GluA1 Levels Research Articles

Loss of SynDIG4/PRRT1 alters distribution of AMPA receptors in Rab4- and Rab11-positive endosomes and impairs basal AMPA receptor recycling.

The transmembrane protein Synapse Differentiation Induced Gene 4 (SynDIG4) functions as an auxiliary factor of AMPA receptors (AMPARs) and plays a critical role in excitatory synapse plasticity as well as hippocampal-dependent learning and memory. Mice lacking SynDIG4 have reduced surface expression of GluA1 and GluA2 and are impaired in single tetanus-induced long-term potentiation and NMDA receptor (NMDAR)-dependent long-term depression. These findings suggest that SynDIG4 may play an important role in regulating AMPAR distribution through intracellular trafficking mechanisms; however, the precise roles by which SynDIG4 governs AMPAR distribution remain unclear. In this study, we characterized the endocytosis and recycling of GluA1-containing AMPARs under basal conditions. We did not observe any change in baseline endocytosis; however, we did observe a significant decrease in recycling of GluA1-containing AMPARs in cultured hippocampal neurons from mice lacking SynDIG4. This resulted in a significant increase in the levels of internal GluA1 and GluA2, along with greater colocalization of these subunits with Rab4-positive recycling endosomes in hippocampal neurons lacking SynDIG4. Notably, the overlap between Rab4- and Rab11-positive vesicles was elevated in hippocampal neurons lacking SynDIG4, suggesting an impairment in the trafficking between Rab4 and Rab11 compartments. Furthermore, our findings revealed a reduction in surface GluA1 within synaptic regions of hippocampal neurons lacking SynDIG4. Collectively, these results indicate that SynDIG4 regulates the distribution of GluA1-containing AMPARs via the Rab4-dependent endosomal recycling pathway, thereby maintaining AMPAR levels at synaptic regions under baseline conditions. This regulatory function of SynDIG4 may contribute to the deficits in GluA1-dependent synaptic plasticity and impairment of hippocampal-dependent learning and memory behaviors observed in SynDIG4 deficient mice.

The effects and mechanisms of chai shao jie yu granules on chronic unpredictable mild stress (CUMS)-induced depressive rats based on network pharmacology.

Chai Shao Jie Yu Granules (CSJY) is a renowned and time-honored formula employed in clinical practice for the management of various conditions, notably depression. Depression, a prevalent psychiatric disorder, poses challenges with limited effective treatment options. Traditional herbal medicines have garnered increasing attention in the realm of combating depression, being perceived as safer alternatives to pharmacotherapy. To explore the effects and mechanisms of CSJY in chronic unpredictable mild stress (CUMS)-induced depression. Rat models of CUMS-induced depression were established, and the rats were randomly allocated into six groups: Control, CUMS, CUMS+Paroxetine (PX), CUMS+CSJY-L, CUMS+CSJY-M, and CUMS+CSJY-H. Throughout the study, the rats' body weight was monitored. Depression-related behaviors were assessed using the sucrose preference test (SPT) and open field test (OFT). High-performance liquid chromatography-mass spectrometry (HPLC-MS) measured monoamine neurotransmitters in the rat cortex and hippocampus. We measured adrenocorticotropic hormone (ACTH), corticosterone (CORT), and corticotropin-release hormone (CRH) levels in rat serum. Additionally, network pharmacology was employed to predict relevant molecular targets and potential mechanisms, followed by in vivo validation. Western blot analysis was conducted to evaluate the protein levels of 5-hydroxytryptamine/serotonin receptor 1A (5-HT1A) and Glutamate (Glu)-related proteins, such as p-GluA1, GluA1, p-GluN1, GluN1, p-GluN2A and GluN2A in the hippocampus. In behavioral assessments, CUMS rats exhibited depressive behaviors, which were ameliorated by CSJY or PX treatment. Moreover, CSJY or PX treatment increased serotonin (5-HT) levels. It reduced the kynurenine/tryptophan (KYN/TRP) and gamma-aminobutyric acid/glutamate (GABA/Glu) in the hippocampus and cortex, as well as reduced serum levels of ACTH, CORT and CRH. Furthermore, CSJY or PX administration enhanced the decreased expression of p-GluN1/GluN1 while upregulating 5-HT1A and p-GluA1/GluA1 levels in the CUMS group. CSJY demonstrated the ability to alleviate depressive behaviors in CUMS-induced depression rats, potentially through the inhibition of the hypothalamic-pituitary-adrenal (HPA) axis, modulation of monoamine neurotransmitters, and glutamatergic neurons. These findings suggest that CSJY could serve as a promising treatment option for depression.

