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In Alzheimer's disease (AD), soluble amyloid-β oligomers (AβOs) trigger neurotoxic signaling, at least partially, via the cellular prion protein (PrPC). However, it is unknown whether other ligands of PrPCcan regulate this potentially toxic interaction. Stress-inducible phosphoprotein 1 (STI1), an Hsp90 cochaperone secreted by astrocytes, binds to PrPCin the vicinity of the AβO binding site to protect neurons against toxic stimuli. Here, we investigated a potential role of STI1 in AβO toxicity. We confirmed the specific binding of AβOs and STI1 to the PrP and showed that STI1 efficiently inhibited AβO binding to PrPin vitro(IC50of ∼70 nm) and also decreased AβO binding to cultured mouse primary hippocampal neurons. Treatment with STI1 prevented AβO-induced synaptic loss and neuronal death in mouse cultured neurons and long-term potentiation inhibition in mouse hippocampal slices. Interestingly, STI1-haploinsufficient neurons were more sensitive to AβO-induced cell death and could be rescued by treatment with recombinant STI1. Noteworthy, both AβO binding to PrPCand PrPC-dependent AβO toxicity were inhibited by TPR2A, the PrPC-interacting domain of STI1. Additionally, PrPC–STI1 engagement activated α7 nicotinic acetylcholine receptors, which participated in neuroprotection against AβO-induced toxicity. We found an age-dependent upregulation of cortical STI1 in the APPswe/PS1dE9 mouse model of AD and in the brains of AD-affected individuals, suggesting a compensatory response. Our findings reveal a previously unrecognized role of the PrPCligand STI1 in protecting neurons in AD and suggest a novel pathway that may help to offset AβO-induced toxicity.

NMDA receptor (NMDAR) antagonists are dissociative anesthetics, drugs of abuse, and are of therapeutic interest in neurodegeneration and neuropsychiatric disease. Many well-known NMDAR antagonists are positively charged, voltage-dependent channel blockers. We recently showed that the hydrophobic anion dipicrylamine (DPA) negatively regulates GABA(A) receptor function by a mechanism indistinguishable from that of sulfated neurosteroids. Because sulfated neurosteroids also modulate NMDARs, here we examined the effects of DPA on NMDAR function. In rat hippocampal neurons DPA inhibited currents gated by 300 µM NMDA with an IC(50) of 2.3 µM. Neither onset nor offset of antagonism exhibited dependence on channel activation but exhibited a noncompetitive profile. DPA antagonism was independent of NMDAR subunit composition and was similar at extrasynaptic and total receptor populations. Surprisingly, similar to cationic channel blockers but unlike sulfated neurosteroids, DPA antagonism was voltage dependent. Onset and offset of DPA antagonism were nearly 10-fold faster than DPA-induced increases in membrane capacitance, suggesting that membrane interactions do not directly explain antagonism. Furthermore, voltage dependence did not derive from association of DPA with a site on NMDARs directly accessible to the outer membrane leaflet, assessed by DPA translocation experiments. Consistent with the expected lack of channel block, DPA antagonism did not interact with permeant ions. Therefore, we speculate that voltage dependence may arise from interactions of DPA with the inherent voltage dependence of channel gating. Overall, we conclude that DPA noncompetitively inhibits NMDA-induced current by a novel voltage-dependent mechanism and represents a new class of anionic NMDAR antagonists.

