AMPA Receptor Subunit Research Articles

Possible involvement of inflammasomes on the post-stroke cognitive impairment in a mouse model of embolic cerebral infarct

Post-stroke cognitive impairment (PSCI) of the complications after stroke has been shown to be involved in brain proinflammatory cytokines such as interleukin (IL)-1β (IL-lβ) and IL-18. In the present study, we examined using acetic acid-induced embolic cerebral infarct (ECI) mice whether post-stroke inflammasome activation is involved in the development of PSCI. In behavioral tests, long-term learning and memory assessed using the passive avoidance test were impaired after ECI. On the other hand, the impairment of short-term learning and memory assessed using the Y-maze test was not observed. Furthermore, the phosphorylated α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit glutamate receptor 1 (GluR1) at Ser 831 and Ser 845 protein was found to be significantly decreased in the dorsal hippocampus of ECI mice. In addition, the expression levels of ionized calcium-binding adapter protein 1 (Iba1), glial fibrillary acidic protein (GFAP), apoptosis-associated speck-like protein containing a caspase recruitment domain / target of methylation-induced silencing 1 (ASC/TMS1), Caspase-1, IL-1β, IL-18 and tumor necrosis factor-α (TNF-α) were significantly increased in the dorsal hippocampus of ECI mice. These results indicate that development of PSCI after embolic cerebral infarction is due to a decrease in AMPA receptor subunit GluR1 at Ser831 and Ser845 through the inflammasome activation pathway in the dorsal hippocampus.

Electrophysiological Assessment of Newly Synthesized 2,3-Benzodiazepine Derivatives for Inhibiting the AMPA Receptor Channel.

Three major subtypes of ionotropic receptors regulate glutamatergic synaptic transmission, one of which is α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPARs). They are tetrameric, cation-permeable ionotropic glutamate receptors found across the brain. Abnormalities in AMPA receptor trafficking and synaptic assembly are linked to cognitive decline and neurological diseases such as Alzheimer's, Parkinson's, and Huntington's. The present study will investigate the effects of four novel 2,3-benzodiazepine derivatives on AMPA receptor subunits by comparing their effects on synaptic responses, desensitization, and deactivation rate in human embryonic kidney cells (HEK293T) recombinant AMPAR subunits using whole-cell patch-clamp electrophysiology. All four 2,3-BDZ compounds showed inhibitory activity against all the homomeric and heteromeric subunits tested. While the desensitization and deactivation rates in 2,3-BDZ-1 and 2,3-BDZ-2 decreased and increased, respectively, in the other two compounds (i.e., 2,3-BDZ-3 and 2,3-BDZ-4), there was no change in the desensitization or deactivation rates. These results contribute to a better understanding of AMPARs by identifying potential 2,3-BDZ drugs that demonstrate inhibitory effects on the AMPAR subunits.

Optogenetic stimulation shapes dendritic trees of infragranular cortical pyramidal cells.

Spontaneous or experimentally evoked activity can lead to changes in length and/or branching of neocortical pyramidal cell dendrites. For instance, an early postnatal overexpression of certain AMPA or kainate glutamate receptor subunits leads to larger amplitudes of depolarizing events driven by spontaneous activity, and this increases apical dendritic complexity. Whether stimulation frequency has a role is less clear. In this study, we report that the expression of channelrhodopsin2-eYFP was followed by a 5-day optogenetic stimulation from DIV 5-10 or 11-15 in organotypic cultures of rat visual cortex-evoked dendritic remodeling. Stimulation at 0.05 Hz, at a frequency range of spontaneous calcium oscillations known to occur in the early postnatal neocortex in vivo until eye opening, had no effect. Stimulation with 0.5 Hz, a frequency at which the cortex in vivo adopts after eye opening, unexpectedly caused shorter and somewhat less branched apical dendrites of infragranular pyramidal neurons. The outcome resembles the remodeling of corticothalamic and callosal projection neurons of layers VI and V, which in the adult have apical dendrites no longer terminating in layer I. Exposure to 2.5 Hz, a frequency not occurring naturally during the time windows, evoked dendritic damage. The results suggested that optogenetic stimulation at a biologically meaningful frequency for the selected developmental stage can influence dendrite growth, but contrary to expectation, the optogenetic stimulation decreased dendritic growth.

Heroin Self-Administration and Extinction Increase Prelimbic Cortical Astrocyte-Synapse Proximity and Alter Dendritic Spine Morphometrics That Are Reversed by N-Acetylcysteine.

