AMPA Subunits Research Articles

Enriched environment ameliorates memory impairments in rats after postsurgery sleep deprivation

Postsurgery sleep deprivation is a common complication that severely deteriorates the quality of life of patients. Here we aim to investigate the effects and mechanism of enriched environment in ameliorating sleep deprivation and memory impairments. Hernia repair surgery was performed on rats to induce sleep deprivation. Enriched environment (EE) was used to treat rats with sleep deprivation, and open field and Y-maze tests were performed to compare behavioral parameters of sleep deprivation rats with or without EE treatments to those of normal rats. To understand the mechanism, neurotrophic and growth factors including BDNF, NGF, NT-3 and GDNF were analyzed using enzyme-linked immunosorbent assay (ELISA). AMPAR subunits, including GluA1-A3, and GABAA receptor α1 subunit expression in hippocampus tissues were assessed using western blot. EE restored normal levels of anxiety index and freezing behavior in open field test and level of alternation in Y-maze test, suggesting the reduction of anxiolytic effects and spatial memory impairment induced by sleep deprivation. EE increased BDNF levels and reduced NT-3 levels in sleep deprivation rats. GluA1/GluA2 ratio was increased by EE. GABAA receptor α1 subunit expression was decreased by EE. EE is effective in ameliorating the detrimental effects of sleep deprivation in spatial memory impairment, and restoring normal levels of neurotrophic factors, which are potentially mediated by attenuating the changes in AMPAR subunit expression and reducing GABAA receptor α1 subunit expression. These data provide supporting evidences for the use of EE to treat adverse outcomes of sleep deprivation induced by surgery.

Unraveling the molecular mechanisms involved in alcohol intake and withdrawal in adolescent mice exposed to alcohol during early life stages

Alcohol interferes with foetal development and prenatal alcohol exposure can lead to adverse effects known as foetal alcohol spectrum disorders. We aimed to assess the underlying neurobiological mechanisms involved in alcohol intake and withdrawal in adolescent mice exposed to alcohol during early life stages, in discrete brain areas. Pregnant C57BL/6 female mice were exposed to binge alcohol drinking from gestation to weaning. Subsequently, alcohol seeking and taking behaviour were evaluated in male adolescent offspring, as assessed in the two-bottle choice and oral self-administration paradigms. Brain area samples were analysed to quantify AMPAR subunits GluR1/2 and pCREB/CREB expression following alcohol self-administration. We measured the expression of mu and kappa opioid receptors both during acute alcohol withdrawal (assessing anxiety alterations by the EPM test) and following reinstatement in the two-bottle choice paradigm. In addition, alcohol metabolism was analysed by measuring blood alcohol concentrations under an acute dose of 3 g/kg alcohol. Our findings demonstrate that developmental alcohol exposure enhances alcohol intake during adolescence, which is associated with a decrease in the pCREB/CREB ratio in the hippocampus, prefrontal cortex and striatum, while the GluR1/GluR2 ratio showed a decrease in the hippocampus. Moreover, PLAE mice showed behavioural alterations, such as increased anxiety-like responses during acute alcohol withdrawal, and higher BAC levels. No significant changes were identified for mu and kappa opioid receptors mRNA expression. The current study highlights that early alcohol exposed mice increased alcohol consumption during late adolescence. Furthermore, a diminished CREB signalling and glutamatergic neuroplasticity are proposed as underpinning neurobiological mechanisms involved in the sensitivity to alcohol reinforcing properties.

Synaptic restoration by cAMP/PKA drives activity-dependent neuroprotection to motoneurons in ALS.

Excessive excitation is hypothesized to cause motoneuron (MN) degeneration in amyotrophic lateral sclerosis (ALS), but actual proof of hyperexcitation in vivo is missing, and trials based on this concept have failed. We demonstrate, by in vivo single-MN electrophysiology, that, contrary to expectations, excitatory responses evoked by sensory and brainstem inputs are reduced in MNs of presymptomatic mutSOD1 mice. This impairment correlates with disrupted postsynaptic clustering of Homer1b, Shank, and AMPAR subunits. Synaptic restoration can be achieved by activation of the cAMP/PKA pathway, by either intracellular injection of cAMP or DREADD-Gs stimulation. Furthermore, we reveal, through independent control of signaling and excitability allowed by multiplexed DREADD/PSAM chemogenetics, that PKA-induced restoration of synapses triggers an excitation-dependent decrease in misfolded SOD1 burden and autophagy overload. In turn, increased MN excitability contributes to restoring synaptic structures. Thus, the decrease of excitation to MN is an early but reversible event in ALS. Failure of the postsynaptic site, rather than hyperexcitation, drives disease pathobiochemistry.