The N-methyl-d-aspartate receptor (NMDAR) coagonists glycine, d-serine and l-proline play crucial roles in NMDAR-dependent neurotransmission and are associated with a range of neuropsychiatric disorders. We conducted the first genome-wide association study of concentrations of these coagonists and their enantiomers in plasma and cerebrospinal fluid (CSF) of human subjects from the general population (N=414). Genetic variants at chromosome 22q11.2, located in and near PRODH (proline dehydrogenase), were associated with l-proline in plasma (β=0.29; P=6.38 × 10-10). The missense variant rs17279437 in the proline transporter SLC6A20 was associated with l-proline in CSF (β=0.28; P=9.68 × 10-9). Suggestive evidence of association was found for the d-serine plasma-CSF ratio at the d-amino-acid oxidase (DAO) gene (β=−0.28; P=9.08 × 10-8), whereas a variant in SRR (that encodes serine racemase and is associated with schizophrenia) constituted the most strongly associated locus for the l-serine to d-serine ratio in CSF. All these genes are highly expressed in rodent meninges and choroid plexus, anatomical regions relevant to CSF physiology. The enzymes and transporters they encode may be targeted to further construe the nature of NMDAR coagonist involvement in NMDAR gating. Furthermore, the highlighted genetic variants may be followed up in clinical populations, for example, schizophrenia and 22q11 deletion syndrome. Overall, this targeted metabolomics approach furthers the understanding of NMDAR coagonist concentration variability and sets the stage for non-targeted CSF metabolomics projects.Molecular Psychiatry advance online publication, 10 February 2015; doi:10.1038/mp.2014.190.

By using multi-probe microdialysis we previously demonstrated that endogenous glutamate differentially regulates the activity of the striatal output pathways in vivo, through N-methyl-D-aspartate (NMDA) receptors containing the GluN2A or GluN2B subunits. Using the same approach, we presently investigate whether reverse dialysis of NMDA in the striatum differentially affects GABA release in the striatum and in striatal target areas, i.e. globus pallidus (GP) and substantia nigra reticulata (SNr). Moreover, we ask whether this control is altered under parkinsonian conditions. Intrastriatal NMDA perfusion (10 min) evoked GABA release more potently in SNr (1–100 μM) than in other regions (10–100 μM), suggesting preferential control over striato-nigral projection neurons. Intrastriatal NMDA more potently stimulated glutamate levels in the striatum (1–100 μM) and SNr (1–10 μM) than in GP (10 μM). Striatal dopamine denervation with 6-hydroxydopamine caused a leftward shift in the NMDA concentration–response curve. Intrastriatal NMDA elevated GABA levels at 0.1 μM (all regions) and 1 μM (striatum and GP only), but not at higher concentrations, indicating that, compared to naive animals, the GABA response in SNr was attenuated. Attenuation of the glutamate response was also observed in SNr (NMDA effective only at 0.1 μM). Conversely, the glutamate response in GP was widened (NMDA effective in the 0.1–1 μM range). We conclude that NMDA preferentially stimulates the activity of the striato-nigral direct pathway under physiological conditions. In Parkinson's disease, dopamine loss compromises the NMDA ability to stimulate striato-nigral neurons, thus shifting the NMDA control towards the striato-pallidal ones.

Glutamate is the main excitatory transmitter in the human brain. Drugs that affect the glutamatergic signaling will alter neuronal excitability. Ethanol inhibits glutamate receptors. We examined the expression level of glutamate receptor subunit mRNAs in human post-mortem samples from alcoholics and compared the results to brain samples from control subjects. RNA from hippocampal dentate gyrus (HP-DG), orbitofrontal cortex (OFC), and dorso-lateral prefrontal cortex (DL-PFC) samples from 21 controls and 19 individuals with chronic alcohol dependence were included in the study. Total RNA was assayed using quantitative RT-PCR. Out of the 16 glutamate receptor subunits, mRNAs encoding two AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid) receptor subunits GluA2 and GluA3; three kainate receptor subunits GluK2, GluK3 and GluK5 and five NMDA (N-methyl-D-aspartate) receptor subunits GluN1, GluN2A, GluN2C, GluN2D and GluN3A were significantly increased in the HP-DG region in alcoholics. In the OFC, mRNA encoding the NMDA receptor subunit GluN3A was increased, whereas in the DL-PFC, no differences in mRNA levels were observed. Our laboratory has previously shown that the expression of genes encoding inhibitory GABA-A receptors is altered in the HP-DG and OFC of alcoholics (Jin et al., 2011). Whether the changes in one neurotransmitter system drives changes in the other or if they change independently is currently not known. The results demonstrate that excessive long-term alcohol consumption is associated with altered expression of genes encoding glutamate receptors in a brain region-specific manner. It is an intriguing possibility that genetic predisposition to alcoholism may contribute to these gene expression changes.

