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Alcohol hangover is defined as the combination of mental and physical symptoms experienced the day after a single episode of heavy drinking, starting when blood alcohol concentration approaches zero. We previously evidenced increments in free radical generation and an imbalance in antioxidant defences in non-synaptic mitochondria and synaptosomes during hangover. It is widely known that acute alcohol exposure induces changes in nitric oxide (NO) production and blocks the binding of glutamate to NMDAR in central nervous system. Our aim was to evaluate the residual effect of acute ethanol exposure (hangover) on NO metabolism and the role of NMDA receptor-PSD95-nNOS pathway in non-synaptic mitochondria and synaptosomes from mouse brain cortex. Results obtained for the synaptosomes fraction showed a 37% decrease in NO total content, a 36% decrease in NOS activity and a 19% decrease in nNOS protein expression. The in vitro addition of glutamate to synaptosomes produced a concentration-dependent enhancement of NO production which was significantly lower in samples from hangover mice than in controls for all the glutamate concentrations tested. A similar patter of response was observed for nNOS activity being decreased both in basal conditions and after glutamate addition. In addition, synaptosomes exhibited a 64% and 15% reduction in NMDA receptor subunit GluN2B and PSD-95 protein expression, respectively. Together with this, glutamate-induced calcium entry was significant decreased in synaptosomes from alcohol-treated mice. On the other hand, in non-synaptic mitochondria, no significant differences were observed in NO content, NOS activity or nNOS protein expression. The expression of iNOS remained unaltered in synaptosomes and non-synaptic mitochondria. Here we demonstrated that hangover effects on NO metabolism are strongly evidenced in synaptosomes probably due to a disruption in NMDAR/PSD-95/nNOS pathway.

Previous midlife estradiol treatment, like continuous treatment, improves memory and results in lasting increases in hippocampal levels of estrogen receptor (ER) α and ER-dependent transcription in ovariectomized rodents. We hypothesized that previous and continuous midlife estradiol act to specifically increase levels of nuclear ERα, resulting in transcriptional regulation of proteins that mediate estrogen effects on memory. Ovariectomized middle-aged rats received estradiol or vehicle capsule implants. After 40 days, rats initially receiving vehicle received another vehicle capsule (Vehicle). Rats initially receiving estradiol received either another estradiol (Continuous Estradiol) or a vehicle (Previous Estradiol) capsule. One month later, hippocampal genes and proteins were analyzed. Continuous and previous estradiol increased levels of nuclear, but not membrane or cytosolic ERα and had no effect on Esr1. Continuous and previous estradiol impacted gene expression and/or protein levels of mediators of estrogenic action on memory including ChAT, BDNF, and PSD-95. Findings demonstrate a long-lasting role for hippocampal ERα as a transcriptional regulator of memory following termination of previous estradiol treatment in a rat model of menopause.

Bright light has been reported to improve spatial memory of diurnal rodents, yet how it will influence the spatial memory of nocturnal rodents is unknown. Here, we found that dynamic changes in spatial memory and anxiety were induced at different time point after bright light treatment. Mice maintained in brighter light exhibited impaired memory in Y maze at one day after bright light exposure, but showed significantly improved spatial memory in the Y maze and Morris water maze at four weeks after bright light exposure. We also found increased anxiety one day after bright light exposure, which could be the reason of impaired memory. However, no change of anxiety was detected after four weeks. Thus, we further explore the underlying mechanism of the beneficial effects of long term bright light on spatial memory. Golgi staining indicated that the structure of dendritic spines changed, accompanied by increased expression of synaptophysin and postsynaptic density 95 in the hippocampus. Further research has found that bright light treatment leads to elevated CaMKII/CREB phosphorylation levels in the hippocampus, which are associated with synaptic function. Moreover, higher expression of brain-derived neurotrophic factor (BDNF) was followed by increased phosphorylated TrkB levels in the hippocampus, indicating that BDNF/TrkB signaling is also activated during this process. Taken together, these findings revealed that bright light exposure with different duration exert different effects on spatial memory in nocturnal rodents, and the potential molecular mechanism by which long term bright light regulates spatial memory was also demonstrated.

The assembly of functional biomolecular condensates often involves liquid-liquid phase separation (LLPS) of proteins with multiple modular domains, which can be folded or conformationally disordered to various degrees. To understand the LLPS-driving domain-domain interactions, a fundamental question is how readily the interactions in the condensed phase can be inferred from interdomain interactions in dilute solutions. In particular, are the interactions leading to LLPS exclusively those underlying the formation of discrete interdomain complexes in homogeneous solutions? We address this question by developing a mean-field LLPS theory of two stoichiometrically constrained solute species. The theory is applied to the neuronal proteins SynGAP and PSD-95, whose complex coacervate serves as a rudimentary model for neuronal postsynaptic densities (PSDs). The predicted phase behaviors are compared with experiments. Previously, a three SynGAP/two PSD-95 ratio was determined for SynGAP/PSD-95 complexes in dilute solutions. However, when this 3:2 stoichiometry is uniformly imposed in our theory encompassing both dilute and condensed phases, the tie-line pattern of the predicted SynGAP/PSD-95 phase diagram differs drastically from that obtained experimentally. In contrast, theories embodying alternate scenarios postulating auxiliary SynGAP-PSD-95 as well as SynGAP-SynGAP and PSD-95-PSD-95 interactions, in addition to those responsible for stoichiometric SynGAP/PSD-95 complexes, produce tie-line patterns consistent with experiment. Hence, our combined theoretical-experimental analysis indicates that weaker interactions or higher-order complexes beyond the 3:2 stoichiometry, but not yet documented, are involved in the formation of SynGAP/PSD-95 condensates, imploring future efforts to ascertain the nature of these auxiliary interactions in PSD-like LLPS and underscoring a likely general synergy between stoichiometric, structurally specific binding and stochastic, multivalent “fuzzy” interactions in the assembly of functional biomolecular condensates.