Stress-induced aggression in heterozygous TPH2 mutant mice is associated with alterations in serotonin turnover and expression of 5-HT6 and AMPA subunit 2A receptors

BackgroundThe contribution of gene-environment interactions that lead to excessive aggression is poorly understood. Environmental stressors and mutations of the gene encoding tryptophan hydroxylase-2 (TPH2) are known to influence aggression. For example, TPH2 null mutant mice (Tph2−/−) are naturally highly aggressive, while heterozygous mice (Tph2+/−) lack a behavioral phenotype and are considered endophenotypically normal. Here we sought to discover whether an environmental stressor would affect the phenotype of the genetically ‘susceptible’ heterozygous mice (Tph2+/−). MethodsTph2+/− male mice or Tph2+/+ controls were subjected to a five-day long rat exposure stress paradigm. Brain serotonin metabolism and the expression of selected genes encoding serotonin receptors, AMPA receptors, and stress markers were studied. ResultsStressed Tph2+/− mice displayed increased levels of aggression and social dominance, whereas Tph2+/+ animals became less aggressive and less dominant. Brain tissue concentrations of serotonin, its precursor hydroxytryptophan and its metabolite 5-hydroxyindoleacetic acid were significantly altered in all groups in the prefrontal cortex, striatum, amygdala, hippocampus and dorsal raphe after stress. Compared to non-stressed animals, the concentration of 5-hydroxytryptophan was elevated in the amygdala though decreased in the other brain structures. The overexpression of the AMPA receptor subunit, GluA2, and downregulation of 5-HT6 receptor, as well as overexpression of c-fos and glycogen-synthase-kinase-3β (GSK3-β), were found in most structures of the stressed Tph2+/− mice. LimitationsRescue experiments would help to verify causal relationships of reported changes. ConclusionsThe interaction of a partial TPH2 gene deficit with stress results in pathological aggression and molecular changes, and suggests that the presence of genetic susceptibility can augment aggression in seemingly resistant phenotypes.

Impairment of Mitochondrial Calcium Buffering Links Mutations in C9ORF72 and TARDBP in iPS-Derived Motor Neurons from Patients with ALS/FTD.

SummaryTDP-43 dysfunction is common to 97% of amyotrophic lateral sclerosis (ALS) cases, including those with mutations in C9orf72. To investigate how C9ORF72 mutations drive cellular pathology in ALS and to identify convergent mechanisms between C9ORF72 and TARDBP mutations, we analyzed motor neurons (MNs) derived from induced pluripotent stem cells (iPSCs) from patients with ALS. C9ORF72 iPSC-MNs have higher Ca2+ release after depolarization, delayed recovery to baseline after glutamate stimulation, and lower levels of calbindin compared with CRISPR/Cas9 genome-edited controls. TARDBP iPS-derived MNs show high glutamate-induced Ca2+ release. We identify here, by RNA sequencing, that both C9ORF72 and TARDBP iPSC-MNs have upregulation of Ca2+-permeable AMPA and NMDA subunits and impairment of mitochondrial Ca2+ buffering due to an imbalance of MICU1 and MICU2 on the mitochondrial Ca2+ uniporter, indicating that impaired mitochondrial Ca2+ uptake contributes to glutamate excitotoxicity and is a shared feature of MNs with C9ORF72 or TARDBP mutations.

