Differential glutamate receptor expression and function in the hippocampus, anterior temporal lobe and neocortex in a pilocarpine model of temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most common form of intractable epilepsy where hyperactive glutamate receptors may contribute to the complex epileptogenic network hubs distributed among different regions. This study was designed to investigate the region-specific molecular alterations of the glutamate receptors and associated excitatory synaptic transmission in pilocarpine rat model of TLE. We recorded spontaneous excitatory postsynaptic currents (EPSCs) from pyramidal neurons in resected rat brain slices of the hippocampus, anterior temporal lobe (ATL) and neocortex. We also performed mRNA and protein expression of the glutamate receptor subunits (NR1, NR2A, NR2B, and GLUR1-4) by qPCR and immunohistochemistry. We observed significant increase in the frequency and amplitude of spontaneous EPSCs in the hippocampal and ATL samples of TLE rats than in control rats. Additionally, the magnitude of the frequency and amplitude was increased in ATL samples compared to that of the hippocampal samples of TLE rats. The mRNA level of NR1 was upregulated in both the hippocampal as well as ATL samples and that of NR2A, NR2B were upregulated only in the hippocampal samples of TLE rats than in control rats. The mRNA level of GLUR4 was upregulated in both the hippocampal as well as ATL samples of TLE rats than in control rats. Immunohistochemical analysis demonstrated that the number of NR1, NR2A, NR2B, and GLUR4 immuno-positive cells were significantly higher in the hippocampal samples whereas number of NR1 and GLUR4 immuno-positive cells were significantly higher in the ATL samples of the TLE rats than in control rats. This study demonstrated the region-specific alterations of glutamate receptor subunits in pilocarpine model of TLE, suggesting possible cellular mechanisms contributing to generation of independent epileptogenic networks in different temporal lobe structures.

An emerging map of glutamate delta 1 receptors in the forebrain.

Glutamate delta 1 (GluD1) and glutamate delta 2 (GluD2) form the delta family of ionotropic glutamate receptors; these proteins plays widespread roles in synaptic architecture, motor behavior, and cognitive function. Though the role of GluD2 at cerebellar parallel fiber-Purkinje cell synapses is well established, attention now turns to the function of GluD receptors in the forebrain. GluD1 regulates synaptic assembly and modulation in multiple higher brain regions, acting as a postsynaptic cell adhesion molecule with effects on both excitatory and inhibitory transmission. Furthermore, variations and mutations in the GRID1 gene, which codes for GluD1, and in genes which code for proteins functionally linked to GluD1, are associated with mental disorders including autism, schizophrenia, bipolar disorder, and major depression. Cerebellin (Cbln) family proteins, the primary binding partners of delta receptors, are secreted C1q-like proteins which also bind presynaptic neurexins (NRXNs), forming a tripartite synaptic bridge. Published research explores this bridge's function in regions including the striatum, hippocampus, cortex, and cerebellum. In this review, we summarize region- and circuit-specific functions and expression patterns for GluD1 and its related proteins, and their implications for behavior and disease.

Metaplastic Effects of Ketamine and MK-801 on Glutamate Receptors Expression in Rat Medial Prefrontal Cortex and Hippocampus.

Ketamine and MK-801 by blocking NMDA receptors may induce reinforcing effects as well as schizophrenia-like symptoms. Recent results showed that ketamine can also effectively reverse depressive signs in patients' refractory to standard therapies. This evidence clearly points to the need of characterization of effects of these NMDARs antagonists on relevant brain areas for mood disorders. The aim of the present study was to investigate the molecular changes occurring at glutamatergic synapses 24 h after ketamine or MK-801 treatment in the rat medial prefrontal cortex (mPFC) and hippocampus (Hipp). In particular, we analyzed the levels of the glutamate transporter-1 (GLT-1), NMDA receptors, AMPA receptors subunits, and related scaffolding proteins. In the homogenate, we found a general decrease of protein levels, whereas their changes in the post-synaptic density were more complex. In fact, ketamine in the mPFC decreased the level of GLT-1 and increased the level of GluN2B, GluA1, GluA2, and scaffolding proteins, likely indicating a pattern of enhanced excitability. On the other hand, MK-801 only induced sparse changes with apparently no correlation to functional modification. Differently from mPFC, in Hipp, both substances reduced or caused no changes of glutamate receptors and scaffolding proteins expression. Ketamine decreased NMDA receptors while increased AMPA receptors subunit ratios, an effect indicative of permissive metaplastic modulation; conversely, MK-801 only decreased the latter, possibly representing a blockade of further synaptic plasticity. Taken together, these findings indicate a fine tuning of glutamatergic synapses by ketamine compared to MK-801 both in the mPFC and Hipp.

