Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia, accounting for 60-70% of cases in subjects over 65 years of age. Several postulates have been put forward that relate AD neuropathology to intellectual and functional impairment. These range from free-radical-induced damage, through cholinergic dysfunction, to beta-amyloid-induced toxicity. However, therapeutic strategies aimed at improving the cognitive symptoms of patients via choline supplementation, cholinergic stimulation or beta-amyloid vaccination, have largely failed. A growing body of evidence suggests that perturbations in systems using the excitatory amino acid L-glutamate (L-Glu) may underlie the pathogenic mechanisms of (e.g.) hypoxia-ischemia, epilepsy, and chronic neurodegenerative disorders such as Huntington's disease and AD. Almost all neurons in the CNS carry the N-methyl-D-aspartate (NMDA) subtype of ionotropic L-glutamate receptors, which can mediate post-synaptic Ca2+ influx. Excitotoxicity resulting from excessive activation of NMDA receptors may enhance the localized vulnerability of neurons in a manner consistent with AD neuropathology, as a consequence of an altered regional distribution of NMDA receptor subtypes. This review discusses mechanisms for the involvement of the NMDA receptor complex and its interaction with polyamines in the pathogenesis of AD. NMDA receptor antagonists have potential for the therapeutic amelioration of AD. (C) 2004 Elsevier Ltd. All rights reserved.

Conotoxins as selective inhibitors of neuronal ion channels, receptors and transporters

Cone snails have evolved a vast array of peptide toxins for prey capture and defence. These peptides are directed against a wide variety of pharmacological targets, making them an invaluable source of ligands for studying the properties of these targets in normal and diseased states. A number of these peptides have shown efficacy in vivo, including inhibitors of calcium channels, the norepinephrine transporter, nicotinic acetylcholine receptors, NMDA receptors and neurotensin receptors, with several having undergone pre-clinical or clinical development for the treatment of pain.

Adenosine triphosphate acts as both a competitive antagonist and a positive allosteric modulator at recombinant N-methyl-D-aspartate receptors

ATP and glutamate are fast excitatory neurotransmitters in the central nervous system acting primarily on ionotropic P2X and glutamate [N-methyl-D-aspartate (NMDA) and non-NMDA] receptors, respectively. Both neurotransmitters regulate synaptic plasticity and long-term potentiation in hippocampal neurons. NMDA receptors are responsible primarily for the modulatory action of glutamate, but the mechanism underlying the modulatory effect of ATP remains uncertain. In the present study, the effect of ATP on recombinant NR1a + 2A, NR1a + 2B, and NR1a + 2C NMDA receptors expressed in Xenopus laevis oocytes was investigated. ATP inhibited NR1a + 2A and NR1a + 2B receptor currents evoked by low concentrations of glutamate but potentiated currents evoked by saturating glutamate concentrations. In contrast, ATP potentiated NR1a + 2C receptor currents evoked by nonsaturating glutamate concentrations. ATP shifted the glutamate concentration-response curve to the right, indicating a competitive interaction at the agonist binding site. ATP inhibition and potentiation of glutamate-evoked currents was voltage-independent, indicating that ATP acts outside the membrane electric field. Other nucleotides, including ADP, GTP, CTP, and UTP, inhibited glutamate-evoked currents with different potencies, revealing that the inhibition is dependent on both the phosphate chain and nucleotide ring structure. At high concentrations, glutamate outcompetes ATP at the agonist binding site, revealing a potentiation of the current. This effect must be caused by ATP binding at a separate site, where it acts as a positive allosteric modulator of channel gating. A simple model of the NMDA receptor, with ATP acting both as a competitive antagonist at the glutamate binding site and as a positive allosteric modulator at a separate site, reproduced the main features of the data.