Hyperfunction of AMPA receptors in the amygdala and hippocampus contributes to enhanced fear memory in diabetic mice

Patients with diabetes mellitus show an elevated prevalence of psychiatric disorders such as anxiety. We have reported that fear memory, a model related to anxiety as reflected in the freezing response, is enhanced in diabetic mice and was ameliorated by an AMPA receptor antagonist. The present study investigated whether functions of AMPA receptors in the amygdala and hippocampus are altered in streptozotocin (STZ)-induced diabetic mice. While protein levels of the GluA1 subunit of AMPA receptors were not altered in the amygdala and hippocampus, protein levels of GluA1 phosphorylated at serine 845 in the amygdala and hippocampus and of GluA1 phosphorylated at serine 831 in the hippocampus were increased in STZ-induced diabetic mice. L-lactate, which is increased in the amygdala and hippocampus of STZ-induced diabetic mice, did not alter these protein levels in either brain area. In contrast, protein levels of phosphorylated protein kinase A (PKA) catalytic subunit and phosphorylated calcium calmodulin kinase II (CaMKII), which are known to phosphorylate serine 845 and serine 831 of GluA1, respectively, were increased in the amygdala and hippocampus of STZ-induced diabetic mice. In the fear memory test, the PKA inhibitor H-89 injected before test sessions and the CaMKII inhibitor KN-62 injected before conditioning or test sessions each reduced the increase in freezing in STZ-induced diabetic mice. These results indicate that the functions of AMPA receptors in the amygdala and hippocampus are enhanced due to increased phosphorylation by PKA and CaMKII, which enhances fear memory in diabetic mice.

AMPA and NMDA Receptors in Hippocampus of Rats with Fluoride-Induced Cognitive Decline.

This experimental study was performed to evaluate the alterations in the expression of a few subunits composing glutamate AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and NMDA (N-methyl-D-aspartate) receptors in the hippocampal cells of Wistar rats in response to long-term fluoride (F-) exposure. The animals were given water with background 0.4 (control), 5, 20, and 50 ppm F- (as NaF) for 12 months. The cognitive capacities of rats were examined by novel object recognition (NOR), Y-maze test, and Morris water maze tests. RT-qPCR and Western blotting techniques were used to evaluate the expression of different AMPA and NMDA subunits at transcriptional and translational levels, respectively. Long-term F- poisoning disturbed the formation of hippocampus-dependent working spatial and long-term non-spatial memory. The expression of Gria1, Gria2, and Gria3 genes encoding different subunits of AMPA receptors were comparable in hippocampi of control and F--exposed animals, although the levels of both Grin2a and Grin2b mRNA increased. Long-term F- intake enhanced the ratio of phospho-GluA1/total-GluA1 proteins in subcellular fraction enriched with cytosolic proteins, while decreased content of GluA2 but elevated level of GluA3 were observed in subcellular fraction enriched with membrane proteins. Such changes were accompanied by increased phosphorylation of GluN2A and GluN2B subunits, higher ratios of GluN2A/GluN1 and GluN2B/GluN1 proteins in the cytosol, and GluN2A/GluN2B ratio in membranes. These changes indicate the predominance of Ca2+-permeable AMPARs in membranes and a shift between different NMDARs subunits in hippocampal cells of F--exposed rats, which is typical for neurodegeneration and can at least partially underly the observed disturbances in cognitive capacities of animals.