Psychosocial Crowding Stress-Induced Changes in Synaptic Transmission and Glutamate Receptor Expression in the Rat Frontal Cortex.

This study demonstrates how exposure to psychosocial crowding stress (CS) for 3, 7, and 14 days affects glutamate synapse functioning and signal transduction in the frontal cortex (FC) of rats. CS effects on synaptic activity were evaluated in FC slices of the primary motor cortex (M1) by measuring field potential (FP) amplitude, paired-pulse ratio (PPR), and long-term potentiation (LTP). Protein expression of GluA1, GluN2B mGluR1a/5, VGLUT1, and VGLUT2 was assessed in FC by western blot. The body's response to CS was evaluated by measuring body weight and the plasma level of plasma corticosterone (CORT), adrenocorticotropic hormone (ACTH), and interleukin 1 beta (IL1B). CS 3 14d increased FP and attenuated LTP in M1, while PPR was augmented in CS 14d. The expression of GluA1, GluN2B, and mGluR1a/5 was up-regulated in CS 3d and downregulated in CS 14d. VGLUTs expression tended to increase in CS 7d. The failure to blunt the effects of chronic CS on FP and LTP in M1 suggests the impairment of habituation mechanisms by psychosocial stressors. PPR augmented by chronic CS with increased VGLUTs level in the CS 7d indicates that prolonged CS exposure changed presynaptic signaling within the FC. The CS bidirectional profile of changes in glutamate receptors' expression seems to be a common mechanism evoked by stress in the FC.

Axon-Myelin Unit Blistering as Early Event in MS Normal Appearing White Matter.

OBJECTIVE: Multiple sclerosis (MS) is a chronic neuroinflammatory and neurodegenerative disease of unknown etiology. Although the prevalent view regards a CD4+ -lymphocyte autoimmune reaction against myelin at the root of the disease, recent studies propose autoimmunity as a secondary reaction to idiopathic brain damage. To gain knowledge about this possibility we investigated the presence of axonal and myelinic morphological alterations, which could implicate imbalance of axon-myelin units as primary event in MS pathogenesis. METHODS: Using high resolution imaging histological brain specimens from patients with MS and non-neurological/non-MS controls, we explored molecular changes underpinning imbalanced interaction between axon and myelin in normal appearing white matter (NAWM), a region characterized by normal myelination and absent inflammatory activity. RESULTS: In MS brains, we detected blister-like swellings formed by myelin detachment from axons, which were substantially less frequently retrieved in non-neurological/non-MS controls. Swellings in MS NAWM presented altered glutamate receptor expression, myelin associated glycoprotein (MAG) distribution, and lipid biochemical composition of myelin sheaths. Changes in tethering protein expression, widening of nodes of Ranvier and altered distribution of sodium channels in nodal regions of otherwise normally myelinated axons were also present in MS NAWM. Finally, we demonstrate a significant increase, compared with controls, in citrullinated proteins in myelin of MS cases, pointing toward biochemical modifications that may amplify the immunogenicity of MS myelin. INTERPRETATION: Collectively, the impaired interaction of myelin and axons potentially leads to myelin disintegration. Conceptually, the ensuing release of (post-translationally modified) myelin antigens may elicit a subsequent immune attack in MS. ANN NEUROL 2021;89:711-725.

Butanol Extract of Tinospora cordifolia Ameliorates Cognitive Deficits Associated with Glutamate-Induced Excitotoxicity: A Mechanistic Study Using Hippocampal Neurons.