SK channels regulate excitatory synaptic transmission and plasticity in the lateral amygdala

At glutamatergic synapses, calcium influx through NMDA receptors (NMDARs) is required for long-term potentiation (LTP); this is a proposed cellular mechanism underlying memory and learning. Here we show that in lateral amygdala pyramidal neurons, SK channels are also activated by calcium influx through synaptically activated NMDARs, resulting in depression of the synaptic potential. Thus, blockade of SK channels by apamin potentiates fast glutamatergic synaptic potentials. This potentiation is blocked by the NMDAR antagonist AP5 (D(-)-2-amino-5-phosphono-valeric acid) or by buffering cytosolic calcium with BAPTA. Blockade of SK channels greatly enhances LTP of cortical inputs to lateral amygdala pyramidal neurons. These results show that NMDARs and SK channels are colocalized at glutamatergic synapses in the lateral amygdala. Calcium influx through NMDARs activates SK channels and shunts the resultant excitatory postsynaptic potential. These results demonstrate a new role for SK channels as postsynaptic regulators of synaptic efficacy.

Chronic smoking and alcoholism change expression of selective genes in the human prefrontal cortex

Background: Alcoholism is commonly associated with chronic smoking. A number of gene expression profiles of regions within the human mesocorticolimbic system have identified potential alcohol-sensitive genes; however, the influence of smoking on these changes was not taken into account. This study addressed the impact of alcohol and smoking on the expression of 4 genes, previously identified as alcoholism-sensitive. in the human prefrontal cortex (PFC). Methods: mRNA expression of apolipoprotein D, tissue inhibitor of the metalloproteinase 3, high-affinity glial glutamate transporter and midkine, was measured in the PFC of alcoholic Subjects and controls with and without smoking comorbidity using real-time polymerase chain reaction. Results: The results show that alcohol affects transcription of some of these genes. Additionally, smoking has a marked influence on gene expression. Conclusion: This study emphasizes the need for careful case selection in future gene expression studies to delineate the adaptive molecular process associated with smoking and alcohol.

Comparative gene expression in brain regions of human alcoholics

The mesocorticolimbic system is the reward centre of the brain and the major target for drugs of abuse including alcohol. Neuroadaptive changes in this region are thought to underlie the process of tolerance and dependence. Recently, several research groups have searched for alcohol-responsive genes using high-throughput microarrays and well-characterized human post-mortem material. Comparison of data from these studies of cortical regions highlights the differences in experimental approach and selection of cases. However, alcohol-responsive gene sets associated with transcription, oxidative stress and energy production were common to these studies. In marked contrast, alcohol-responsive genes in the nucleus accumbens and the ventral tegmental area are primarily associated with changes in neurotransmission and signal transduction. These data support the concept that, within cortical regions, changes in gene expression are associated with alcoholism-related pathology. In the dopaminergic tract of the mesocorticolimbic system, alcohol-responsive gene sets suggest long-term neuroplastic changes in synaptic transmission.

Genes and gene expression in the brains of human alcoholics

Chronic alcohol misuse by human subjects leads to neuronal loss in regions such as the superior frontal cortex (SFC). Propensity to alcoholism is associated with several genes. γ-Aminobutyric acid (GABA)A receptor expression differs between alcoholics and controls, whereas glutamate receptor differences are muted. We determined whether genotype differentiated the regional presentation of GABAA and glutamate-NMDA (N-methyl-d-aspartate) receptors in SFC. Autopsy tissue was obtained from alcoholics without comorbid disease, alcoholics with liver cirrhosis, and matched controls. ADH1C, DRD2B, EAAT2, and APOE genotypes modulated GABAA-β subunit protein expression in SFC toward a less-effective form of the receptor. Most genotypes did not divide alcoholics and controls on glutamate-NMDA receptor pharmacology, although gender and cirrhosis did. Genotype may affect amino acid transmission locally to influence neuronal vulnerability.