Prenatal ethanol exposure impairs hippocampal plasticity and cognition in adolescent mice

BackgroundPrenatal alcohol exposure (PAE) induces a wide range of neurodevelopmental disabilities that are grouped under the term ‘fetal alcohol spectrum disorders’ (FASD). The effects of PAE on brain development are dependent on complex neurochemical events, including modification of AMPA receptors (AMPARs). We have recently found that chronic ethanol (EtOH) exposure decreases AMPA-mediated neurotransmission and expression through the overexpression of the specific microRNA (miR)137 and 501-3p, which target GluA1 AMPA subunit, in the developing hippocampus in vitro. Here, we explored how PAE mice may alter AMPAergic synapses in the hippocampus, and its effects on behavior. MethodsTo model PAE, we exposed C57Bl/6 pregnant mice to 10 % EtOH during during the first 10 days of gestation (GD 0–10; equivalent to the first trimester of pregnancy in humans). AMPA subunits postsynaptic expression in the hippocampus, electrical properties of CA1 neurons, memory recognition, and locomotor functions were then analyzed in adolescent PAE-exposed offspring. ResultsPAE adolescent mice showed dysregulation of AMPAergic neurotransmission, and increased miR 501-3p expression, associated with a significant reduction of spontaneous AMPA currents and intrinsic somatic excitability. In addition, PAE reduced the phosphorylation of AMPAR-containing GluA1 subunit, despite an increase in its total levels. Of note, the total levels of GluA2 and GluA3 AMPA receptors were enhanced as well. Consistently, at behavioral level, PAE reduced object recognition without altering locomotor activity. ConclusionsOur study shows that PAE leads to dysfunctional formation of AMPAergic synapses that could be responsible for neurobehavioral impairments, contributing to the understanding of the pathogenesis of FASD.

BETA-HYDROXYBUTYRATE COUNTERACTS THE DELETERIOUS EFFECTS OF A SATURATED HIGH-FAT DIET ON SYNAPTIC AMPA RECEPTORS AND COGNITIVE PERFORMANCE.

The ketogenic diet, characterized by high fat and low carbohydrates, has gained popularity not only as a strategy for managing body weight but also for its efficacy in delaying cognitive decline associated with neurodegenerative diseases and the aging process. Since this dietary approach stimulates the liver's production of ketone bodies, primarily β-hydroxybutyrate (BHB), which serves as an alternative energy source for neurons, we investigated whether BHB could mitigate impaired AMPA receptor trafficking, synaptic dysfunction, and cognitive decline induced by metabolic challenges such as saturated fatty acids. Here, we observe that, in cultured primary cortical neurons, exposure to palmitic acid (200μM) decreased surface levels of glutamate GluA1-containing AMPA receptors, whereas unsaturated fatty acids, such as oleic acid and ω-3 docosahexaenoic acid (200μM), and BHB (5mM) increased them. Furthermore, BHB countered the adverse effects of palmitic acid on synaptic GluA1 levels in hippocampal neurons, as well as excitability and plasticity in hippocampal slices. Additionally, daily intragastric administration of BHB (100 mg/kg/day) for two months reversed cognitive impairment induced by a saturated high-fat diet (49% of calories from fat) in a mouse experimental model of obesity. In summary, our findings underscore the significant impact of fatty acids and ketone bodies on AMPA receptors abundance, synaptic function and neuroplasticity, shedding light on the potential use of BHB to delay cognitive impairments associated with metabolic diseases.

Effects of non-rapid eye movement sleep on the cortical synaptic expression of GluA1-containing AMPA receptors.

Converging electrophysiological, molecular and ultrastructural evidence supports the hypothesis that sleep promotes a net decrease in excitatory synaptic strength, counteracting the net synaptic potentiation caused by ongoing learning during waking. However, several outstanding questions about sleep-dependent synaptic weakening remain. Here, we address some of these questions by using two established molecular markers of synaptic strength, the levels of the AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors containing the GluA1 subunit and the phosphorylation of GluA1 at serine 845 (p-GluA1(845)). We previously found that, in the rat cortex and hippocampus, these markers are lower after 6-8h of sleep than after the same time spent awake. Here, we measure GluA1 and p-GluA1(845) levels in synaptosomes of mouse cortex after 5h of either sleep, sleep deprivation, recovery sleep after sleep deprivation or selective REM sleep deprivation (32 C57BL/B6 adult mice, 16 females). We find that relative to after sleep deprivation, these synaptic markers are lower after sleep independent of whether the mice were allowed to enter REM sleep. Moreover, 5h of recovery sleep following acute sleep deprivation is enough to renormalize their expression. Thus, the renormalization of GluA1 and p-GluA1(845) expression crucially relies on NREM sleep and can occur in a few hours of sleep after acute sleep deprivation.

Ketamine's Amelioration of Fear Extinction in Adolescent Male Mice Is Associated with the Activation of the Hippocampal Akt-mTOR-GluA1 Pathway.

