition was thus examined before and after the neuromuscular blockade of atenolol-treated rats. In spontaneously breathing rats, GLU microinjections reversed PHE-induced bradycardia to tachycardia, thereby producing a 153% inhibition of reflex bradycardia (from -38 bpm to +20 bpm). In contrast, the baroreflex inhibition was attenuated in only 53% after neuromuscular blockade (from -34 to -16 bpm). Data are the first evidence of the contribution of pulmonary stretch receptor feedback in DPAG-evoked inhibition of reflex bradycardia.

Cortactin-binding protein 2 modulates the mobility of cortactin and regulates dendritic spine formation and maintenance.

Dendritic spines, the actin-rich protrusions emerging from dendrites, are the locations of excitatory synapses in mammalian brains. Many molecules that regulate actin dynamics also influence the morphology and/or density of dendritic spines. Since dendritic spines are neuron-specific subcellular structures, neuron-specific proteins or signals are expected to control spinogenesis. In this report, we characterize the distribution and function of neuron-predominant cortactin-binding protein 2 (CTTNBP2) in rodents. An analysis of an Expressed Sequence Tag database revealed three splice variants of mouse CTTNBP2: short, long, and intron. Immunoblotting indicated that the short form is the dominant CTTNBP2 variant in the brain. CTTNBP2 proteins were highly concentrated at dendritic spines in cultured rat hippocampal neurons as well as in the mouse brain. Knockdown of CTTNBP2 in neurons reduced the density and size of dendritic spines. Consistent with these morphological changes, the frequencies of miniature EPSCs in CTTNBP2 knockdown neurons were lower than those in control neurons. Cortactin acts downstream of CTTNBP2 in spinogenesis, as the defects caused by CTTNBP2 knockdown were rescued by overexpression of cortactin but not expression of a CTTNBP2 mutant protein lacking the cortactin interaction. Finally, immunofluorescence staining demonstrated that, unlike cortactin, CTTNBP2 stably resided at dendritic spines even after glutamate stimulation. Fluorescence recovery after photobleaching further suggested that CTTNBP2 modulates the mobility of cortactin in neurons. CTTNBP2 may thus help to immobilize cortactin in dendritic spines and control the density of dendritic spines.

Sympathetic premotor neurones project to and are influenced by neurones in the contralateral rostral ventrolateral medulla of the rat in vivo.

The tonic activity of bulbospinal neurones in the rostral ventrolateral medulla (RVLM) is thought to underlie basal sympathetic nerve activity. A key research objective is to delineate the mechanisms that contribute to the firing of these neurones. In the current study we investigate the hypothesis that inputs arising in the contralateral RVLM converge on barosensitive bulbospinal neurones and contribute to their discharge pattern. Extracellular recordings were made from 24 barosensitive bulbospinal neurones in urethane anaesthetised, vagotomised and artificially ventilated rats during activation (glutamate or D,L-homocysteic acid microinjection, 50 nl, 50mM, or monopolar electrical stimulation) or inhibition (microinjection of GABA receptor agonists muscimol or isoguvacine, 50 nl, 10mM) of the contralateral RVLM. Chemical RVLM activation strongly increased (10/17) or inhibited (6/17) the spontaneous activity of neurones recorded in the contralateral RVLM. Electrical RVLM stimulation evoked a combination of short latency (median 6 ms) inhibitory and longer latency (median 9.1 ms, P<0.01) excitatory orthodromic responses in contralateral sympathetic premotor neurones and in some cases evoked antidromic action potentials that collided with spontaneous spikes. RVLM inhibition increased the discharge rate of sympathetic premotor neurones in the contralateral brainstem by 21 ± 13% (P<0.05) and reduced the variability of unit firing by 37 ± 12% (n=5, p<0.05). These findings indicate that sympathetic premotor neurones receive inhibitory and excitatory input from the contralateral RVLM, that inhibitory inputs predominate under baseline conditions, and that a population of sympathetic premotor neurones project to the contralateral RVLM in addition to their spinal targets.

Microarray analysis of rat hippocampus exposed to excitotoxicity: reversal Na(+)/Ca(2+) exchanger NCX3 is overexpressed in glial cells.

Multiple factors are involved in the glutamate-induced excitotoxicity phenomenon, such as overload of ionotropic and metabotropic receptors, excess Ca(2+) influx, nitric oxide synthase activation, oxidative damage due to increase in free radicals, and release of endogenous polyamine, among others. In order to attempt a more integrated approach to address this issue, we established, by microarray analysis, the hippocampus gene expression profiles under glutamate-induced excitotoxicity conditions. Increased gene expression is mainly related to excitotoxicity (CaMKII, glypican 2, GFAP, NCX3, IL-2, and Gmeb2) or with cell damage response (dynactin and Ecel1). Several genes that augmented their expression are related to glutamatergic system modulation, in particular with NMDA receptor modulation and calcium homeostasis (IL-2, CaMKII, acrosin, Gmeb2, hAChE, Slc83a, and SP1 factor). Conversely, among genes that diminished their expression, we found the Syngap 1, which is downregulated by CaMKII, and the MHC II, which is downregulated by glutamate. Changes observed in gene expression induced by monosodium glutamate (MSG) neonatal treatment in the hippocampus are consistent with the activation of the mechanisms that modulate NMDA receptor function as well as with the implementation of plastic response to cell damage and intracellular calcium homeostasis. Regarding this aspect, we report here that NCX3/Slc8a3, a Na(+)/Ca(2+) membrane exchanger, is highly expressed in astrocytes, both in vitro and in vivo, in response to glutamate-induced excitotoxicity. Hence, the results of this analysis present a broad view of the expression profile elicited by MSG neonatal treatment, and lead us to suggest the possible molecular pathways of action and reaction involved under this experimental model of excitotoxicity.