Investigation of GluA1 and GluA2 AMPA receptor subtype distribution in the hippocampus and anterior cingulate cortex of Long Evans rats during development

Preadolescent development is characterized by a reorganization of connectivity within and between brain regions that coincides with the emergence of complex behaviors. During the preadolescent period, the rodent hippocampus and regions of the frontal cortex are remodelled as the brain strengthens active connections and eliminates others. In the developing and mature brain, changes in the properties of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAr)-mediated synaptic responses contribute to experience-dependent changes in neural organization and function. AMPAr are made up of 4 subunits, of which GluA1 and GluA2 have been shown to play the most prominent role in functional plasticity. In this study, we sought to determine whether levels of these two subunits changed during the course of pre-adolescent development in the hippocampus and anterior cingulate cortex (ACC). To investigate the developmental changes in GluA1 and GluA2 AMPAr subunits, Western blotting and immunohistochemistry were performed on the ACC and hippocampus from P18 - P30 and compared to adult (P50) levels and distribution. Within the hippocampus, protein levels of GluA1 and GluA2 peaked around P26−30 whereby localized staining in the dentate gyrus reflected this pattern. GluA1 and GluA2 levels within the ACC showed little variation during this developmental period. These results indicate that changes in AMPAr subunits within the hippocampus coincide with developmental modifications that underlie the shift from juvenile- to adult-like capabilities. However, changes in AMPAr distribution in the ACC might not mediate changes that reflect preadolescent developmental shifts.

Effects of “Junk‐Food” on Excitatory Transmission and NMDA Receptor Subunit Expression in the NAc of Females

Eating diets high in fats and sugars (i.e., “junk‐food”) produces alterations in the function of brain regions like the nucleus accumbens (NAc) that promote food‐seeking and over‐eating. For example, consumption of “junk‐food” increases the expression and function of high conductance calcium permeable AMPA receptors (CP‐AMPARS) in the NAc of obesity‐prone, but not obesity‐resistant male rats. These alterations persist for up to 30 days after initial “junk‐food” exposure. In addition, blockade of CP‐AMPARs within the NAc prevents enhanced food‐seeking in obesity‐prone rats. Thus, obesity‐prone rats are more sensitive to diet‐induced enhancements in NAc glutamatergic transmission that promote food‐seeking. However, whether similar experience‐induced alterations occur in females is unknown. Therefore, the goal of the studies presented here is to determine the effects of “junk‐food” on NAc glutamate receptor expression and function in females. Males were included for comparison and to replicate initial findings described above. Rats were given the “junk‐food” diet (10 days) followed by a return to ad lib standard chow for 14 days (i.e., “junk‐food” deprivation) in order to examine long‐lasting effects. Controls remained on standard chow throughout. Biotinylation or BS3 crosslinking followed by western blotting was then used to measure changes in the expression of AMPAR and NMDA receptor (NMDAR) subunits. In addition, whole‐cell patch clamp recordings from ex vivo brain slices were used to measure effects on AMPAR and NMDAR‐mediated transmission. Furthermore, the estrus cycle was monitored throughout and recordings were conducted in the metestrus/diestrus phase of the cycle. For biochemical studies, post hoc analyses are used to assess potential effects of the cycle on protein expression. In males, we found increases in GluA1, but not GluA2 surface expression and enhanced CP‐AMPAR‐mediated transmission following “junk‐food” consumption, replicating previous results. However, in females no effects on GluA1 or GluA2 surface expression or on AMPAR‐mediated transmission (CP‐AMPARs or non‐CP‐AMPARs) were found. However, there was a “junkfood” induced increase in the AMPA/NMDA ratio in females that was due to a reduction in NMDAR‐mediated transmission in the absence of any change in AMPAR‐mediated transmission. Thus, initial results show that there are sex differences in “junk‐food” induced alterations in CP‐AMPARs, and suggest a reduction in NMDA‐mediated transmission in females. Ongoing studies are examining effects of “junk‐food” on NMDAR subunit expression (GluN1, GluN2, GluN3). This will provide insights into which NMDAR populations may be affected. Together, these data shed light on potential sex specific mechanisms by which “junkfood” diet alters NAc function.Support or Funding InformationNIH R01 DK106188 (CRF)NIH F99 NS108549 (YAC)

Origins of Parkinson's Disease in Brain Development: Insights From Early and Persistent Effects of LRRK2-G2019S on Striatal Circuits.