Fear-related disorders, including post-traumatic stress disorder (PTSD), and anxiety disorders are pervasive psychiatric conditions marked by persistent fear, stemming from its dysregulated acquisition and extinction. The primary treatment for these disorders, exposure therapy (ET), relies heavily on fear extinction (FE) principles. Adolescence, a vulnerable period for developing psychiatric disorders, is characterized by neurobiological changes in the fear circuitry, leading to impaired FE and increased susceptibility to relapse following ET. Ketamine, known for relieving anxiety and reducing PTSD symptoms, influences fear-related learning processes and synaptic plasticity across the fear circuitry. Our study aimed to investigate the effects of ketamine (10 mg/kg) on FE in adolescent male C57 BL/6 mice at the behavioral and molecular levels. We analyzed the protein and gene expression of synaptic plasticity markers in the hippocampus (HPC) and prefrontal cortex (PFC) and sought to identify neural correlates associated with ketamine's effects on adolescent extinction learning. Ketamine ameliorated FE in the adolescent males, likely affecting the consolidation and/or recall of extinction memory. Ketamine also increased the Akt and mTOR activity and the GluA1 and GluN2A levels in the HPC and upregulated BDNF exon IV mRNA expression in the HPC and PFC of the fear-extinguished mice. Furthermore, ketamine increased the c-Fos expression in specific brain regions, including the ventral HPC (vHPC) and the left infralimbic ventromedial PFC (IL vmPFC). Providing a comprehensive exploration of ketamine's mechanisms in adolescent FE, our study suggests that ketamine's effects on FE in adolescent males are associated with the activation of hippocampal Akt-mTOR-GluA1 signaling, with the vHPC and the left IL vmPFC as the proposed neural correlates.

Enhancing Sleep Quantity and Quality with a Novel GABAergic Dietary Supplement

Purpose: The primary objective of this investigation was to evaluate the effects of BH_SLP_01 (Bonafide Health Sleep Product) on sleep quality and brain wave patterns using a caffeine-induced insomnia model, and on sleep duration and latency using a pentobarbital-induced sleep model. Methods: Five male BALB/c mice were allocated per treatment arm (age: 8 weeks, body mass: 180 ± 20 g). Treatment arms included control, caffeine or phenobarbital (active) control, and BH_SLP_01. Caffeine treatment model. To induce sleep disturbance, 7.5 mg/kg caffeine was injected intraperitoneally at the 15th minute (min) of recordings, and second injections (saline or combinations) were performed at the 30th minute. Brain electrical activity was monitored and recorded for a total of two hours using electrocorticography (ECoG) recording. Serum melatonin, serotonin, and dopamine were measured by ELISA. Brain levels of MDA were measured by HPLC, and protein concentrations were determined using Western Blot analysis. Phenobarbital treatment model Groups were administered saline or BH_SLP_01, and then, 45 min later, pentobarbital (42 mg/kg) was injected. After injection, sleep latency and duration were measured. Study treatments were compared using an ANOVA and Student's unpaired t-test. Significance levels were set at p<0.05. Results: Analysis of ECoG recording data revealed notable findings. In the caffeine-pretreated group, a significant increase in amplitude was observed during the 30-45 min interval, compared to control. Caffeine administration also significantly increased frequency at 75-90 and 90-105 min compared to control (p<0.05). Conversely, the group treated with BH_SLP_01 displayed no significant changes in amplitude and frequency, except for a significant change in frequency at 90-105 min compared to control (p<0.05). Both sleep duration and sleep latency were significantly improved for BH_SLP_01 compared to control and phenobarbital control (p<0.05). In addition, serotonin, dopamine, melatonin, GABAA R2, GABAB R1, GABAB R2, 5-HT1A, GluA1, GluN1, GluN2A, Bax, Bcl-2, and Caspase-3 levels were significantly improved by BH_SLP_01 compared to control and caffeine control (p<0.05). Conclusion: The results of these studies provide evidence for the effcacy of BH_SLP_01 to improve impaired sleep, leading to notable enhancements in markers of deep sleep. This conclusion is supported by the significant upregulation of GABA, glutamine receptors, and endogenous melatonin, all of which collectively contribute to the augmentation of both sleep quantity and quality. These findings underscore the potential of BH_SLP_01 as a promising therapeutic intervention for optimizing sleep parameters and promoting overall sleep wellness. This research was supported by Bonafide Health, LLC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Investigating the Effects of Perampanel on Autophagy-mediated Regulation of GluA2 and PSD95 in Epilepsy.