Overstimulation of glutamate receptors leads to development of excitotoxicity, which is implicated as final destructive pathway in neurodegenerative diseases. Development of alternative therapeutic strategies effective against glutamate-induced excitotoxicity is much in demand. Herbal drug development is emerging as a major research area for the treatment of various debilitating diseases due to multimodal action and least side effects of herbal products. The current study was aimed to investigate neuroprotective potential of butanol extract of Tinospora cordifolia (B-TCE) against glutamate-induced excitotoxicity using primary hippocampal neurons as in vitro and Wistar strain albino rats as in vivo model systems. Molecular and behavioral parameters were studied to elucidate the underlying mechanism of beneficial effects of B-TCE. B-TCE treatment was also effective in prevention of anxiety, cognition, and motor-coordination deficits induced by glutamate. B-TCE pre-treatment protected the hippocampal neurons from glutamate-induced neurodegeneration and impaired plasticity. At molecular level, B-TCE was observed to attenuate overactivation of glutamate receptors. B-TCE promoted upregulation of ERK and AKT pathways of synaptic plasticity and cell survival in the hippocampus region of brain. This study provides first evidence of neuroprotective potential of B-TCE against glutamate-induced excitotoxicity in hippocampus region and suggests that B-TCE may act as a potential candidate for neuroprotective therapeutic approaches. A single compound 'tinosporicide' was further isolated from B-TCE, which was found to be effective at 800× lower concentration against glutamate-induced neurodegeneration under in vitro conditions.

Bupropion, a possible antidepressant without negative effects on alcohol relapse.

RATIONALE: the role that antidepressants play on alcohol consumption is not well understood. Previous studies have reported that treatment with a Selective Serotonin Reuptake Inhibitor (SSRIs) increases alcohol consumption in an animal model of relapse, however it is unknown whether this effect holds for other antidepressants such as the atypical dopamine/norepinephrine reuptake inhibitors (SNDRI). OBJECTIVES: the main goal of the present study was to compare the effects of two classes of antidepressants drugs, bupropion (SNDRI) and fluoxetine (SSRI), on alcohol consumption during relapse. Since glutamatergic and endocannabinoid signaling systems plays an important role in alcohol abuse and relapse, we also evaluated the effects of both antidepressants onthe expression of the main important genes and proteins of both systems in the prefrontal cortex, a critical brain region in alcohol relapse. METHODS: rats were trained to self-administered alcohol. During abstinence, rats received a 14d-treatment with vehicle, fluoxetine (10 mg/kg) or bupropion (20 mg/kg), and we evaluated alcohol consumption during relapse for 3 weeks. Samples of prefrontal cortex were taken to evaluate the mRNA and protein expression of the different components of glutamatergic and endocannabinoid signaling systems. RESULTS: fluoxetine treatment induced a long-lasting increase in alcohol consumption during relapse, an effect that was not observed in the case of bupropion treatment. The observed increases in alcohol consumption were accompanied by distinct alterations in the glutamate and endocannabinoid systems. CONCLUSIONS: our results suggest that SSRIs can negatively impact alcohol consumption in relapse while SNDRIs have no effects. The observed increase in alcohol consumption are accompanied by functional alterations in the glutamatergic and endocannabinoid systems. This finding could open new strategies for the treatment of depression in patients with alcohol use disorders.

Toll-like receptor 3 activation impairs excitability and synaptic activity via TRIF signalling in immature rat and human neurons.

Toll like receptor 3 (TLR3) belongs to a family of pattern recognition receptors that recognise molecules found on pathogens referred to as pathogen associated molecular patterns (PAMPs). Its involvement in innate immunity is well known but despite its presence in the central nervous system (CNS), our knowledge of its function is limited. Here, we have investigated whether TLR3 activation modulates synaptic activity in primary hippocampal cultures and induced pluripotent stem cell (iPSC)-derived neurons. Synaptically driven spontaneous action potential (AP) firing was significantly reduced by the TLR3 specific activator, poly I:C, in a concentration-dependent manner following both short (5 min) and long exposures (1h) in rat hippocampal cultures. Notably, the consequence of TLR3 activation on neuronal function was reproduced in iPSC-derived cortical neurons, with poly I:C (25 µg/ml, 1h) significantly inhibiting sAP firing. We examined the mechanisms underlying these effects, with poly I:C significantly reducing peak sodium current, an effect dependent on the MyD88-independent TRIF dependent pathway. Furthermore, poly I:C (25 µg/ml, 1h) resulted in a significant reduction in miniature excitatory postsynaptic potential (mEPSC) frequency and amplitude and significantly reduced surface AMPAR expression. These novel findings reveal that TLR3 activation inhibits neuronal excitability and synaptic activity through multiple mechanisms, with this being observed in both rat and human iPSC-derived neurons. These data might provide further insight into how TLR3 activation may contribute to neurodevelopmental disorders following maternal infection and in patients with increased susceptibility to herpes simplex encephalitis.

RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation.

Local regulation of synaptic efficacy is thought to be important for proper networking of neurons and memory formation. Dysregulation of global translation influences long-term memory in mice, but the relevance of the regulation specific for local translation by RNA granules remains elusive. Here, we demonstrate roles of RNG105/caprin1 in long-term memory formation. RNG105 deletion in mice impaired synaptic strength and structural plasticity in hippocampal neurons. Furthermore, RNG105-deficient mice displayed unprecedentedly severe defects in long-term memory formation in spatial and contextual learning tasks. Genome-wide profiling of mRNA distribution in the hippocampus revealed an underlying mechanism: RNG105 deficiency impaired the asymmetric somato-dendritic localization of mRNAs. Particularly, RNG105 deficiency reduced the dendritic localization of mRNAs encoding regulators of AMPAR surface expression, which was consistent with attenuated homeostatic AMPAR scaling in dendrites and reduced synaptic strength. Thus, RNG105 has an essential role, as a key regulator of dendritic mRNA localization, in long-term memory formation.

Maternal Immune Activation Causes Behavioral Impairments and Altered Cerebellar Cytokine and Synaptic Protein Expression.

Emerging epidemiology studies indicate that maternal immune activation (MIA) resulting from inflammatory stimuli such as viral or bacterial infections during pregnancy serves as a risk factor for multiple neurodevelopmental disorders including autism spectrum disorders and schizophrenia. Although alterations in the cortex and hippocampus of MIA offspring have been described, less evidence exists on the impact on the cerebellum. Here, we report altered expression of cytokines and chemokines in the cerebellum of MIA offspring, including increase in the neuroinflammatory cytokine TNFα and its receptor TNFR1. We also report reduced expression of the synaptic organizing proteins cerebellin-1 and GluRδ2. These synaptic protein alterations are associated with a deficit in the ability of cerebellar neurons to form synapses and an increased number of dendritic spines that are not in contact with a presynaptic terminal. These impairments are likely contributing to the behavioral deficits in the MIA exposed offspring.

Differential effects between one week and four weeks exposure to same mass of SO2 on synaptic plasticity in rat hippocampus.

Sulfur dioxide (SO2 ) is a ubiquitous air pollutant. The previous studies have documented the adverse effects of SO2 on nervous system health, suggesting that acutely SO2 inhalation at high concentration may be associated with neurotoxicity and increase risk of hospitalization and mortality of many brain disorders. However, the remarkable features of air pollution exposure are lifelong duration and at low concentration; and it is rarely reported that whether there are different responses on synapse when rats inhaled same mass of SO2 at low concentration with a longer term. In this study, we evaluated the synaptic plasticity in rat hippocampus after exposure to same mass of SO2 at various concentrations and durations (3.5 and 7 mg/m(3) , 6 h/day, for 4 weeks; and 14 and 28 mg/m(3) , 6 h/day, for 1 week). The results showed that the mRNA level of synaptic plasticity marker Arc, glutamate receptors (GRIA1, GRIA2, GRIN1, GRIN2A, and GRIN2B) and the protein expression of memory related kinase p-CaMKпα were consistently inhibited by SO2 both in 1 week and 4 weeks exposure cases; the protein expression of presynaptic marker synaptophysin, postsynaptic density protein 95 (PSD-95), protein kinase A (PKA), and protein kinase C (PKC) were increased in 1 week exposure case, and decreased in 4 weeks exposure case. Our results indicated that SO2 inhalation caused differential synaptic injury in 1 week and 4 weeks exposure cases, and implied the differential effects might result from different PKA- and/or PKC-mediated signal pathway. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 820-829, 2016.

Sildenafil reduces neuroinflammation and restores spatial learning in rats with hepatic encephalopathy: underlying mechanisms.