Late-onset Parkinson’s disease (PD) is dominated clinically and experimentally by a focus on dopamine neuron degeneration and ensuing motor system abnormalities. There are, additionally, a number of non-motor symptoms – including cognitive and psychiatric – that can appear much earlier in the course of the disease and also significantly impair quality of life. The neurobiology of such cognitive and psychiatric non-motor symptoms is poorly understood. The recognition of genetic forms of late-onset PD, which are clinically similar to idiopathic forms in both motor and non-motor symptoms, raises the perspective that brain cells and circuits – and the behaviors they support – differ in significant ways from normal by virtue of the fact that these mutations are carried throughout life, including especially early developmental critical periods where circuit structure and function is particularly susceptible to the influence of experience-dependent activity. In this focused review, we support this central thesis by highlighting studies of LRRK2-G2019S mouse models. We describe work that shows that in G2019S mutants, corticostriatal activity and plasticity are abnormal by P21, the end of a period of excitatory synaptogenesis in striatum. Moreover, by young adulthood, impaired striatal synaptic and non-synaptic forms of plasticity likely underlie altered and variable performance by mutant mice in validated tasks that test for depression-like and anhedonia-like behaviors. Mechanistically, deficits in cellular, synaptic and behavioral plasticity may be unified by mutation-linked defects in trafficking of AMPAR subunits and other membrane channels, which in turn may reflect impairment in the function of the Rab family of GTPases, a major target of LRRK2 phosphorylation. These findings underscore the need to better understand how PD-related mutant proteins influence brain structure and function during an extended period of brain development, and offer new clues for future therapeutic strategies to target non-motor cognitive or psychiatric symptoms of PD.

Low doses of Perampanel protect striatal and hippocampal neurons against in vitro ischemia by reversing the ischemia-induced alteration of AMPA receptor subunit composition

Energy depletion caused by ischemic brain insults may result in persistent neuronal depolarization accompanied by hyper-stimulation of ionotropic glutamate receptors and excitotoxic phenomena, possibly leading to cell death. The use of glutamate receptor antagonists, such as the AMPARs antagonist Perampanel (PER), might be a pharmacological approach to counteract the excessive over-activation of glutamate receptors providing neuroprotective effects. Using electrophysiological and molecular analyses, we investigated the effect of PER against in vitro ischemia obtained by oxygen and glucose deprivation (OGD) in rat slices of two brain structures particularly sensitive to ischemic insults, the nucleus striatum and the hippocampus. We found that in these regions PER was able to avoid the OGD-induced neuronal suffering, at low doses not reducing basal excitatory synaptic transmission and not altering long-term potentiation (LTP) induction. Furthermore, in both the analysed regions, PER blocked a pathological form of LTP, namely ischemic LTP (iLTP). Finally, we hypothesized that the protective effect of PER against OGD was due to its capability to normalize the altered synaptic localization and function of AMPAR subunits, occuring after an ischemic insult. Taken together these findings support the idea that PER is a drug potentially effective to counteract ischemic damage.

GRP receptor and AMPA receptor cooperatively regulate itch-responsive neurons in the spinal dorsal horn

Gastrin-releasing peptide (GRP) receptor-expressing (GRPR)+ neurons have a central role in the spinal transmission of itch. Because their fundamental regulatory mechanisms are not yet understood, it is important to determine how such neurons are excited and integrate itch sensation. In this study, we investigated the mechanisms for the activation of itch-responsive GRPR+ neurons in the spinal dorsal horn (SDH). GRPR+ neurons expressed the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) containing the GluR2 subunit. In mice, peripherally elicited histaminergic and non-histaminergic itch was prevented by intrathecal (i.t.) administration of the AMPAR antagonist NBQX, which was consistent with the fact that firing of GRPR+ neurons in SDH under histaminergic and non-histaminergic itch was completely blocked by NBQX, but not by the GRPR antagonist RC-3095. Because GRP+ neurons in SDH contain glutamate, we investigated the role of GRP+ (GRP+/Glu+) neurons in regulating itch. Chemogenetic inhibition of GRP+ neurons suppressed both histaminergic and non-histaminergic itch without affecting the mechanical pain threshold. In nonhuman primates, i.t. administration of NBQX also attenuated peripherally elicited itch without affecting the thermal pain threshold. In a mouse model of diphenylcyclopropenone (DCP)-induced contact dermatitis, GRP, GRPR, and AMPAR subunits were upregulated in SDH. DCP-induced itch was prevented by either silencing GRP+ neurons or ablation of GRPR+ neurons. Altogether, these findings demonstrate that GRP and glutamate cooperatively regulate GRPR+ AMPAR+ neurons in SDH, mediating itch sensation. GRP–GRPR and the glutamate–AMPAR system may play pivotal roles in the spinal transmission of itch in rodents and nonhuman primates.