Neurobiological alterations in the human alcoholic brain: effect of genotype

Long-term alcohol abuse by human subjects leads to selective brain damage that is restricted in extent and variable in severity. Within the cerebral cortex, neuronal loss is most marked in the superior frontal cortex and relatively mild in motor cortex. Cirrhotic alcoholics and subjects with alcohol-related Wernicke-Korsakoff syndrome show more severe and more extensive damage than do uncomplicated cases. Accumulating evidence suggests that the likelihood of developing alcohol dependency is associated with one or more genetic markers. In previous work we showed that GABAA receptor functionality, and the subunit isoform expression that underlies this, differed in region- and disease-specific ways between alcoholics and controls. By contrast, glutamate receptor (NMDA, KA, AMPA) differences were muted or absent. Here we asked if genotype differentiated the form, pharmacology, or expression of glutamate and GABA receptors in pathologically vulnerable and spared cortical regions, with a view to determining whether such subject factors might influence the severity of alcohol-induced brain damage. Cerebrocortical tissue was obtained at autopsy under informed, written consent from uncomplicated and alcoholic-cirrhotic Caucasian (predominantly Anglo-Celtic) cases, together with matched controls and cases with cirrhosis of non-alcoholic origin. All subjects had pathological confirmation of liver and brain diagnosis; none had been polydrug abusers. Samples were processed for synaptic membrane receptor binding, mRNA analysis by quantitative RT-PCR, and protein analysis by Western blot. Genotyping was performed by PCR methods, in the main using published primers. Several genetic markers differentiated between our alcoholic and control subjects, including the GABAA receptor 2 subunit (GABB2) gene ( 2 (3) 10.329, P 0.01), the dopamine D2 receptor B1 (DRD2B) allele ( 2 (3) 10.109, P 0.01) and a subset of the alcohol dehydrogenase-3 (ADH3) alleles ( 2 (2) 4.730, P 0.05). Although neither the type-2 glutamate transporter (EAAT2) nor the serotonin transporter (5HTT) genes were significantly associated with alcoholism, only EAAT2 heterozygotes showed a significant association between ADH3 genotype and alcoholism ( 2 (3) 7.475, P 0.05). Other interactions between genotypes were also observed. DRD2A, DRD2B, GABB2, EAAT2 and 5HTT genotypes did not divide alcoholic cases and controls on NMDA receptor parameters, although in combined subjects there was a significant DRD2B X Area Interaction with glutamateNMDA receptor efficacy (F(1,57) 4.67; P 0.05), measured as the extent of glutamate-enhanced MK801 binding. In contrast, there was a significant Case-group X ADH3 X Area Interaction with glutamateNMDA receptor efficacy (F(3,57) 2.97; P 0.05). When GABAA receptor subunit isoform expression was examined, significant Case-group X Genotype X Area X Isoform interactions were found for EAAT2 with subunit mRNA (F(1,37) 4.22; P0.05), for GABB2 with isoform protein (F(1,37) 5.69; P 0.05), and for DRD2B with isoform protein (F(2,34)5.69; P0.05). The results suggest that subjects’ genetic makeup may modulate the effectiveness of amino acid-mediated transmission in different cortical regions, and thereby influence neuronal vulnerability to excitotoxicity.

Genes and gene expression in human alcoholic brain

Chronic alcoholism leads to localized brain damage, which is prominent in superior frontal cortex but mild in motor cortex. The likelihood of developing alcohol dependence is associated with genetic markers. GABA-A receptor expression differs between alcoholics and controls, whereas glutamate receptor differences are muted. We determined whether genotype differentiated the localized expression of glutamate N-methyl-D-aspartate (NMDA) and GABA-A receptors to influence the severity of alcohol-induced brain damage. Cerebral cortex tissue was obtained at autopsy from alcoholics without disease comorbid with alcoholics, alcoholics with cirrhosis, and matched controls. DRD2A, DRD2B, GABRB2, SLC1A2, and 5HTT genotypes did not divide alcoholic cases and controls on NMDA receptor parameters. In contrast, a specific alcohol dehydrogenase (ADHIC) genotype interacted significantly with NMDA efficacy and affinity in a region-specific manner SLC1A2 (glutamate transporter-2) genotype interacted significantly with local GABAA receptor b subunit mRNA expression, and ADHIC, DRD2B, SLC1A2, and APOE genotypes with b subunit isoform protein expression. In the latter instance, possession of the alcoholism- associated allele altered b isoform protein expression patterns toward a less-efficacious form of the GABA-A receptor in the pathologically vulnerable region. GABRB2 and GRIN2B (NMDA receptor 2B subunit} Genotypes were associated with significant regional difference in the pattern of b subunit protein isoform expression, but this was not influenced by alcoholism status. Genotype may modulate amino acid transmission locally so as to mediate neuronal vulnerability. This has implications for the effectiveness of pharmacological interventions aimed at ameliorating brain damage and, possibly, dependence.