Clinical and preclinical studies indicate that adaptations in corticostriatal neurotransmission significantly contribute to heroin relapse vulnerability. In animal models, heroin self-administration and extinction produce cellular adaptations in both neurons and astrocytes within the nucleus accumbens (NA) core that are required for cue-induced heroin seeking. Specifically, decreased glutamate clearance and reduced association of perisynaptic astrocytic processes with NAcore synapses allow glutamate release from prelimbic (PrL) cortical terminals to engage synaptic and structural plasticity in NAcore medium spiny neurons. Normalizing astrocyte glutamate homeostasis with drugs like the antioxidant N-acetylcysteine (NAC) prevents cue-induced heroin seeking. Surprisingly, little is known about heroin-induced alterations in astrocytes or pyramidal neurons projecting to the NAcore in the PrL cortex (PrL-NAcore). Here, we observe functional adaptations in the PrL cortical astrocyte following heroin self-administration (SA) and extinction as measured by the electrophysiologically evoked plasmalemmal glutamate transporter 1 (GLT-1)-dependent current. We likewise observed the increased complexity of the glial fibrillary acidic protein (GFAP) cytoskeletal arbor and increased association of the astrocytic plasma membrane with synaptic markers following heroin SA and extinction training in the PrL cortex. Repeated treatment with NAC during extinction reversed both the enhanced astrocytic complexity and synaptic association. In PrL-NAcore neurons, heroin SA and extinction decreased the apical tuft dendritic spine density and enlarged dendritic spine head diameter in male Sprague-Dawley rats. Repeated NAC treatment during extinction prevented decreases in spine density but not dendritic spine head expansion. Moreover, heroin SA and extinction increased the co-registry of the GluA1 subunit of AMPA receptors in both the dendrite shaft and spine heads of PrL-NAcore neurons. Interestingly, the accumulation of GluA1 immunoreactivity in spine heads was further potentiated by NAC treatment during extinction. Finally, we show that the NAC treatment and elimination of thrombospondin 2 (TSP-2) block cue-induced heroin relapse. Taken together, our data reveal circuit-level adaptations in cortical dendritic spine morphology potentially linked to heroin-induced alterations in astrocyte complexity and association at the synapses. Additionally, these data demonstrate that NAC reverses PrL cortical heroin SA-and-extinction-induced adaptations in both astrocytes and corticostriatal neurons.

Schedule-induced alcohol intake during adolescence sex dependently impairs hippocampal synaptic plasticity and spatial memory

In a previous study, we demonstrated that intermittent ethanol administration in male adolescent animals impaired hippocampus-dependent spatial memory, particularly under conditions of excessive ethanol administration. In this current study, we subjected adolescent male and female Wistar rats an alcohol schedule-induced drinking (SID) procedure to obtain an elevated rate of alcohol self-administration and assessed their hippocampus-dependent spatial memory. We also studied hippocampal synaptic transmission and plasticity, as well as the expression levels of several genes involved in these mechanisms. Both male and female rats exhibited similar drinking patterns throughout the sessions of the SID protocol reaching similar blood alcohol levels in all the groups. However, only male rats that consumed alcohol showed spatial memory deficits which correlated with inhibition of hippocampal synaptic plasticity as long-term potentiation. In contrast, alcohol did not modify hippocampal gene expression of AMPA and NMDA glutamate receptor subunits, although there are differences in the expression levels of several genes relevant to synaptic plasticity mechanisms underlying learning and memory processes, related to alcohol consumption as Ephb2, sex differences as Pi3k or the interaction of both factors such as Pten.In conclusion, elevated alcohol intake during adolescence seems to have a negative impact on spatial memory and hippocampal synaptic plasticity in a sex dependent manner, even both sexes exhibit similar blood alcohol concentrations and drinking patterns.

Ubiquitination of the GluA1 Subunit of AMPA Receptors Is Required for Synaptic Plasticity, Memory, and Cognitive Flexibility.

Activity-dependent changes in the number of AMPA-type glutamate receptors (AMPARs) at the synapse underpin the expression of LTP and LTD, cellular correlates of learning and memory. Post-translational ubiquitination has emerged as a key regulator of the trafficking and surface expression of AMPARs, with ubiquitination of the GluA1 subunit at Lys-868 controlling the post-endocytic sorting of the receptors into the late endosome for degradation, thereby regulating their stability at synapses. However, the physiological significance of GluA1 ubiquitination remains unknown. In this study, we generated mice with a knock-in mutation in the major GluA1 ubiquitination site (K868R) to investigate the role of GluA1 ubiquitination in synaptic plasticity, learning, and memory. Our results reveal that these male mice have normal basal synaptic transmission but exhibit enhanced LTP and deficits in LTD. They also display deficits in short-term spatial memory and cognitive flexibility. These findings underscore the critical roles of GluA1 ubiquitination in bidirectional synaptic plasticity and cognition in male mice.SIGNIFICANCE STATEMENT Subcellular targeting and membrane trafficking determine the precise number of AMPA-type glutamate receptors at synapses, processes that are essential for synaptic plasticity, learning, and memory. Post-translational ubiquitination of the GluA1 subunit marks AMPARs for degradation, but its functional role in vivo remains unknown. Here we demonstrate that the GluA1 ubiquitin-deficient mice exhibit an altered threshold for synaptic plasticity accompanied by deficits in short-term memory and cognitive flexibility. Our findings suggest that activity-dependent ubiquitination of GluA1 fine-tunes the optimal number of synaptic AMPARs required for bidirectional synaptic plasticity and cognition in male mice. Given that increases in amyloid-β cause excessive ubiquitination of GluA1, inhibiting that GluA1 ubiquitination may have the potential to ameliorate amyloid-β-induced synaptic depression in Alzheimer's disease.