AMPA receptor subunit localization in schizophrenia anterior cingulate cortex

The glutamate hypothesis of schizophrenia suggests that altered glutamatergic transmission occurs in this illness, although precise mechanisms of dysregulation remain elusive. AMPA receptors (AMPARs), a subtype of ionotropic glutamate receptor, are the main facilitators of fast, excitatory neurotransmission in the brain, and changes in AMPAR number or composition at synapses can regulate synaptic strength and plasticity. Prior evidence of abnormal expression of transmembrane AMPAR regulatory proteins (TARPs) in schizophrenia suggests defective trafficking of AMPARs, which we propose could lead to altered AMPAR expression at excitatory synapses. To test this hypothesis, we isolated subcellular fractions enriched for endoplasmic reticulum (ER) and synapses from anterior cingulate cortex (ACC) from schizophrenia (N = 18) and comparison (N = 18) subjects, and measured glutamate receptor subunits (GluA1, GluA2, GluA3, GluA4, NR1, NR2A, NR2B, and NR3A) and TARP member γ2 (stargazin) in homogenates and subcellular fractions by western blot analysis. We found decreased expression of stargazin and an increased ratio of GluA2:stargazin in ACC homogenates, while in the synapse fraction we identified a decrease in GluA1 and reduced ratios of GluA1:stargazin and GluA1:GluA2 in schizophrenia. The amount of stargazin in the ER fraction was not different, but the relative amount of ER/Total stargazin was increased in schizophrenia. Together, these findings suggest that associations between stargazin and AMPA subunits are abnormal, potentially affecting forward trafficking or synaptic stability of GluA1-containing AMPARs. These data provide evidence that altered interactions with trafficking proteins may contribute to glutamate dysregulation in schizophrenia.

Transcriptional modulation of calcium-permeable AMPA receptor subunits in glioblastoma by MEK-ERK1/2 inhibitors and their role in invasion.

Glioblastoma is the most common primary brain tumor. Glioblastoma cells secrete a significant amount of glutamate, which serve as a potential growth factor in glioma pathobiology through their specific receptor subtypes including α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Glioblastoma express AMPAR subunits; however, its regulation and activation with downstream intracellular signaling are not well-understood. Phosphorylated-extracellular signaling-regulated kinase (ERK)1/2 is known to regulate the ionotropic glutamate receptors in cortical neurons. The mitogen-activated protein kinase cascade is frequently activated in several tumors, including glioma. Nonetheless, the association of ERK signaling with AMPAR subunits in glioblastoma is undetermined. Here, we demonstrated potential role of AMPAR in invasion, and the modulation of AMPAR subunits at transcript level by ERK signaling in glioblastoma cells. Inhibition of ERK signaling specifically downregulated the expression of calcium-permeable AMPAR subunits, GluA1 and GluA4, and upregulated calcium-impermeable AMPAR subunit GluA2 implying differential regulation of the expression of calcium-permeable AMPAR subunits of glioblastoma. Concomitantly, it significantly decreased the invasion of U87MG cells. Taken together, these findings suggest that the AMPAR enhances invasion of glioblastoma, and ERK signaling modulates the differential expression of calcium-permeable AMPAR phenotype that might play a crucial role in the invasive propensity of glioblastoma cells.

Dysregulation of AMPA receptor subunit expression in sporadic ALS post-mortem brain.