Sustained neuronal activity generated by glial plasticity

Astrocytes release gliotransmitters, notably glutamate, that can affect neuronal and synaptic activity. In particular, astrocytic glutamate release results in the generation of NMDA receptor (NMDA-R)-mediated slow inward currents (SICs) in neurons. However, factors underlying the emergence of SICs and their physiological roles are essentially unknown. Here we show that, in acute slices of rat somatosensory thalamus, stimulation of lemniscal or cortical afferents results in a sustained increase of SICs in thalamocortical (TC) neurons that outlasts the duration of the stimulus by 1 h. This long-term enhancement of astrocytic glutamate release is induced by group I metabotropic glutamate receptors and is dependent on astrocytic intracellular calcium. Neuronal SICs are mediated by extrasynaptic NR2B subunit-containing NMDA-Rs and are capable of eliciting bursts. These are distinct from T-type Ca2+ channel-dependent bursts of action potentials and are synchronized in neighboring TC neurons. These findings describe a previously unrecognized form of excitatory, nonsynaptic plasticity in the CNS that feeds forward to generate local neuronal firing long after stimulus termination.

Investigating the effects of antiepileptic drugs on the electrophysiology of vision

The principal aim of this work was to examine the effects of antiepileptic drugs (AEDs) on vision. Vigabatrin acts by increasing GABA at brain inhibitory synapses by irreversibly binding to GABA-transaminase. Remacemide is a novel non-competitive NMDA receptor antagonist and fast sodium channel inhibitor that results in the inhibition of the NMDA receptors located in the neuronal membrane calcium channels increasing glutamate in the brain. Vigabatrin has been shown to cause a specific pattern of visual field loss, as one in three adults taking vigabatrin have shown a bilateral concentric constriction. Remacemide has unknown effects on vision. The majority of studies of the effects of AEDs on vision have not included the paediatric population due to difficulties assessing visual field function using standard perimetry testing. Evidently an alternative test is required to establish and monitor visual field problems associated with AEDs both in children and in adults who cannot comply with perimetry. In order to test paediatric patients exposed to vigabatrin, a field-specific visual evoked potential was developed. Other tests performed on patients taking either vigabatrin or remacemide were electroretinograms, electro-oculograms, multifocal VEPs and perimetry. Comparing these tests to perimetry results from vigabatrin patients the field specific VEP was found to have a high sensitivity and specificity, as did the 30Hz flicker amplitude. The modified VEP was also found to provide useful results in vigabatrin patients. Remacemide did not produce a similar visual field loss to vigabatrin although macular vision was affected. The field specific VEP is a useful method for detecting vigabatrin associated visual field loss that is well tolerated by young children. This technique combined with the ERG under light adapted (30Hz flicker) condition is presently the superior method for detecting vigabatrin-attributed peripheral field defects present in children below the developmental age of 9. The effects of AEDs on vision should be monitored carefully and the use of multifocal stimulation allows for specific areas of the retina and visual pathway to be monitored.