The excitatory transmission from basolateral nuclues of amygdala to nucleus accumbens shell regulates propofol self-administration through AMPA receptors.

Propofol addictive properties have been demonstrated in humans and rats. The glutamatergic transmission from basolateral nucleus of amygdala (BLA) to the nucleus accumbens (NAc) modulates reward-seeking behaviour; especially, NAc shell (NAsh) is implicated in reward-seeking response. Previous studies indicated the interactions between AMPA receptors (AMPARs) and dopamine D1 receptor (D1R) in NAc mediated drug addiction, but whether the circuit of BLA-to-NAsh and AMPARs regulate propofol addiction remains unclear. We trained adult male Sprague-Dawley rats for propofol self-administration to examine the changes of action potentials (APs) and spontaneous excitatory postsynaptic currents (sEPSCs) in the NAsh. Thereafter, optogenetic stimulation with adeno-associated viral vectors microinjections in BLA was used to explore the effect of BLA-to-NAsh on propofol self-administration behaviour (1.7mg/kg/injection). The pretreatment effects with NBQX (0.25-1.0μg/0.3μl/site) or vehicle in the NAsh on propofol self-administration behaviour, the expressions of AMPARs subunits and D1R/ERK/CREB signalling pathway in the NAc were detected. The results showed that the number of APs, amplitude and frequency of sEPSCs were enhanced in propofol self-administrated rats. Propofol self-administration was inhibited in the NpHR3.0-EYFP group, but in the ChR2-EYFP group, there was a promoting effect, which could be weakened by NBQX pretreatment. NBQX pretreatment also significantly decreased the expressions of GluA2 subunit and D1R in the NAc but did not change the expressions of GluA1 and ERK/CREB signalling pathway. The evidence supports a vital role of BLA-to-NAsh circuit in regulating propofol self-administration and suggests this central reward processing may function through the interaction between AMPARs and D1R in the NAsh.

GPER1 deficiency causes sex-specific dysregulation of hippocampal plasticity and cognitive function.

Estrogens regulate synaptic properties and influence hippocampus-related learning and memory via estrogen receptors, which include the G-protein-coupled estrogen receptor 1 (GPER1). Studying mice, in which the GPER1 gene is dysfunctional (GPER1-KO), we here provide evidence for sex-specific roles of GPER1 in these processes. GPER1-KO males showed reduced anxiety in the elevated plus maze, whereas the fear response ('freezing') was specifically increased in GPER1-KO females in a contextual fear conditioning paradigm. In the Morris water maze, spatial learning and memory consolidation was impaired by GPER1 deficiency in both sexes. Notably, in the females, spatial learning deficits and the fear response were more pronounced if mice were in a stage of the estrous cycle, in which E2 serum levels are high (proestrus) or rising (diestrus). On the physiological level, excitability at Schaffer collateral synapses in CA1 increased in GPER1-deficient males and in proestrus/diestrus ('E2 high') females, concordant with an increased hippocampal expression of the AMPA-receptor subunit GluA1 in GPER1-KO males and females as compared to wildtype males. Further changes included an augmented early long-term potentiation (E-LTP) maintenance specifically in GPER1-KO females and an increased hippocampal expression of spinophilin in metestrus/estrus ('E2 low') GPER1-KO females. Our findings suggest modulatory and sex-specific functions of GPER1 in the hippocampal network, which reduce rather than increase neuronal excitability. Dysregulation of these functions may underlie sex-specific cognitive deficits or mood disorders.