Amyotrophic lateral sclerosis (ALS) is characterised by progressive motor neuron degeneration. Although there are over 40 genes associated with causal monogenetic mutations, the majority of ALS patients are not genetically determined. Causal ALS mutations are being increasingly mechanistically studied, though how these mechanisms converge and diverge between the multiple known familial causes of ALS (fALS) and sporadic forms of ALS (sALS) and furthermore between different neuron types, is poorly understood. One common pathway that is implicated in selective motor neuron death is enhanced α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate (AMPAR)‐mediated excitoxicity. Specifically, human in vitro and pathological evidence has linked the C9orf72 repeat expansion mutation to a relative increase in the Ca2+‐permeable AMPAR population due to AMPAR subunit dysregulation. Here, we provide the first comparative quantitative assessment of the expression profile of AMPAR subunit transcripts, using BaseScope, in post‐mortem lower motor neurons (spinal cord, anterior horn), upper motor neurons (motor cortex) and neurons of the pre‐frontal cortex in sALS and fALS due to mutations in SOD1 and C9orf72. Our data indicated that AMPAR dysregulation is prominent in lower motor neurons in all ALS cases. However, sALS and mutant C9orf72 cases exhibited GluA1 upregulation whereas mutant SOD1 cases displayed GluA2 down regulation. We also showed that sALS cases exhibited widespread AMPAR dysregulation in the motor and pre‐frontal cortex, though the exact identity of the AMPAR subunit being dysregulated was dependent on brain region. In contrast, AMPAR dysregulation in mutant SOD1 and C9orf72 cases was restricted to lower motor neurons only. Our data highlight the complex dysregulation of AMPAR subunit expression that reflects both converging and diverging mechanisms at play between different brain regions and between ALS cohorts. © 2019 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Anterior insular cortex mediates hyperalgesia induced by chronic pancreatitis in rats

Central sensitization plays a pivotal role in the maintenance of chronic pain induced by chronic pancreatitis (CP), but cortical modulation of painful CP remains elusive. This study was designed to examine the role of anterior insular cortex (aIC) in the pathogenesis of hyperalgesia in a rat model of CP. CP was induced by intraductal administration of trinitrobenzene sulfonic acid (TNBS). Abdomen hyperalgesia and anxiety were assessed by von Frey filament and open field tests, respectively. Two weeks after surgery, the activation of aIC was indicated by FOS immunohistochemical staining and electrophysiological recordings. Expressions of VGluT1, NMDAR subunit NR2B and AMPAR subunit GluR1 were analyzed by immunoblottings. The regulatory roles of aIC in hyperalgesia and pain-related anxiety were detected via pharmacological approach and chemogenetics in CP rats. Our results showed that TNBS treatment resulted in long-term hyperalgesia and anxiety-like behavior in rats. CP rats exhibited increased FOS expression and potentiated excitatory synaptic transmission within aIC. CP rats also showed up-regulated expression of VGluT1, and increased membrane trafficking and phosphorylation of NR2B and GluR1 within aIC. Blocking excitatory synaptic transmission significantly attenuated abdomen mechanical hyperalgesia. Specifically inhibiting the excitability of insular pyramidal cells reduced both abdomen hyperalgesia and pain-related anxiety. In conclusion, our findings emphasize a key role for aIC in hyperalgesia and anxiety of painful CP, providing a novel insight into cortical modulation of painful CP and shedding light on aIC as a potential target for neuromodulation interventions in the treatment of CP.

Inhibition and assessment of the biophysical gating properties of GluA2 and GluA2/A3 AMPA receptors using curcumin derivatives.

The development of efficacious and safe drugs for the treatment of neurological diseases related to glutamate toxicity has been a focus in neuropharmacological research. Specifically, discovering antagonists to modulate the activity and kinetics of AMPA receptors, which are the fastest ligand-gated ion channels involved in excitatory neurotransmission in response to glutamate. Thus, the current study investigated novel curcumin derivatives on the biophysical properties of AMPA receptors, specifically on the homomeric GluA2 and the heteromeric GluA2/A3 subunits and assessed for inhibitory actions. The biophysical parameter (i.e., desensitization, deactivation, and peak currents) were measured by using whole-cell patch clamp electrophysiology with and without the administration of the derivatives onto HEK293 cells. CR-NN, CR-NNPh, CR-MeNH, and CR-NO of the tested derivatives showed inhibition on all AMPA receptors up to 6 folds. Moreover, the inhibitory derivatives also increased desensitization and deactivation, which further intensifies the compounds’ neuroprotective effects. However, CR-PhCl, CR-PhF, and CR-PhBr did not show any significant changes on the peak current, deactivation or desensitization rates. By comparison to other discovered and widely used antagonist, the prepared curcumin derivatives are not selective to a specific AMPA subunit, instead implement its effect in the same way between all types of AMPA receptors. Additionally, the obtained results provide derivatives that not only noncompetitively inhibit AMPARs but also decrease its biophysical kinetics, specifically desensitization and deactivation rates. Hence, to potentially serve as a new AMPAR inhibitor with therapeutic potential, the current study provides compounds that are non-selective and non-competitive antagonist, which also effect the desensitization and deactivation rates of the receptor.