Excitation-induced ataxin-3 aggregation in neurons from patients with Machado-Joseph disease.

Machado-Joseph disease (MJD; also called spinocerebellar ataxia type 3) is a dominantly inherited late-onset neurodegenerative disorder caused by expansion of polyglutamine (polyQ)-encoding CAG repeats in the MJD1 gene (also known as ATXN3). Proteolytic liberation of highly aggregation-prone polyQ fragments from the protective sequence of the MJD1 gene product ataxin 3 (ATXN3) has been proposed to trigger the formation of ATXN3-containing aggregates, the neuropathological hallmark of MJD. ATXN3 fragments are detected in brain tissue of MJD patients and transgenic mice expressing mutant human ATXN3(Q71), and their amount increases with disease severity, supporting a relationship between ATXN3 processing and disease progression. The formation of early aggregation intermediates is thought to have a critical role in disease initiation, but the precise pathogenic mechanism operating in MJD has remained elusive. Here we show that L-glutamate-induced excitation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons initiates Ca(2+)-dependent proteolysis of ATXN3 followed by the formation of SDS-insoluble aggregates. This phenotype could be abolished by calpain inhibition, confirming a key role of this protease in ATXN3 aggregation. Aggregate formation was further dependent on functional Na(+) and K(+) channels as well as ionotropic and voltage-gated Ca(2+) channels, and was not observed in iPSCs, fibroblasts or glia, thereby providing an explanation for the neuron-specific phenotype of this disease. Our data illustrate that iPSCs enable the study of aberrant protein processing associated with late-onset neurodegenerative disorders in patient-specific neurons.

Physiological evidence that D-aspartate activates a current distinct from ionotropic glutamate receptor currents in Aplysia californica neurons.

D-Aspartate (D-Asp) activates an excitatory current in neurons of Aplysia californica. Although D-Asp is presumed to activate a subset of L-glutamate (L-Glu) channels, the identities of putative d-Asp receptors and channels are unclear. Whole cell voltage- and current-clamp studies using primary cultures of Aplysia buccal S cluster (BSC) neurons were executed to characterize D-Asp-activated ion channels. Both D-Asp and L-Glu evoked currents with similar current-voltage relationships, amplitudes, and relatively slow time courses of activation and inactivation when agonists were pressure applied. D-Asp-induced currents, however, were faster and desensitized longer, requiring 40 s to return to full amplitude. Of cells exposed to both agonists, 25% had D-Asp- but not L-Glu-induced currents, suggesting a receptor for D-Asp that was independent of l-Glu receptors. D-Asp channels were permeable to Na(+) and K(+), but not Ca²âº, and were vulnerable to voltage-dependent Mg²âº block similarly to vertebrate NMDA receptor (NMDAR) channels. d-Asp may activate both NMDARs and non-l-Glu receptors in the nervous system of Aplysia.

Glutamatergic modulation of separation distress: profound emotional effects of excitatory amino acids in chicks.

Pre-clinical models of brain affective circuits provide relevant evidence for understanding the brain systems that figure heavily in psychiatric disorders. Social isolation and the resulting separation distress contribute to the onset of depression. In this work, the effects of excitatory amino acids (EAA) on isolation-induced distress vocalization (DV) were assessed in young domestic chicks. Both glutamate and quisqualate (QA) produced dose-dependent reductions in DVs, while N-methyl-d-aspartate (NMDA) and kainate (KA) increased DVs. Such a differential pattern of responsiveness may indicate the presence of reciprocal or interacting EAA systems in the brain control of separation distress. Administration of either the NMDA receptor antagonist 2-amino-5-phosphonovalerate (APV) or the broad-spectrum antagonist gamma-d-glutamylglycine (DGG) greatly reduced DVs, as did the antagonist 2-amino-4-phosphonobutyrate (APB). APV did not attenuate the increase in vocalizations seen after NMDA or KA administration. DGG, however, was able to block the increase in calling produced by either of these agonists, suggesting a KA receptor mechanism. KA treatment inhibited the ability of other chicks, or auditory and somatosensory information, to suppress DVs. KA-treated animals exhibited a hyperemotional behavior pattern during which a variety of motivated behaviors were disrupted including reactions to novel objects, approaching the flock, and foraging. They could not sustain a coherent flock-like social cohesion, but exhibited strong fixed-action patterns of flight interspersed with hiding and crouching behaviors. The evident behavioral changes suggest that glutamatergic synapses directly influence sensory, motor and emotional processes in the brain and may be especially important in the integration of environmental stimuli with emotional central state processes of animals. Considering that unresolved social loss and grief have been deemed to be among the main precipitating causes of depression, and glutamate plays a large role in the production of negative effect related to separation distress, these results are consistent with the emerging work targeting glutamate blockade as a way to produce rapid anti-depressant effects.