Ketamine’s rapid antidepressant effects are mediated by Ca2+-permeable AMPA receptors

Ketamine is shown to enhance excitatory synaptic drive in multiple brain areas, which is presumed to underlie its rapid antidepressant effects. Moreover, ketamine’s therapeutic actions are likely mediated by enhancing neuronal Ca2+ signaling. However, ketamine is a noncompetitive NMDA receptor (NMDAR) antagonist that reduces excitatory synaptic transmission and postsynaptic Ca2+ signaling. Thus, it is a puzzling question how ketamine enhances glutamatergic and Ca2+ activity in neurons to induce rapid antidepressant effects while blocking NMDARs in the hippocampus. Here, we find that ketamine treatment in cultured mouse hippocampal neurons significantly reduces Ca2+ and calcineurin activity to elevate AMPA receptor (AMPAR) subunit GluA1 phosphorylation. This phosphorylation ultimately leads to the expression of Ca2+-Permeable, GluA2-lacking, and GluA1-containing AMPARs (CP-AMPARs). The ketamine-induced expression of CP-AMPARs enhances glutamatergic activity and glutamate receptor plasticity in cultured hippocampal neurons. Moreover, when a sub-anesthetic dose of ketamine is given to mice, it increases synaptic GluA1 levels, but not GluA2, and GluA1 phosphorylation in the hippocampus within 1 hr after treatment. These changes are likely mediated by ketamine-induced reduction of calcineurin activity in the hippocampus. Using the open field and tail suspension tests, we demonstrate that a low dose of ketamine rapidly reduces anxiety-like and depression-like behaviors in both male and female mice. However, when in vivo treatment of a CP-AMPAR antagonist abolishes the ketamine’s effects on animals’ behaviors. We thus discover that ketamine at the low dose promotes the expression of CP-AMPARs via reduction of calcineurin activity, which in turn enhances synaptic strength to induce rapid antidepressant actions.

The effect of Lavandula Coronopifolia essential oil on the biophysical properties of desensitization and deactivation gating currents in ionotropic receptors

The rising incidence of cancer and the lack of effective therapeutic interventions for many neurological illnesses like Alzheimer's and epilepsy has prompted us to investigate the composition and effects of the Lavandula coronopifolia oil from Palestine on cancer cells and AMPA receptor subunits in the brain due to the vast range of beneficial properties of Lavandula coronopifolia essential oil (EO). GC/MS was used to analyze L. coronopifolia's EO chemistry. EO's cytotoxicity and biophysical effects on AMPA receptors were investigated using MTS and electrophysiological techniques. The GC–MS results revealed that L. coronopifolia EO has a high content of eucalyptol (77.23%), β-pinene (6.93%), and α-pinene (4.95%). The EO showed more significant antiproliferative selectivity activities against HepG2 cancer cell lines than HEK293T cell lines with IC50 values of 58.51 and 133.22 µg/mL, respectively. The EO of L. coronopifolia affected AMPA receptor kinetics (desensitization and deactivation) and preferred homomeric GluA1 and heteromeric GluA1/A2 receptors. These findings indicate the potential therapeutic use of L. coronopifolia EO in the selective treatment of HepG2 cancer cell lines and neurodegenerative diseases.

ICA69 regulates activity-dependent synaptic strengthening and learning and memory.

Long-term potentiation (LTP) is one of the major cellular mechanisms for learning and memory. Activity-dependent increases in surface AMPA receptors (AMPARs) are important for enhanced synaptic efficacy during LTP. Here, we report a novel function of a secretory trafficking protein, ICA69, in AMPAR trafficking, synaptic plasticity, and animal cognition. ICA69 is first identified as a diabetes-associated protein well characterized for its function in the biogenesis of secretory vesicles and trafficking of insulin from ER, Golgi to post-Golgi in pancreatic beta cells. In the brain, ICA69 is found in the AMPAR protein complex through its interaction with PICK1, which binds directly to GluA2 or GluA3 AMPAR subunits. Here, we showed that ICA69 regulates PICK1's distribution in neurons and stability in the mouse hippocampus, which in turn can impact AMPAR function in the brain. Biochemical analysis of postsynaptic density (PSD) proteins from hippocampi of mice lacking ICA69 (Ica1 knockout) and their wild-type littermates revealed comparable AMPAR protein levels. Electrophysiological recording and morphological analysis of CA1 pyramidal neurons from Ica1 knockout also showed normal AMPAR-mediated currents and dendrite architecture, indicating that ICA69 does not regulate synaptic AMPAR function and neuron morphology at the basal state. However, genetic deletion of ICA69 in mice selectively impairs NMDA receptor (NMDAR)-dependent LTP but not LTD at Schaffer collateral to CA1 synapses, which correlates with behavioral deficits in tests of spatial and associative learning and memory. Together, we identified a critical and selective role of ICA69 in LTP, linking ICA69-mediated synaptic strengthening to hippocampus-dependent learning and memory.

Intermittent treatment with Apremilast, a phosphodiesterase-4 inhibitor, ameliorates Alzheimer's-like pathology and symptoms through multiple targeting actions in aged T2D rats.