Targeting the inhibition of fatty acid amide hydrolase ameliorate the endocannabinoid-mediated synaptic dysfunction in a valproic acid-induced rat model of Autism

Autism spectrum disorder (ASD) is a neurodevelopmental disorder, characterized by social interaction impairment, stereotypical/repetitive behaviors and emotional deregulation. The endocannabinoid (eCB) system plays a crucial role in modulating the behavioral traits that are typically core symptoms of ASD. The major molecular mechanisms underlying eCB-dependent long-term depression (eCB-LTD) are mediated by group 1 metabotropic glutamate receptor (mGluR)-induced removal of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). Recently, modulation of anandamide (AEA), one of the main endocannabinoids in the brain, has been reported to alter social behaviors in genetic models of ASD. On this basis, we investigated the effects of treatment and the synaptic mechanism underlying AEA-mediated signaling in prenatal exposure to valproic acid (VPA) in rats. We found that the social deficits, repetitive behaviors and abnormal emotion-related behaviors in VPA-exposed offspring were improved after treatment with an inhibitor of AEA degrading enzyme, URB597. Using an integrative approach combing electrophysiological and cellular mechanisms, the results showed that the impaired eCB-LTD, abnormal mGluR-mediated LTD (mGluR-LTD) and decreased removal of AMPAR subunits GluA1 and GluA2 were reversed by URB597 in the prefrontal cortex (PFC) of VPA-exposed offspring. Taken together, these results provide the first evidence that rescue of the ASD-like phenotype by URB597 is mediated by enhancing the mechanism of removal of AMPAR subunits GluA1/2 underlying AEA signaling in the PFC in a VPA-induced model of ASD.

Different periods of forced abstinence after instrumental learning for food reward of different macronutrient value on responding for conditioned cues and AMPAr subunit levels

Food craving can be viewed as an intense desire for a specific food that propagates seeking and consuming behavior. Prolonged forced abstinence from rewarding foods can result in escalated food-seeking behavior as measured via elevated responding for food-paired cues in the absence of the primary reward. Palatable food consumption and food-seeking is associated with changes in the abundance and composition of AMPA receptors in the nucleus accumbens (NAc) but differing results have been reported. The present study examined whether different food types could produce escalated food-seeking behavior after various abstinence periods and whether this was associated with changes in AMPA receptor protein levels. Rats were trained for 10 days to bar press for purified, sucrose, or chocolate-flavored sucrose pellets. Rats were tested at 24 hrs, 7 d or 14 d whereby bar pressing resulted in presentation of cues paired with food but no food reward was delivered. Western blotting was used to determine protein levels of GluR1, GluR1pSer845, and GluR2 in the NAc. Three separate groups were assessed: 1) a group that was trained on the operant task and tested for conditioned responding (tested group); 2) a group that was trained on the operant task but not tested (non-tested group); 3) a group that was neither trained nor tested (control). The purified food group showed a time-dependent elevation in conditioned bar pressing over the 3 abstinence periods. GluR1 AMPAr subunit levels were higher in the purified and sucrose groups tested at 24 hours compared to the non-tested and control values. GluR1 levels subsequently declined at the 7- and 14-day abstinence periods in the purified and sucrose tested and non-tested groups compared to control values. GluR2 and pSer845 Glur1 levels were similar across all groups and abstinence periods. These results show that food-seeking behavior associated with forced abstinence from different food rewards may depend on the macronutrient composition of the food reward or the food type given during the abstinence period. A clear link with AMPAr subunit levels in this model was not established.