Astrocyte-neuron signalling by synaptic stimulation in the ventrobasal thalamus

In the Ventrobasal (VB) thalamus, astrocytes are known to elicit NMDA-receptor mediated slow inward currents (SICs) spontaneously in neurons. Fluorescence imaging of astrocytes and patch clamp recordings from the thalamocortical (TC) neurons in the VB of 6-23 day old Wistar rats were performed. TC neurons exhibit spontaneous SICs at low frequencies (~0.0015Hz) that were inhibited by NMDA-receptor antagonists D-AP5 (50µM), and were insensitive to TTX (1µM) suggesting a non-neuronal origin. The effect of corticothalamic (CT) and sensory (Sen) afferent stimulation on astrocyte signalling was assessed by varying stimulus parameters. Moderate synaptic stimulation elicited astrocytic Ca2+ increases, but did not affect the incidence of spontaneous SICs. Prolonged synaptic stimulation induced a 265% increase in SIC frequency. This increase lasted over one hour after the cessation of synaptic stimulation, so revealing a Long Term Enhancement (LTE) of astrocyte-neuron signalling. LTE induction required group I mGluR activation. LTE SICs targeted NMDA-receptors located at extrasynaptic sites. LTE showed a developmental profile: from weeks 1-3, the SIC frequency was increased by an average 50%, 240% and 750% respectively. Prolonged exposure to glutamate (200µM) increased spontaneous SIC frequency by 1800%. This “chemical” form of LTE was prevented by the broad-spectrum excitatory amino acid transporter (EAAT) inhibitor TBOA (300µM) suggesting that glutamate uptake was a critical factor. My results therefore show complex glutamatergic signalling interactions between astrocytes and neurons. Furthermore, two previously unrecognised mechanisms of enhancing SIC frequency are described. The synaptically induced LTE represents a form of non-synaptic plasticity and a glial “memory” of previous synaptic activity whilst enhancement after prolonged glutamate exposure may represent a pathological glial signalling mechanism.

Glutamatergic input-output properties of thalamic astrocytes

Astrocytes in the somatosensory ventrobasal (VB) thalamus of rats respond to glutamatergic synaptic input with metabotropic glutamate receptor (mGluR) mediated intracellular calcium ([Ca²?](i)) elevations. Astrocytes in the VB thalamus also release the gliotransmitter (GT) glutamate in a Ca²?-dependent manner. The tripartite synapse hypothesis posits that astrocytic [Ca²?](i) elevations resulting from synaptic input releases gliotransmitters that then feedback to modify the synapse. Understanding the dynamics of this process and the conditions under which it occurs are therefore important steps in elucidating the potential roles and impact of GT release in particular brain activities. In this study, we investigated the relationship between VB thalamus afferent synaptic input and astrocytic glutamate release by recording N-methyl-D-aspartate (NMDA) receptor-mediated slow inward currents (SICs) elicited in neighboring neurons. We found that Lemniscal or cortical afferent stimulation, which can elicit astrocytic [Ca²?](i) elevations, do not typically result in the generation of SICs in thalamocortical (TC) neurons. Rather, we find that the spontaneous emergence of SICs is largely resistant to acute afferent input. The frequency of SICs, however, is correlated to long-lasting afferent activity. In contrast to short-term stimulus-evoked GT release effects reported in other brain areas, astrocytes in the VB thalamus do not express a straightforward input-output relationship for SIC generation but exhibit integrative characteristics.

Spontaneous astrocytic Ca2+ oscillations in situ drive NMDAR-mediated neuronal excitation

Astrocytes respond to chemical, electrical and mechanical stimuli with transient increases in intracellular calcium concentration ([Ca2+]i). We now show that astrocytes in situ display intrinsic [Ca2+]i oscillations that are not driven by neuronal activity. These spontaneous astrocytic oscillations can propagate as waves to neighboring astrocytes and trigger slowly decaying NMDA receptor-mediated inward currents in neurons located along the wave path. These findings show that astrocytes in situ can act as a primary source for generating neuronal activity in the mammalian central nervous system.

Ketamine alters oscillatory coupling in the hoppocampus

Recent studies show that higher order oscillatory interactions such as cross-frequency coupling are important for brain functions that are impaired in schizophrenia, including perception, attention and memory. Here we investigated the dynamics of oscillatory coupling in the hippocampus of awake rats upon NMDA receptor blockade by ketamine, a pharmacological model of schizophrenia. Ketamine (25, 50 and 75 mg/kg i.p.) increased gamma and high-frequency oscillations (HFO) in all depths of the CA1-dentate axis, while theta power changes depended on anatomical location and were independent of a transient increase of delta oscillations. Phase coherence of gamma and HFO increased across hippocampal layers. Phase-amplitude coupling between theta and fast oscillations was markedly altered in a dose-dependent manner: ketamine increased hippocampal theta-HFO coupling at all doses, while theta-gamma coupling increased at the lowest dose and was disrupted at the highest dose. Our results demonstrate that ketamine alters network interactions that underlie cognitively relevant theta-gamma coupling.