Apremilast (Apre), a novel phosphodiesterase-4 (PDE4) inhibitor, has been shown to have anti-inflammatory, immunomodulator, neuroprotective and senolytic properties, therefore, Apre like other PDE4 inhibitors may be a promising candidate for treatment of Alzheimer's disease (AD). To evaluate the effectiveness of Apre on Alzheimer's like pathology and symptoms in an animal model. The effects of Apre and cilostazol, a reference drug, on the behavioral, biochemical, and pathological features of Alzheimer's disease induced by a high-fat/high-fructose diet combined with low-dose streptozotocin (HF/HFr/l-STZ) were investigated. Apre 5mg/kg IP/day for 3 consecutive days per week for 8weeks attenuated memory and learning deficits tested by novel object recognition, Morris water maze and passive avoidance tests. Apre treatment significantly decreased the number of degenerating cells, and abnormal suppression of gene expression of AMPA and NMDA receptor subunits in the cortex and hippocampus of the AD rat model compared to rats that received vehicle. A significant decrease in elevated levels of hippocampal amyloid beta, tau-positive cell count, cholinesterase activity, and hippocampal caspase-3, a biomarker of neurodegeneration, was also observed after treatment with Apre in AD rats compared to rats that received placebo. Furthermore, a significant decrease in pro-inflammatory cytokines, oxidative stress, insulin resistance and GSK-3 was demonstrated in AD aged rats treated by Apre. Our findings demonstrate that intermittent treatment with Apre can enhance cognitive function in HF/HFr/l-STZ rats which may be related to decreased pro-inflammatory cytokines, oxidative stress, insulin resistance and GSK-3β.

Fingolimod Administration Following Hypoxia Induced Neonatal Seizure Can Restore Impaired Long-term Potentiation and Memory Performance in Adult Rats

Neonatal seizures commonly caused by hypoxia can lead to long-term neurological outcomes. Early inflammation plays an important role in the pathology of these outcomes. Therefore, in the current study, we explored the long-term effects of Fingolimod (FTY720), an analog of sphingosine and potent sphingosine 1-phosphate (S1P) receptors modulator, as an anti-inflammatory and neuroprotective agent in attenuating anxiety, memory impairment, and possible alterations in gene expression of hippocampal inhibitory and excitatory receptors following hypoxia-induced neonatal seizure (HINS). Seizure was induced in 24 male and female pups (6 in each experimental group) at postnatal day 10 (P10) by premixed gas (5% oxygen/ 95% nitrogen) in a hypoxic chamber for 15 minutes. Sixty minutes after the onset of hypoxia, FTY720 (0.3 mg/kg) or saline (100 µl) was administered for 12 days (from P10 up to P21). Anxiety-like behavior and hippocampal memory function were assessed at P90 by elevated plus maze (EPM) and novel object recognition (NOR), respectively. Long-term potentiation (LTP) was recorded from hippocampal dentate gyrus region (DG) following stimulation of perforant pathway (PP). In addition, the hippocampal concentration of superoxide dismutase activity (SOD), malondialdehyde (MDA), and thiol as indices of oxidative stress were evaluated. Finally, the gene expression of NR2A subunit of N-Methyl-D-aspartic acid (NMDA) receptor, GluR2 subunit of (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) AMPA receptor and γ2 subunit of γ-Aminobutyric acid (GABAA) receptor were assessed at P90 by the quantitative real-time PCR. FTY720 significantly reduced later-life anxiety-like behavior, ameliorated object recognition memory and increased the amplitude and slope of the field excitatory postsynaptic potential (fEPSP) in the rats following HINS. These effects were associated with restoration of the hippocampal thiol content to the normal values and the regulatory role of FTY720 in the expression of hippocampal GABA and glutamate receptors subunits. In conclusion, FTY720 could restore the dysregulated gene expression of excitatory and inhibitory receptors. It also increased the reduced hippocampal thiol content, which was accompanied with attenuation of HINS-induced anxiety, reduced the impaired hippocampal related memory, and prevented hippocampal LTP deficits in later life following HINS.

A maternal high-fat diet during pregnancy and lactation disrupts short-term memory functions via altered hippocampal glutamatergic signaling in female rat offspring

A maternal high-fat diet (HFD) provokes changes in the offspring's brain's structure, function, and development. These changes may cause neuropsychiatric disorders in the early life of offspring the basis of which may be memory impairment. In this study, the effects of maternal HFD during pregnancy and lactation on the short-term memory in adolescent and young adult offspring were evaluated. We analyzed the expression of genes encoding the glutamatergic transporters in the hippocampus to verify the association between changes in glutamatergic transporters and behavioral changes in offspring. Next, we examined whether maternal diet-induced changes in the mRNA levels of genes encoding the NMDA receptor subunits and the AMPA receptor subunits, as well as BDNF in this structure in offspring. All significant changes were validated at the protein level. We found that a maternal HFD during pregnancy and lactation disrupts short-term memory in adolescent and young adult females. The latter change is likely related to the dysregulation of hippocampal levels of GluN2B subunit of NMDA receptors and of reduced levels of BDNF. In summary, we showed that a maternal HFD during pregnancy and lactation triggered several changes within the glutamatergic system in the hippocampus of rat offspring, which may be related to producing behavioral changes in offspring.