Epilepsy is a chronic neurological disorder characterized by recurrent seizures. Despite various treatment approaches, a significant number of patients continue to experience uncontrolled seizures, leading to refractory epilepsy. The emergence of novel anti-epileptic drugs, such as perampanel (PER), has provided promising options for effective epilepsy treatment. However, the specific mechanisms underlying the therapeutic effects of PER remain unclear. This study aimed to investigate the intrinsic molecular regulatory mechanisms involved in the downregulation of GluA2, a key subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, following epileptic seizures. Primary mouse hippocampal neurons were cultured and subjected to an epilepsy cell model. The expression levels of GluA2 and autophagy-related proteins were assessed using Western blotting and real-time fluorescent quantitative PCR. Immunofluorescence and immunohistochemistry techniques were employed to investigate the nuclear translocation of CREB-regulated transcriptional coactivator 1 (CRTC1). Additionally, status epilepticus animal models were established to further validate the findings. The epilepsy cell model exhibited a significant decrease in GluA2 expression, accompanied by elevated levels of autophagy-related proteins. Immunofluorescence analysis revealed the nuclear translocation of CRTC1, which correlated with the expression of autophagy-related genes. Treatment with an autophagy inhibitor reversed the decreased expression of GluA2 in the epilepsy cell model. Furthermore, the calcium/calmodulin-dependent protein phosphatase inhibitor FK506 and CaN overexpression affected the dephosphorylation and nuclear translocation of CRTC1, consequently influencing GluA2 expression. Animal model results further supported the involvement of these molecular mechanisms in epilepsy. Our findings suggest that the downregulation of GluA2 following epileptic seizures involves the activation of autophagy and the regulation of CRTC1 nuclear translocation. These intrinsic molecular regulatory mechanisms provide potential targets for developing novel therapeutic strategies to alleviate refractory epilepsy and preserve cognitive functions in patients.

RIPK1 activation in Mecp2-deficient microglia promotes inflammation and glutamate release in RTT

Rett syndrome (RTT) is a devastating neurodevelopmental disorder primarily caused by mutations in the methyl-CpG binding protein 2 (Mecp2) gene. Here, we found that inhibition of Receptor-Interacting Serine/Threonine-Protein Kinase 1 (RIPK1) kinase ameliorated progression of motor dysfunction after onset and prolonged the survival of Mecp2-null mice. Microglia were activated early in myeloid Mecp2-deficient mice, which was inhibited upon inactivation of RIPK1 kinase. RIPK1 inhibition in Mecp2-deficient microglia reduced oxidative stress, cytokines production and induction of SLC7A11, SLC38A1, and GLS, which mediate the release of glutamate. Mecp2-deficient microglia release high levels of glutamate to impair glutamate-mediated excitatory neurotransmission and promote increased levels of GluA1 and GluA2/3 proteins in vivo, which was reduced upon RIPK1 inhibition. Thus, activation of RIPK1 kinase in Mecp2-deficient microglia may be involved both in the onset and progression of RTT.

Tacrolimus Induces Pain by Upregulating the Synaptic Expression of AMPA Receptors in the Spinal Cord Dorsal Horn