BACKGROUND: There are no specific treatments for the neurological alterations of cirrhotic patients with minimal hepatic encephalopathy (MHE). Rats with MHE due to portacaval shunt (PCS) show impaired spatial learning. The underlying mechanisms remain unknown. The aims of this work were to assess: (a) whether PCS rats show neuroinflammation in hippocampus, (b) whether treatment with sildenafil reduces neuroinflammation and restores spatial learning in PCS rats, and (c) analyze the underlying mechanisms. METHODS: Neuroinflammation was assessed by determining inflammatory markers by Western blot. Phosphorylation of MAP-kinase p38 was assessed by immunohistochemistry. Membrane expression of GABA and glutamate receptors was analyzed using BS3 cross-linker. Spatial learning was analyzed using the radial and Morris water mazes. To assess if sildenafil reverses the alterations, rats were treated with sildenafil in the drinking water. RESULTS: PCS rats show increased IL-1ß and TNF-α levels and phosphorylation (activity) of p38 in hippocampus. Membrane expression of subunits α1 of GABAA receptor and GluR2 of AMPA receptor are increased in PCS rats, while subunits GluR1 of AMPA receptors and NR1 and NR2a of NMDA receptors are reduced. PCS rats show reduced spatial learning in the radial and Morris water mazes. Sildenafil treatment normalizes IL-1ß and TNF-α levels, p38 phosphorylation, and membrane expression of GABAA, AMPA, and NMDA receptors and restores spatial learning. CONCLUSIONS: Increased IL-1ß alters GABAergic and glutamatergic neurotransmission in hippocampus and impairs spatial learning in rats with MHE. Sildenafil reduces neuroinflammation and restores learning. Phosphodiesterase-5 inhibitors may be useful to improve cognitive function in patients with MHE.

A Presynaptic Regulatory System Acts Transsynaptically via Mon1 to Regulate Glutamate Receptor Levels in Drosophila.

Mon1 is an evolutionarily conserved protein involved in the conversion of Rab5 positive early endosomes to late endosomes through the recruitment of Rab7. We have identified a role for Drosophila Mon1 in regulating glutamate receptor levels at the larval neuromuscular junction. We generated mutants in Dmon1 through P-element excision. These mutants are short-lived with strong motor defects. At the synapse, the mutants show altered bouton morphology with several small supernumerary or satellite boutons surrounding a mature bouton; a significant increase in expression of GluRIIA and reduced expression of Bruchpilot. Neuronal knockdown of Dmon1 is sufficient to increase GluRIIA levels, suggesting its involvement in a presynaptic mechanism that regulates postsynaptic receptor levels. Ultrastructural analysis of mutant synapses reveals significantly smaller synaptic vesicles. Overexpression of vglut suppresses the defects in synaptic morphology and also downregulates GluRIIA levels in Dmon1 mutants, suggesting that homeostatic mechanisms are not affected in these mutants. We propose that DMon1 is part of a presynaptically regulated transsynaptic mechanism that regulates GluRIIA levels at the larval neuromuscular junction.

Sex differences in glutamate receptor gene expression in major depression and suicide.

Accumulating data indicate that the glutamate system is disrupted in major depressive disorder (MDD), and recent clinical research suggests that ketamine, an antagonist of the N-methyl-d-aspartate (NMDA) glutamate receptor (GluR), has rapid antidepressant efficacy. Here we report findings from gene expression studies of a large cohort of postmortem subjects, including subjects with MDD and controls. Our data reveal higher expression levels of the majority of glutamatergic genes tested in the dorsolateral prefrontal cortex (DLPFC) in MDD (F21,59=2.32, P=0.006). Posthoc data indicate that these gene expression differences occurred mostly in the female subjects. Higher expression levels of GRIN1, GRIN2A-D, GRIA2-4, GRIK1-2, GRM1, GRM4, GRM5 and GRM7 were detected in the female patients with MDD. In contrast, GRM5 expression was lower in male MDD patients relative to male controls. When MDD suicides were compared with MDD non-suicides, GRIN2B, GRIK3 and GRM2 were expressed at higher levels in the suicides. Higher expression levels were detected for several additional genes, but these were not statistically significant after correction for multiple comparisons. In summary, our analyses indicate a generalized disruption of the regulation of the GluRs in the DLPFC of females with MDD, with more specific GluR alterations in the suicides and in the male groups. These data reveal further evidence that, in addition to the NMDA receptor, the AMPA, kainate and the metabotropic GluRs may be targets for the development of rapidly acting antidepressant drugs.