Alterations in the properties of the glutamatergic system of the rat hippocampus in a lithium-pilocarpine model of temporal lobe epilepsy

Status epilepticus (SE) triggers many pathological changes in the nervous system that are not yet fully understood and may lead to the development of epilepsy. In this work, we studied the effects of SE on the properties of excitatory glutamatergic transmission in the hippocampus in a rat model of lithium-pilocarpine temporal lobe epilepsy. Studies were performed 1 day (acute phase of the model), 3 and 7 days (latent phase), and 30 to 80 days (chronic phase) after SE. Using real-time PCR, we found that in the latent phase there is a decrease in gene expression of GluA1 and GluA2 AMPA receptor subunits, which may also be accompanied by an increased proportion of calcium-permeable AMPA receptors, which play an essential role in the pathogenesis of many CNS diseases. In acute brain slices we found a decrease in the efficiency of excitatory synaptic neurotransmission in all phases of the model when recording field responses in the CA1 region of the hippocampus in response to stimulation of Schaffer collaterals by electric currents of different intensities. However, in the chronic phase we found an increase in the frequency of spontaneous excitatory postsynaptic potentials, indicating an increased background activity of the glutamatergic system in epilepsy. This is also supported by a decrease in the threshold of hind limb extension in the test of maximal electroshock seizure in rats with temporal lobe epilepsy compared to control animals. The results obtained indicate the presence of a number of functional alterations in the glutamatergic system related to epilepsy. These findings can be used to develop antiepileptogenic therapy.

Sex differences in glutamate AMPA receptor subunits mRNA with fast gating kinetics in the mouse cochlea.

Evidence shows that females have increased supra-threshold peripheral auditory processing compared to males. This is indicated by larger auditory brainstem responses (ABR) wave I amplitude, which measures afferent spiral ganglion neuron (SGN)-auditory nerve synchrony. However, the underlying molecular mechanisms of this sex difference are mostly unknown. We sought to elucidate sex differences in ABR wave I amplitude by examining molecular markers known to affect synaptic transmission kinetics. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) mediate fast excitatory transmission in mature SGN afferent synapses. Each AMPAR channel is a tetramer composed of GluA2, 3, and 4 subunits (Gria2, 3, and 4 genes), and those lacking GluA2 subunits have larger currents, are calcium-permeable, and have faster gating kinetics. Moreover, alternatively spliced flip and flop isoforms of each AMPAR subunit affect channel kinetics, having faster kinetics those AMPARs containing Gria3 and Gria4 flop isoforms. We hypothesized that SGNs of females have more fast-gating AMPAR subunit mRNA than males, which could contribute to more temporally precise synaptic transmission and increased SGN synchrony. Our data show that the index of Gria3 relative to Gria2 transcripts on SGN was higher in females than males (females: 48%; males: 43%), suggesting that females have more SGNs with higher Gria3 mRNA relative to Gria2. Analysis of the relative abundance of the flip and flop alternatively spliced isoforms showed that females have a 2-fold increase in fast-gating Gria3 flop mRNA, while males have more slow-gating (2.5-fold) of the flip. We propose that Gria3 may in part mediate greater SGN synchrony in females. Significance Statement: Females of multiple vertebrate species, including fish and mammals, have been reported to have enhanced sound-evoked synchrony of afferents in the auditory nerve. However, the underlying molecular mediators of this physiologic sex difference are unknown. Elucidating potential molecular mechanisms related to sex differences in auditory processing is important for maintaining healthy ears and developing potential treatments for hearing loss in both sexes. This study found that females have a 2-fold increase in Gria3 flop mRNA, a fast-gating AMPA-type glutamate receptor subunit. This difference may contribute to greater neural synchrony in the auditory nerve of female mice compared to males, and this sex difference may be conserved in all vertebrates.

Impaired AMPARs Translocation into Dendritic Spines with Motor Skill Learning in the Fragile X Mouse Model.