The glutamatergic neurotransmission system and the N-methyl-D-aspartate receptor (NMDAR) have been implicated in smoking and alcohol consumption behavior. Preclinical studies have demonstrated that nicotine and ethanol influence NMDAR functionality, which may have a role in tendencies to consume these substances. Nonetheless, little is known about concentrations of NMDAR coagonists in the cerebrospinal fluid (CSF) and plasma of individuals who smoke or consume alcohol. Glycine and L- and D-stereoisomers of alanine, serine, and proline were therefore measured using ultra-high-performance liquid chromatography-tandem mass spectrometry in 403 healthy subjects. Nicotine and alcohol consumption were quantified using questionnaires. Possible differences in NMDAR coagonist concentrations in plasma and CSF were investigated using ANCOVA with age, body mass index, and storage duration as covariates. The significance threshold was Bonferroni corrected (alpha = 0.00625). Compared with non-smokers, smokers displayed lower levels of D-proline in plasma (p = 0.0027, Cohen's d = -0.41) and D-proline in CSF (p = 0.0026, Cohen's d = -0.43). D-Serine in CSF was higher in smokers than in non-smokers (p = 0.0052, Cohen's d = 0.41). After subdividing participants based on smoking quantity, dose-dependent decreases were demonstrated in smokers for D-proline in plasma (F = 5.65, p = 0.0039) and D-proline in CSF (F = 5.20, p = 0.0060). No differences in NMDAR coagonist levels between alcohol consumption groups were detected. To our knowledge, this is the first report to implicate D-amino acids in smoking behavior of humans. Whether such concentration differences lie at the root of or result from smoking habits may be addressed in prospective studies.

The dysfunction and loss of basal forebrain cholinergic neurons and their cortical projections are among the earliest pathological events in the pathogenesis of Alzheimer's disease (AD). The evidence pointing to cholinergic impairments come from studies that report a decline in the activity of choline acetyltransferase (ChAT) and acetylcholine esterase (AChE), acetylcholine (ACh) release and the levels of nicotinic and muscarinic receptors, and loss of cholinergic basal forebrain neurons in the AD brain. Alzheimer's disease pathology is characterized by an extensive loss of synapses and neuritic branchings which are the dominant scenario as compared to the loss of the neuronal cell bodies themselves. The appearance of cholinergic neuritic dystrophy, i.e. aberrant fibers and fiber swelling are more and more pronounced during brain aging and widely common in AD. When taking amyloid-beta (A beta) deposition as the ultimate causal factor of Alzheimer's disease the role of A beta in cholinergic dysfunction should be considered. In that respect it has been stated that ACh release and synthesis are depressed, axonal transport is inhibited, and that ACh degradation is affected in the presence of A beta peptides. beta-Amyloid peptide 1-42, the principal constituent of the neuritic plagues seen in AD patients, is known to trigger excess amount of glutamate in the synaptic cleft by inhibiting the astroglial glutamate transporter and to increase the intracellular Ca(2+) level. Based on the glutamatergic overexcitation theory of AD progression, the function of NMDA receptors and treatment with NMDA antagonists underlie some recent therapeutic applications. Memantine, a moderate affinity uncompetitive NMDA receptor antagonist interacts with its target only during states of pathological activation but does not interfere with the physiological receptor functions. In this study the neuroprotective effect of memantine on the forebrain cholinergic neurons against A beta 42 oligomers-induced toxicity was studied in an in vivo rat dementia model. We found that memantine rescued the neocortical cholinergic fibers originating from the basal forebrain cholinergic neurons, attenuated microglial activation around the intracerebral lesion sides, and improved attention and memory of A beta 42-injected rats exhibiting impaired learning and loss of cholinergic innervation of neocortex. (C) 2010 Elsevier B.V. All rights reserved.