Incubation of methamphetamine craving is associated with selective increases in expression of Bdnf and trkb, glutamate receptors, and epigenetic enzymes in cue-activated fos-expressing dorsal striatal neurons.

Cue-induced methamphetamine seeking progressively increases after withdrawal (incubation of methamphetamine craving), but the underlying mechanisms are largely unknown. We determined whether this incubation is associated with alterations in candidate genes in dorsal striatum (DS), a brain area implicated in cue- and context-induced drug relapse. We first measured mRNA expression of 24 candidate genes in whole DS extracts after short (2 d) or prolonged (1 month) withdrawal in rats following extended-access methamphetamine or saline (control condition) self-administration (9 h/d, 10 d). We found minimal changes. Next, using fluorescence-activated cell sorting, we compared gene expression in Fos-positive dorsal striatal neurons, which were activated during "incubated" cue-induced drug-seeking tests after prolonged withdrawal, with nonactivated Fos-negative neurons. We found significant increases in mRNA expression of immediate early genes (Arc, Egr1), Bdnf and its receptor (Trkb), glutamate receptor subunits (Gria1, Gria3, Grm1), and epigenetic enzymes (Hdac3, Hdac4, Hdac5, GLP, Dnmt3a, Kdm1a) in the Fos-positive neurons only. Using RNAscope to determine striatal subregion and cell-type specificity of the activated neurons, we measured colabeling of Fos with Drd1 and Drd2 in three DS subregions. Fos expression was neither subregion nor cell-type specific (52.5 and 39.2% of Fos expression colabeled with Drd1 and Drd2, respectively). Finally, we found that DS injections of SCH23390 (C17H18ClNO), a D1-family receptor antagonist known to block cue-induced Fos induction, decreased incubated cue-induced methamphetamine seeking after prolonged withdrawal. Results demonstrate a critical role of DS in incubation of methamphetamine craving and that this incubation is associated with selective gene-expression alterations in cue-activated D1- and D2-expressing DS neurons.

Quantitative Analysis of Glutamate Receptors in Glial Cells from the Cortex of GFAP/EGFP Mice Following Ischemic Injury: Focus on NMDA Receptors.

Cortical glial cells contain both ionotropic and metabotropic glutamate receptors. Despite several efforts, a comprehensive analysis of the entire family of glutamate receptors and their subunits present in glial cells is still missing. Here, we provide an overall picture of the gene expression of ionotropic (AMPA, kainate, NMDA) and the main metabotropic glutamate receptors in cortical glial cells isolated from GFAP/EGFP mice before and after focal cerebral ischemia. Employing single-cell RT-qPCR, we detected the expression of genes encoding subunits of glutamate receptors in GFAP/EGFP-positive (GFAP/EGFP(+)) glial cells in the cortex of young adult mice. Most of the analyzed cells expressed mRNA for glutamate receptor subunits, the expression of which, in most cases, even increased after ischemic injury. Data analyses disclosed several classes of GFAP/EGFP(+) glial cells with respect to glutamate receptors and revealed in what manner their expression correlates with the expression of glial markers prior to and after ischemia. Furthermore, we also examined the protein expression and functional significance of NMDA receptors in glial cells. Immunohistochemical analyses of all seven NMDA receptor subunits provided direct evidence that the GluN3A subunit is present in GFAP/EGFP(+) glial cells and that its expression is increased after ischemia. In situ and in vitro Ca(2+) imaging revealed that Ca(2+) elevations evoked by the application of NMDA were diminished in GFAP/EGFP(+) glial cells following ischemia. Our results provide a comprehensive description of glutamate receptors in cortical GFAP/EGFP(+) glial cells and may serve as a basis for further research on glial cell physiology and pathophysiology.

Methyl-donor deficiency in adolescence affects memory and epigenetic status in the mouse hippocampus.