Motor skill learning induces changes in synaptic structure and function in the primary motor cortex (M1). In the fragile X syndrome (FXS) mouse model an impairment in motor skill learning and associated formation of new dendritic spines was previously reported. However, whether modulation of synaptic strength through trafficking of AMPA receptors (AMPARs) with motor skill training is impaired in FXS is not known. Here, we performed in vivo imaging of a tagged AMPA receptor subunit, GluA2, in layer (L)2/3 neurons in the primary motor cortex of wild-type (WT) and Fmr1 knock-out (KO) male mice at different stages of learning a single forelimb-reaching task. Surprisingly, in the Fmr1 KO mice, despite impairments in learning there was no deficit in motor skill training-induced spine formation. However, the gradual accumulation of GluA2 in WT stable spines, which persists after training is completed and past the phase of spine number normalization, is absent in the Fmr1 KO mouse. These results demonstrate that motor skill learning not only reorganizes circuits through formation of new synapses, but also strengthens existing synapses through accumulation of AMPA receptors and GluA2 changes are better associated with learning than new spine formation.

Alterations in the Properties of the Rat Hippocampus Glutamatergic System in the Lithium-Pilocarpine Model of Temporal Lobe Epilepsy

Status epilepticus (SE) triggers many not yet fully understood pathological changes in the nervous system that can lead to the development of epilepsy. In this work, we studied the effects of SE on the properties of excitatory glutamatergic transmission in the hippocampus in the lithium-pilocarpine model of temporal lobe epilepsy in rats. The studies were performed 1 day (acute phase), 3 and 7 days (latent phase), and 30 to 80 days (chronic phase) after SE. According to RT-qPCR data, expression of the genes coding for the AMPA receptor subunits GluA1 and GluA2 was downregulated in the latent phase, which may lead to the increased proportion of calcium-permeable AMPA receptors that play an essential role in the pathogenesis of many CNS diseases. The efficiency of excitatory synaptic neurotransmission in acute brain slices was decreased in all phases of the model, as determined by recording field responses in the CA1 region of the hippocampus in response to the stimulation of Schaffer collaterals by electric current of different strengths. However, the frequency of spontaneous excitatory postsynaptic potentials increased in the chronic phase, indicating an increased background activity of the glutamatergic system in epilepsy. This was also evidenced by a decrease in the threshold current causing hindlimb extension in the maximal electroshock seizure threshold test in rats with temporal lobe epilepsy compared to the control animals. The results suggest a series of functional changes in the properties of glutamatergic system associated with the epilepsy development and can be used to develop the antiepileptogenic therapy.

Regulation of AMPA receptor on propofol induced hippocampal mitochondrial injury in neonatal rats