We have studied the effect of intrahippocampal administration of quinolinic acid (QUIN) on the temporal expression of mRNAs encoding the immediate early genes (IEGs) c-fos and NGFI-A, by in situ hybridization histochemistry. After administration of QUIN to the left hippocampus, expression of mRNA of both IEGs was transiently stimulated. Maximal expression was found between 1 and 3 h. mRNA of both IEGs was simultaneously expressed in the ipsilateral and contralateral sides in the granule cell layer of the dentate gyrus, the pyramidal cell layer of the CA1 and CA3 fields as well as in the cortex. After pretreatment with the non-competitive NMDA antagonist MK-801 (2 mg/kg i.p. - 30 min) the increased expression of both IEGs was partially prevented in the hippocampus and completely in the cortex. No inhibition was observed after treatment with the AMPA antagonist NBQX (30 mg/kg i.p. - 15, - 5 and + 10 min). Additional delayed expression of both IEGs was observed in the ipsilateral hippocampus. This expression was related to cell damage. Twelve h after QUIN administration, c-fos and NGFI-A mRNAs were present in the dentate gyrus. After 4 days, only c-fos mRNA was observed in the dentate gyrus and CA1 field while no NGFI-A mRNA was detected. The present results show that the effect of QUIN is mediated by NMDA and not by AMPA receptors.

We have studied the effect of intrahippocampal administration of quinolinic acid (QUIN) on the temporal expression of mRNAs encoding the immediate early genes (IEGs) c-fos and NGFI-A, by in situ hybridization histochemistry. After administration of QUIN to the left hippocampus, expression of mRNA of both IEGs was transiently stimulated. Maximal expression was found between 1 and 3 h. mRNA of both IEGs was simultaneously expressed in the ipsilateral and contralateral sides in the granule cell layer of the dentate gyrus, the pyramidal cell layer of the CA1 and CA3 fields as well as in the cortex. After pretreatment with the non-competitive NMDA antagonist MK-801 (2 mg/kg i.p. - 30 min) the increased expression of both IEGs was partially prevented in the hippocampus and completely in the cortex. No inhibition was observed after treatment with the AMPA antagonist NBQX (30 mg/kg i.p. - 15, - 5 and + 10 min). Additional delayed expression of both IEGs was observed in the ipsilateral hippocampus. This expression was related to cell damage. Twelve h after QUIN administration, c-fos and NGFI-A mRNAs were present in the dentate gyrus. After 4 days, only c-fos mRNA was observed in the dentate gyrus and CA1 field while no NGFI-A mRNA was detected. The present results show that the effect of QUIN is mediated by NMDA and not by AMPA receptors.

Among the various molecular events that have been proposed to contribute to the mechanisms of long-term potentiation (LTP), one of the most cited possibilities has been the activation of protein kinase C (PKC). Here we review various aspects of the cellular actions of PKC activationa and inhibition, with special emphasis on the effects of the kinase on synaptic transmission and the N-methyl-D-aspartate (NMDA) and non-NMDA receptor-mediated components of synaptic responses. We discuss the implications of these effects for interpretations of the role of PKC in the mechanisms of LTP induction and maintenance.

Among the various molecular events that have been proposed to contribute to the mechanisms of long-term potentiation (LTP), one of the most cited possibilities has been the activation of protein kinase C (PKC). Here we review various aspects of the cellular actions of PKC activationa and inhibition, with special emphasis on the effects of the kinase on synaptic transmission and the N-methyl-D-aspartate (NMDA) and non-NMDA receptor-mediated components of synaptic responses. We discuss the implications of these effects for interpretations of the role of PKC in the mechanisms of LTP induction and maintenance.