To investigate the effects of long-term administration of tacrolimus (also known as FK506) on the pain-related behaviors in mice and to study the underlying mechanism of pain induced by FK506 via measuring the effect of FK506 on the synaptic expression and phosphorylation of alpha-amino-3-hyroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the spinal cord dorsal horn of mice. 1) A total of 24 mice were evenly and randomly assigned to two groups, a FK506 group and a Saline group. The FK506 group was given daily intraperitoneal injection of FK506 and the Saline group received normal saline. Both groups received injection once a day for 7 days in a row. Some of the mice ( n=6 in each group) were monitored for the changes in the paw withdrawal threshold (PWT), the paw withdrawal latency (PWL), and the spontaneous pain behaviors to establish the pain model. The other mice ( n=6 in each group) of each group underwent isolation of the dorsal horn when obvious pain symptoms were induced on day 7 of injection. Then, immunoblotting was performed to determine the synaptic expression and phosphorylation levels of GluA1 and GluA2 subunits of AMPA receptors. 2) The mice were randomly divided into two groups, FK506+calcineurin (CaN) group and FK506+Saline group ( n=6 in each group). After the pain model was constructed, the mice were given intrathecal injection of recombinant CaN (also know as 33 U) or normal saline. Then, 60 minutes later, the PWT and the PWL of the mice were measured to investigate the role of CaN in FK506-induced pain. 3) Another18 mice were selected. The mice were randomly and evenly assigned to three groups, a control group (receiving intraperitoneal injection of normal saline followed by intrathecal injection of normal saline), FK506+Saline group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of normal saline) and FK506+CaN group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of CaN). Then, 60 minutes later, the spinal cords were isolated and subjected to immunoblotting assay to determine the role of CaN in FK506-induced AMPA receptor modification. 1) After 7 consecutive days of intraperitoneal injection of FK506, the PWT and PWL of mice dropped significantly, reaching on day 7 as low as 22.3%±0.05% and 66.6%±0.05% of the control group, respectively ( P<0.01). The FK506-treated mice displayed evident spontaneous pain behavior, presenting significantly increased licking activities ( P<0.01). These results indicated that FK506-induced pain model was successfully established. Immunoblotting assay showed that the total expressions of GluA1 and GluA2 subunits in the spinal dorsal horn of the FK506 group remained unchanged in comparison with those of the Saline group. However, FK506 specifically induced an increase in the synaptic expression of GluA1. In addition, the phosphorylation levels of GluA1 at Ser845 and Ser831 in FK506-treated mice were significantly increased in comparison with those of the control group ( P<0.05). 2) Compared with those of the mice in the FK506+Saline group, the PWT and the PWL of mice in the FK506+CaN group were significantly increased ( P<0.05). 3) Compared with those of the FK506+Saline group, the synaptic expression of GluA1 were decreased in FK506+CaN group ( P<0.01) and the phosphorylation levels of GluA1 at Ser845 and Ser831 were significantly downregulated ( P<0.001). The hyper-expression and hyperphosphorylation of GluA1 subunit in the spinal cord dorsal horn resulting from CaN inhibition contributes to the FK506-induced pain syndrome. FK506 induces the synaptic hyper-expression and hyperphosphorylation of GluA1 in the dorsal horn of the spinal cord through CaN inhibition, thereby inducing pain.

TARPγ2 Is Required for Normal AMPA Receptor Expression and Function in Direction-Selective Circuits of the Mammalian Retina.

AMPA receptors (AMPARs) are the major mediators of fast excitatory neurotransmission in the retina as in other parts of the brain. In most neurons, the synaptic targeting, pharmacology, and function of AMPARs are influenced by auxiliary subunits including the transmembrane AMPA receptor regulatory proteins (TARPs). However, it is unclear which TARP subunits are present at retinal synapses and how they influence receptor localization and function. Here, we show that TARPɣ2 (stargazin) is associated with AMPARs in the synaptic layers of the mouse, rabbit, macaque, and human retina. In most species, TARPɣ2 expression was high where starburst amacrine cells (SACs) ramify and transcriptomic analyses suggest correspondingly high gene expression in mouse and human SACs. Synaptic expression of GluA2, GluA3, and GluA4 was significantly reduced in a mouse mutant lacking TARPɣ2 expression (stargazer mouse; stg), whereas GluA1 levels were unaffected. AMPAR-mediated light-evoked EPSCs in ON-SACs from stg mice were ∼30% smaller compared with heterozygous littermates. There was also loss of a transient ON pathway-driven GABAergic input to ON-SACs in stg mutants. Direction-selective ganglion cells in the stg mouse showed normal directional tuning, but their surround inhibition and thus spatial tuning was reduced. Our results indicate that TARPɣ2 is required for normal synaptic expression of GluA2, GluA3, and GluA4 in the inner retina. The presence of residual AMPAR expression in the stargazer mutant suggests that other TARP subunits may compensate in the absence of TARPɣ2.

Long-Term Treatment with Cannabidiol-Enriched Cannabis Extract Induces Synaptic Changes in the Adolescent Rat Hippocampus.