DNA methylation is one of the essential factors in the control of gene expression. Alteration of the DNA methylation pattern has been linked to various neurological, behavioral and neurocognitive dysfunctions. Recent studies have pointed out the importance of epigenetics in brain development and functions including learning and memory. Nutrients related to one-carbon metabolism are known to play important roles in the maintenance of genomic DNA methylation. Previous studies have shown that the long-term administration of a diet lacking essential one-carbon nutrients such as methionine, choline and folic acid (methyl donors) caused global DNA hypermethylation in the brain. Therefore, the long-term feeding of a methyl-donor-deficient diet may cause abnormal brain development including learning and memory. To confirm this hypothesis, 3-week-old mice were maintained on a folate-, methionine- and choline-deficient (FMCD) or control (CON) diet for 3 weeks. We found that the methyl-donor deficiency impaired both novel object recognition and fear extinction after 3 weeks of treatment. The FMCD group showed spontaneous recovery of fear that differed from that in CON. In addition, we found decreased Gria1 gene expression and specific CpG hypermethylation of the Gria1 promoter region in the FMCD hippocampus. Our data suggest that a chronic dietary lack of methyl donors in the developmental period affects learning, memory and gene expressions in the hippocampus.

Early-onset autosomal recessive cerebellar ataxia associated with retinal dystrophy: new human hotfoot phenotype caused by homozygous GRID2 deletion.

PURPOSE: The aim of this study was to identify the genetic cause of early-onset autosomal recessive cerebellar ataxia associated with retinal dystrophy in a consanguineous family. METHODS: An affected 6-month-old child underwent neurological and ophthalmological examinations. Genetic analyses included homozygosity mapping, copy number analysis, conventional polymerase chain reaction, Sanger sequencing, quantitative polymerase chain reaction, and whole-exome sequencing. Expression analysis of GRID2 was performed by quantitative polymerase chain reaction and immunohistochemistry. RESULTS: A homozygous deletion of exon 2 of GRID2 (p.Gly30_Glu81del) was identified in the proband. GRID2 encodes an ionotropic glutamate receptor known to be selectively expressed in cerebellar Purkinje cells. Here, we demonstrated GRID2 expression in human adult retina and retinal pigment epithelium. In addition, Grid2 expression was demonstrated in different stages of murine retinal development. GRID2 immunostaining was shown in murine and human retina. Whole-exome sequencing in the proband did not provide arguments for other disease-causing mutations, supporting the idea that the phenotype observed represents a single clinical entity. CONCLUSION: We identified GRID2 as an underlying disease gene of early-onset autosomal recessive cerebellar ataxia with retinal dystrophy, expanding the clinical spectrum of GRID2 deletion mutants. We demonstrated for the first time GRID2 expression and localization in human and murine retina, providing evidence for a novel functional role of GRID2 in the retina.

Enriched expression of GluD1 in higher brain regions and its involvement in parallel fiber-interneuron synapse formation in the cerebellum.

Of the two members of the δ subfamily of ionotropic glutamate receptors, GluD2 is exclusively expressed at parallel fiber-Purkinje cell (PF-PC) synapses in the cerebellum and regulates their structural and functional connectivity. However, little is known to date regarding cellular and synaptic expression of GluD1 and its role in synaptic circuit formation. In the present study, we investigated this issue by producing specific and sensitive histochemical probes for GluD1 and analyzing cerebellar synaptic circuits in GluD1-knock-out mice. GluD1 was widely expressed in the adult mouse brain, with high levels in higher brain regions, including the cerebral cortex, striatum, limbic regions (hippocampus, nucleus accumbens, lateral septum, bed nucleus stria terminalis, lateral habenula, and central nucleus of the amygdala), and cerebellar cortex. In the cerebellar cortex, GluD1 mRNA was expressed at the highest level in molecular layer interneurons and its immunoreactivity was concentrated at PF synapses on interneuron somata. In GluD1-knock-out mice, the density of PF synapses on interneuron somata was significantly reduced and the size and number of interneurons were significantly diminished. Therefore, GluD1 is common to GluD2 in expression at PF synapses, but distinct from GluD2 in neuronal expression in the cerebellar cortex; that is, GluD1 in interneurons and GluD2 in PCs. Furthermore, GluD1 regulates the connectivity of PF-interneuron synapses and promotes the differentiation and/or survival of molecular layer interneurons. These results suggest that GluD1 works in concert with GluD2 for the construction of cerebellar synaptic wiring through distinct neuronal and synaptic expressions and also their shared synapse-connecting function.

Cell-specific cre recombinase expression allows selective ablation of glutamate receptors from mouse horizontal cells.

In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57), a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99%) and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl). In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼ 50%) in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼ 75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less excitatory input, can thus be used to analyze the contribution of horizontal cells to retinal processing.