The spatial organization of long-term synaptic plasticity [long-term potentiation (LTP) and long-term depression (LTD)] is supposed to play a critical role for distributed signal processing in neuronal networks, but its nature remains undetermined in most central circuits. By using multielectrode array recordings, we have reconstructed activation maps of the granular layer in cerebellar slices. LTP and LTD induced by theta-burst stimulation appeared in patches organized in such a way that, on average, LTP was surrounded by LTD. The sign of long-term synaptic plasticity in a given granular layer region was directly correlated with excitation and inversely correlated with inhibition: the most active areas tended to generate LTP, whereas the least active areas tended to generate LTD. Plasticity was almost entirely prevented by application of the NMDA receptor blocker, APV. This suggests that synaptic inhibition, through a control of membrane depolarization, effectively regulates NMDA channel unblock, postsynaptic calcium entry, and the induction of bidirectional synaptic plasticity at the mossy fiber–granule cell relay (Gall et al., 2005). By this mechanism, LTP and LTD could regulate the geometry and contrast of network computations, preprocessing the mossy fiber input to be conveyed to Purkinje cells and molecular layer interneurons.

We have studied the effect of intrahippocampal administration of quinolinic acid (QUIN) on the temporal expression of mRNAs encoding the immediate early genes (IEGs) c-fos and NGFI-A, by in situ hybridization histochemistry. After administration of QUIN to the left hippocampus, expression of mRNA of both IEGs was transiently stimulated. Maximal expression was found between 1 and 3 h. mRNA of both IEGs was simultaneously expressed in the ipsilateral and contralateral sides in the granule cell layer of the dentate gyrus, the pyramidal cell layer of the CA1 and CA3 fields as well as in the cortex. After pretreatment with the non-competitive NMDA antagonist MK-801 (2 mg/kg i.p. - 30 min) the increased expression of both IEGs was partially prevented in the hippocampus and completely in the cortex. No inhibition was observed after treatment with the AMPA antagonist NBQX (30 mg/kg i.p. - 15, - 5 and + 10 min). Additional delayed expression of both IEGs was observed in the ipsilateral hippocampus. This expression was related to cell damage. Twelve h after QUIN administration, c-fos and NGFI-A mRNAs were present in the dentate gyrus. After 4 days, only c-fos mRNA was observed in the dentate gyrus and CA1 field while no NGFI-A mRNA was detected. The present results show that the effect of QUIN is mediated by NMDA and not by AMPA receptors.

Tottering mice present a useful experimental model of genetically determined generalized epilepsy of the absence type. In electrophysiological recordings from hippocampal slices in vitro we found that the postsynaptic excitability (firing threshold) of pyramidal neurons in the CA1 area of tg/tg slices was significantly higher than that of normal slices. In spite of this hyperexcitability, in vitro epileptiform discharges were not observed spontaneously, or upon provocation by intracellular depolarizing pulses, or in response to moderate elevations (+2 mM) in extracellular potassium. The latter elevations actually induced significantly smaller increases in the CA1 synaptic responses of tg/tg as compared to normal slices. The hyperexcitability of tottering neurons could not be explained in terms of altered membrane electrical properties or any reduction of synaptic inhibition or increased capacity for long-term potentiation. Responses to noradrenaline, histamine and adenosine, as well as to the release of N-methyl-D-asparate channels - by eliminating Mg2+ - were comparable in tg/tg and normal slices. These studies show that hyperexcitability can be co-inherited with epilepsy and in this model its expression can be maintained in vitro. The neuronal mechanism of this expression remains elusive, as it does not appear to include some features known to be shared by experimental models of chemically or electrically induced epilepsy. Journal of Neural Transmission-General Section

Among the various molecular events that have been proposed to contribute to the mechanisms of long-term potentiation (LTP), one of the most cited possibilities has been the activation of protein kinase C (PKC). Here we review various aspects of the cellular actions of PKC activationa and inhibition, with special emphasis on the effects of the kinase on synaptic transmission and the N-methyl-D-aspartate (NMDA) and non-NMDA receptor-mediated components of synaptic responses. We discuss the implications of these effects for interpretations of the role of PKC in the mechanisms of LTP induction and maintenance.