The endocannabinoid system (eCS) is widely distributed in mammalian tissues and it is classically formed by cannabinoid receptors, endogenous bioactive lipids and its synthesis and degradation enzymes. Due to the modulatory role of eCS in synaptic activity in the Central Nervous System (CNS), phytocannabinoids have been increasingly used for the treatment of neurological disorders, even though little is known in terms of the long-term effect of these treatments on CNS development, mainly in the timeframe that comprises childhood and adolescence. Furthermore, an increased number of clinical trials using full-spectrum Cannabis extracts has been seen, rather than the isolated form of phytocannabinoids, when exploring the therapeutical benefits of the Cannabis plant. Thus, this study aims to evaluate the effect of cannabidiol (CBD)-enriched Cannabis extract on synaptic components in the hippocampus of rats from adolescence to early adulthood (postnatal day 45 to 60). Oral treatment of healthy male Wistar rats with a CBD-enriched Cannabis extract (3 mg/kg/day CBD) during 15 days did not affect food intake and water balance. There was also no negative impact on locomotor behaviour and cognitive performance. However, the hippocampal protein levels of GluA1 and GFAP were reduced in animals treated with the extract, whilst PSD95 levels were increased, which suggests rearrangement of glutamatergic synapses and modulation of astrocytic features. Microglial complexity was reduced in CA1 and CA3 regions, but no alterations in their phagocytic activity have been identified by Iba-1 and LAMP2 co-localization. Collectively, our data suggest that CBD-enriched Cannabis treatment may be safe and well-tolerated in healthy subjects, besides acting as a neuroprotective agent against hippocampal alterations related to the pathogenesis of excitatory and astrogliosis-mediated disorders in CNS.

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 autism-associated loss of δ-catenin functions disrupts social behavior

δ-catenin is expressed in excitatory synapses and functions as an anchor for the glutamatergic AMPA receptor (AMPAR) GluA2 subunit in the postsynaptic density. The glycine 34 to serine (G34S) mutation in the δ-catenin gene has been found in autism spectrum disorder (ASD) patients and results in loss of δ-catenin functions at excitatory synapses, which is presumed to underlie ASD pathogenesis in humans. However, how the G34S mutation causes loss of δ-catenin functions to induce ASD remains unclear. Here, using neuroblastoma cells, we identify that the G34S mutation increases glycogen synthase kinase 3β (GSK3β)-dependent δ-catenin degradation to reduce δ-catenin levels, which likely contributes to the loss of δ-catenin functions. Synaptic δ-catenin and GluA2 levels in the cortex are significantly decreased in mice harboring the δ-catenin G34S mutation. The G34S mutation increases glutamatergic activity in cortical excitatory neurons while it is decreased in inhibitory interneurons, indicating changes in cellular excitation and inhibition. δ-catenin G34S mutant mice also exhibit social dysfunction, a common feature of ASD. Most importantly, pharmacological inhibition of GSK3β activity reverses the G34S-induced loss of δ-catenin function effects in cells and mice. Finally, using δ-catenin knockout mice, we confirm that δ-catenin is required for GSK3β inhibition-induced restoration of normal social behavior in δ-catenin G34S mutant animals. Taken together, we reveal that the loss of δ-catenin functions arising from the ASD-associated G34S mutation induces social dysfunction via alterations in glutamatergic activity and that GSK3β inhibition can reverse δ-catenin G34S-induced synaptic and behavioral deficits.

Deubiquitinase CYLD regulates excitatory synaptic transmission and short-term plasticity in the hippocampus

The fate of proteins is determined by the addition of various forms of polyubiquitin during ubiquitin-mediated proteasomal degradation. Cylindromatosis (CYLD), a K63-specific deubiquitinase, is enriched in postsynaptic density fractions of the rodent central nervous system (CNS), but the synaptic role of CYLD in the CNS is poorly understand. Here we show that CYLD deficiency (Cyld−/−) results in reduced intrinsic hippocampal neuronal firing, a decrease in the frequency of spontaneous excitatory postsynaptic currents and a decrease in the amplitude of field excitatory postsynaptic potentials. Moreover, Cyld−/− hippocampus shows downregulated levels of presynaptic vesicular glutamate transporter 1 (vGlut1) and upregulated levels of postsynaptic GluA1, a subunit of the AMPA receptor, together with an altered paired-pulse ratio (PPR). We also found increased activation of astrocytes and microglia in the hippocampus of Cyld−/− mice. The present study suggests a critical role for CYLD in mediating hippocampal neuronal and synaptic activity.