To investigate whether propofol can cause injury to hippocampal mitochondria in neonatal rats and the regulation of excitatory amino acid receptor AMPA receptor. Forty-eight Sprague-Dawley (SD) rats aged 7 days were randomly divided into control group, propofol group, propofol+AMPA receptor agonist AMPA group (propofol+AMPA group) and propofol+AMPA receptor inhibitor CNQX group (propofol+CNQX group), with 12 rats in each group. The rats in the propofol groups were intraperitoneally injected with 30 mg/kg propofol, while in control group with 3 mg/kg normal saline. Each group was given 1/2 of the first dose every 20 minutes after the first administration, three times a day, for three consecutive days. The rats in the propofol+AMPA group and the propofol+CNQX group were injected with 1 g/L AMPA or CNQX 5 μL through left ventricle after the first administration. Three days after administration, the rats were sacrificed to obtain brain tissue. Western blotting was used to determine the expression of AMPA receptor glutamate receptors (GluR1, GluR2) subunit totally (T) and on membrane (M) in hippocampus. The expression of dynamin-related protein-1 (DRP-1) and phosphorylated-DRP-1 (p-DRP-1) and mitofusin 2 (Mfn2) related to mitochondrial fission and fusion were determined. The adenosine triphosphate (ATP) content and ATPase activity were determined. Compared with the control group, GluR1 expression and its M/T ratio were significantly increased after treatment of propofol, GluR2 expression and its M/T ratio were significantly decreased, the ATP content and ATP-related enzyme activity were decreased significantly, while the expression of DRP-1 and its phosphorylation was significantly increased, and the expression of Mfn2 was significantly decreased. The changes indicated that repeated intraperitoneal injection of 30 mg/kg propofol leading to the injury of mitochondria in neural cells. Compared with the propofol group, the GluR1 expression and its M/T ratio further increased after AMPA agonist administration [T-GluR1 protein (T-GluR1/β-actin): 2.41±0.29 vs. 1.72±0.11, M-GluR1 protein (M-GluR1/β-actin): 1.18±0.15 vs. 0.79±0.09, M/T ratio: 0.78±0.12 vs. 0.46±0.08, all P < 0.01], GluR2 expression was significantly increased [T-GluR2 protein (T-GluR2/β-actin): 0.65±0.13 vs. 0.30±0.14, P < 0.01; M-GluR2 protein (M-GluR2/β-actin): 0.17±0.05 vs. 0.13±0.07, P > 0.05], but its M/T ratio was further decreased (0.27±0.10 vs. 0.41±0.08, P < 0.05). The ATP-related enzyme activity was further decreased, and the ATP content was further decreased (μmol/g: 0.32±0.07 vs. 0.70±0.10, P < 0.01). Mitochondria DRP-1 expression and its phosphorylation were further increased [DRP-1 protein (DRP-1/GAPDH): 2.75±0.36 vs. 1.70±0.19, p-DRP-1 protein (p-DRP-1/GAPDH): 0.99±0.14 vs. 0.76±0.15, both P < 0.05], and Mfn2 expression was further decreased (Mfn2/GAPDH: 0.23±0.12 vs. 0.54±0.12, P < 0.05). This indicated that the AMPA agonist increased the expression of the AMPA receptor GluR1 subunit on the cell membrane and shifted the GluR2 into the cell, thus increasing the mitochondrial injury caused by propofol. Compared with the propofol group, the GluR1 expression and its M/T ratio decreased significantly after AMPA inhibitor administration [T-GluR1 protein (T-GluR1/β-actin): 0.99±0.14 vs. 1.72±0.11, M-GluR1 protein (M-GluR1/β-actin): 0.21±0.07 vs. 0.79±0.09, M/T ratio: 0.21±0.07 vs. 0.46±0.08, all P < 0.01], the change of GluR2 expression was not significant, but its M/T ratio was significantly increased (0.59±0.09 vs. 0.41±0.08, P < 0.05). The ATP-related enzyme activity was increased significantly, and the ATP content was increased significantly (μmol/g: 0.87±0.12 vs. 0.70±0.10, P < 0.05). Mitochondria DRP-1 expression and its phosphorylation were significantly decreased [DRP-1 protein (DRP-1/GAPDH): 1.18±0.17 vs. 1.70±0.19, p-DRP-1 protein (p-DRP-1/GAPDH): 0.37±0.10 vs. 0.76±0.10, both P < 0.05], and Mfn2 expression was significantly increased (Mfn2/GAPDH: 0.78±0.10 vs. 0.54±0.12, P < 0.05). This indicated that AMPA inhibitor promoted the movement to the cell membrane of GluR2 subunits meanwhile inhibited the expression of GluR1 subunits, thus alleviating the injury of mitochondrial caused by propofol in the brain. Repeated intraperitoneal injection of 30 mg/kg propofol for 3 days can increase the expression of GluR1 subunits of AMPA receptor in 7-day neonatal rats hippocampus mainly distributing in the cell membrane, decrease the expression of GluR2 subunits moving into the cell, thus causing injury of mitochondrial function and dynamics, which can be aggravated by AMPA receptor agonist and alleviated by AMPA receptor inhibitors.

Investigating role of ASIC2 in synaptic and behavioral responses to drugs of abuse.

Drugs of abuse produce rearrangements at glutamatergic synapses thought to contribute to drug-reinforced behaviors. Acid-Sensing Ion Channels (ASICs) have been suggested to oppose these effects, largely due to observations in mice lacking the ASIC1A subunit. However, the ASIC2A and ASIC2B subunits are known to interact with ASIC1A, and their potential roles in drugs of abuse have not yet been investigated. Therefore, we tested the effects of disrupting ASIC2 subunits in mice exposed to drugs of abuse. We found conditioned place preference (CPP) to both cocaine and morphine were increased in Asic2 -/- mice, which is similar to what was observed in Asic1a -/- mice. Because nucleus accumbens core (NAcc) is an important site of ASIC1A action, we examined expression of ASIC2 subunits there. By western blot ASIC2A was readily detected in wild-type mice, while ASIC2B was not, suggesting ASIC2A is the predominant subunit in nucleus accumbens core. An adeno-associated virus vector (AAV) was used to drive recombinant ASIC2A expression in nucleus accumbens core of Asic2 -/- mice, resulting in near normal protein levels. Moreover, recombinant ASIC2A integrated with endogenous ASIC1A subunits to form functional channels in medium spiny neurons (MSNs). However, unlike ASIC1A, region-restricted restoration of ASIC2A in nucleus accumbens core was not sufficient to affect cocaine or morphine conditioned place preference, suggesting effects of ASIC2 differ from those of ASIC1A. Supporting this contrast, we found that AMPA receptor subunit composition and the ratio of AMPA receptor-mediated current to NMDA receptor-mediated current (AMPAR/NMDAR) were normal in Asic2 -/- mice and responded to cocaine withdrawal similarly to wild-type animals. However, disruption of ASIC2 significantly altered dendritic spine morphology, and these effects differed from those reported previously in mice lacking ASIC1A. We conclude that ASIC2 plays an important role in drug-reinforced behavior, and that its mechanisms of action may differ from ASIC1A.