Changes in excitatory and inhibitory receptor expression and network activity during induction and establishment of epilepsy in the rat Reduced Intensity Status Epilepticus (RISE) model

The RISE model is an effective system to study the underlying molecular and cellular mechanisms involved in the initiation and maintenance of epilepsy in vivo. Here we profiled the expression of excitatory and inhibitory neurotransmitter receptor subunits and synaptic scaffolding proteins in the hippocampus and temporal lobe and compared these changes with alterations in network activity at specific timepoints during epileptogenesis. Significant changes occurred in all of the ionotropic glutamate receptor subunits tested during epilepsy induction and progression and the profile of these changes differed between the hippocampus and temporal lobe. Notably, AMPAR subunits were dramatically decreased during the latent phase of epilepsy induction, matched by a profound decrease in the network response to kainate application in the hippocampus. Moreover, decreases in the GABAAβ3 subunit are consistent with a loss of inhibitory input contributing to the perturbation of excitatory/inhibitory balance and seizure generation. These data highlight the synaptic reorganisation that mediates the relative hypoexcitability prior to the manifestation of seizures and subsequent hyperexcitability when spontaneous seizures develop. These patterns of changes give new insight into the mechanisms underpinning epilepsy and provide a platform for future investigations targeting particular receptor subunits to reduce or prevent seizures.

Glutamate Stimulation Dysregulates AMPA Receptors-Induced Signal Transduction Pathway in Leber's Inherited Optic Neuropathy Patient-Specific hiPSC-Derived Retinal Ganglion Cells.

The mitochondrial genetic disorder, Leber’s hereditary optic neuropathy (LHON), is caused by a mutation in MT-ND4 gene, encoding NADH dehydrogenase subunit 4. It leads to the progressive death of retinal ganglion cells (RGCs) and causes visual impairment or even blindness. However, the precise mechanisms of LHON disease penetrance and progression are not completely elucidated. Human-induced pluripotent stem cells (hiPSCs) offer unique opportunities to investigate disease-relevant phenotypes and regulatory mechanisms underlying LHON pathogenesis at the cellular level. In this study, we successfully generated RGCs by differentiation of LHON patient-specific hiPSCs. We modified the protocol of differentiation to obtain a more enriched population of single-cell RGCs for LHON study. Based on assessing morphology, expression of specific markers and electrophysiological activity, we found that LHON-specific hiPSC-derived were more defective in comparison with normal wild-type RGCs. Based on our previous study, whereby by using microarray analysis we identified that the components of glutamatergic synapse signaling pathway were significantly downregulated in LHON-specific RGCs, we focused our study on glutamate-associated α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. We found that the protein expression levels of the subunits of the AMPA receptor, GluR1 and GluR2, and their associated scaffold proteins were decreased in LHON-RGCs. By performing the co-immunoprecipitation assay, we found several differences in the efficiencies of interaction between AMPA subunits and scaffold proteins between normal and LHON-specific RGCs.

The inhibitory role of curcumin derivatives on AMPA receptor subunits and their effect on the gating biophysical properties

Curcumin is a natural polyphenol that has a broad spectrum of therapeutic characters, including neuroprotective actions against various neurological diseases. However, the molecular mechanism behind its neuroprotective properties remains obscure. The current study investigated the neuroprotective properties of 7 different curcumin derivatives on the gating biophysical properties of AMPA receptors, specifically on the calcium-permeable homomeric GluA1 and calcium impermeable heteromeric GluA1/A2 subunits. Due to the association between excessive activation of AMPARs and neurotoxicity linked to numerous pathologies, we aim to target and manipulate the kinetics of AMPARs through these derivatives. The current study used patch-clamp electrophysiology to measure the whole-cell currents in the presence and absence of the curcumin derivatives onto HEK293 cells expressing AMPA subunits. Our results showed that some of the curcumin derivatives showed an inhibitory effect and altered the gating biophysical properties, namely, deactivation and desensitization. In the presence of those derivatives, the peak current measured was significantly reduced, and the desensitization and deactivation rates decreased as well, achieving slower kinetics of the receptor and depressing its activity. These results suggest that the two most promising derivatives have inhibitory actions and act as allosteric modulators. Many neurological diseases like epilepsy, ALS, and strokes are associated with overactivation of AMPA receptors. We can potentially synthesize a more potent neuroprotective drug to treat those neurological diseases, by understanding the most stable chemical interaction between the derivative and the receptor underlying the reported neuronal depressive properties.