Neural mechanism underlies CYLD modulation of morphology and synaptic function of medium spiny neurons in dorsolateral striatum.

Although the deubiquitinase cylindromatosis (CYLD), an abundant protein in the postsynaptic density fraction, plays a crucial role in mediating the synaptic activity of the striatum, the precise molecular mechanism remains largely unclear. Here, using a Cyld-knockout mouse model, we demonstrate that CYLD regulates dorsolateral striatum (DLS) neuronal morphology, firing activity, excitatory synaptic transmission, and plasticity of striatal medium spiny neurons via, likely, interaction with glutamate receptor 1 (GluA1) and glutamate receptor 2 (GluA2), two key subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). CYLD deficiency reduces levels of GluA1 and GluA2 surface protein and increases K63-linked ubiquitination, resulting in functional impairments both in AMPAR-mediated excitatory postsynaptic currents and in AMPAR-dependent long-term depression. The results demonstrate a functional association of CYLD with AMPAR activity, which strengthens our understanding of the role of CYLD in striatal neuronal activity.

Methylmercury Decreases AMPA Receptor Subunit GluA2 Levels in Cultured Rat Cortical Neurons.

Methylmercury (MeHg) is a well-known environmental pollutant that has harmful effects on the central nervous systems of humans and animals. The molecular mechanisms of MeHg-induced neurotoxicity at low concentrations are not fully understood. Here, we investigated the effects of low-concentration MeHg on the cell viability, Ca2+ homeostasis, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA2 levels, which determine Ca2+ permeability of AMPA receptors, in rat primary cortical neurons. Exposure of cortical neurons to 100 and 300 nM MeHg for 7 d resulted in a decrease in GluA2 levels, an increase in basal intracellular Ca2+ concentration, increased phosphorylation levels of extracellular signal-regulated kinase (ERK)1/2 and p38, and decreased cell viability. Moreover, glutamate stimulation exacerbated the decrease in cell viability and increased intracellular Ca2+ levels in MeHg-treated neurons compared to control neurons. MeHg-induced neuronal cell death was ameliorated by 1-naphthyl acetyl spermine, a specific antagonist of Ca2+-permeable, GluA2-lacking AMPA receptors. Our findings raise the possibility that decreased neuronal GluA2 levels and the subsequent increase in intracellular Ca2+ concentration may contribute to MeHg-induced neurotoxicity.

Chronic Δ9-tetrahydrocannabinol impact on plasticity, and differential activation requirement for CB1-dependent long-term depression in ventral tegmental area GABA neurons in adult versus young mice.

The ventral tegmental area (VTA) mediates incentive salience and reward prediction error through dopamine (DA) neurons that are regulated by local VTA GABA neurons. In young mice, VTA GABA cells exhibit a form of synaptic plasticity known as long-term depression (LTD) that is dependent on cannabinoid 1 (CB1) receptors preceded by metabotropic glutamate receptor 5 (mGluR5) signaling to induce endocannabinoid production. This LTD was eliminated following chronic (7-10 consecutive days) exposure to the marijuana derived cannabinoid Δ9 -tetrahydrocannabinol (THC). We now examine the mechanism behind THC-induced elimination of LTD in adolescents as well as plasticity induction ability in adult versus young male and female mice using whole-cell electrophysiology experiments of VTA GABA cells. Chronic THC injections in adolescents resulted in a loss of CB1 agonist-mediated depression, illustrating chronic THC likely desensitizes or removes synaptic CB1. We noted that seven days withdrawal from chronic THC restored LTD and CB1 agonist-induced depression, suggesting reversibility of THC-induced changes. Adult mice continue to express functional mGluR5 and CB1, but require a doubling of the synaptic stimulation compared to young mice to induce LTD, suggesting a quantitative difference in CB1-dependent plasticity between young and adult mice. One potential rationale for this difference is changes in AMPA and NMDA glutamate receptors. Indeed, AMPA/NMDA ratios were increased in in adults compared to young mice. Lastly, we performed quantitative reverse-transcription PCR and identified that CB1, DAGLα, and GluA1 levels increased following chronic THC exposure. Collectively, our data demonstrate the first age-dependent GABA neuron plasticity in the VTA, which could have implications for decreased THC dependence capacity in adults, as well as the mechanism behind chronic THC-induced synaptic alterations in young mice.