Neuropathic pain is a very complex chronic pain state, the detailed molecular mechanisms of which remain unclear. In the present study, Shank3 was found to play an important role in neuropathic pain in rats following spared nerve injury (SNI). Shank3 was upregulated in the spinal dorsal horn of rats subjected to SNI, and mechanical hypersensitivity to noxious stimuli in these rats could be alleviated by knock down of Shank3. Shank3 also interacted with hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) and promoted the expression of HCN2 in central neurons of the spinal dorsal. Together with the SNI-dependent increase of HCN2, we also found that the postsynaptic protein of excitatory synapse (PSD95) was increased in rats following SNI. Taken together, our results showed that Shank3 modulated neuropathic pain by facilitating the SNI-dependent increase of HCN2 and the expression of PSD95 in spinal dorsal horn neurons. Our findings revealed new synaptic remodeling mechanisms linking Shank3 with neuropathic pain.

Anesthesia of neonates with propofol induces persistent behavioral abnormalities in adulthood. Although propofol-triggered apoptosis of neurons in the developing brain may contribute to the development of cognitive deficits, the mechanism of neurotoxicity induced by neonatal exposure to propofol remains unclear. In this study, the effects of neonatal propofol anesthesia on synaptic plasticity and neurocognitive function were investigated. Postnatal day 7 (PND-7) Sprague-Dawley rats were intraperitoneally injected with fat emulsion or 20, 40 or 60 mg/kg propofol for three consecutive days. The expression of brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrkB) and postsynaptic density protein 95 (PSD-95) in the rat hippocampus at PND-10 and PND-12 was measured by Western blotting. The number of dendritic branches, total dendritic length and dendritic spine density were observed by Golgi-Cox staining 24 h and 72 h after the last propofol administration. Long-term potentiation (LTP) was measured electrophysiologically in hippocampus of PND-60 rats to evaluate the synaptic function. The learning and memory abilities of rats were evaluated by Morris water maze (MWM) experiments, Novel object recognition test (NORT) and Object location test (OLT) at PND-60. Our results showed that neonatal exposure to propofol significantly inhibited the expression of BDNF, TrkB and PSD-95 in the rat hippocampus. The number of dendritic branches, total dendritic length and dendritic spine density of neurons in the rat hippocampus were markedly reduced after neonatal propofol anesthesia. LTP was significantly diminished in hippocampus of PND-60 rats after repeated exposure to propofol in the neonatal period. Morris water maze experiments showed that repeated neonatal exposure to propofol significantly prolonged the escape latency and decreased the time spent in the target quadrant and the number of platform crossings. NORT and OLT showed that repeated neonatal exposure to propofol markedly reduced the Investigation Time for novel object or location. All of the results above indicate that repeated exposure to propofol in the neonatal period can impair hippocampal synaptic plasticity and the recognition function of rats in adulthood.

The health effects of prolonged exposure to embedded metal fragments, such as those found in shrapnel wounds sustained by an increasing number of military personnel, are not well known. As part of a large collaborative effort to expand this knowledge, we use an animal model of shrapnel wounds originally developed to investigate effects of embedded depleted uranium to investigate effects of military-relevant metals tungsten, nickel, cobalt, iron, copper, aluminum, lead, and depleted uranium compared to an inert control, tantalum. Rats are surgically implanted with pellets of one of the metals of interest in the gastrocnemius (leg) muscle and tracked until 1 month, 3 months, 6 months, or 12 months from the time of implant, at which point they are euthanized and multiple organs and tissue samples are collected for inspection. Here we focus on four regions of the brain: frontal cortex, hippocampus, amygdala, and cerebellum. We examined changes in accumulated metal concentration in each region as well as changes in expression of proteins related to blood brain barrier tight junction formation, occludin and ZO-1, and synapse function, PSD95, spinophilin, and synaptotagmin. We report few changes in metal accumulation or blood brain barrier protein expression, but a large number of synapse proteins have reduced expression levels, particularly within the first 6 months of exposure, but there are regional and metal-specific differences in effects.

We recently demonstrated that NMDA receptors (NMDARs) are capable of ion-flux independent signaling through conformational change in the NMDAR intracellular domain resulting in long-term depression of synaptic transmission (LTD). Here we show that PSD-95 overexpression blocks agonist induced conformational movement in the NMDAR intracellular domain as well as LTD that is NMDAR-dependent and ion-flux independent. Interestingly, previous studies indicate that overexpressed PSD-95 does not block NMDAR-dependent LTD. These data support a model where ion-flux independent LTD is predominant in young animals, which have synapses with low amounts of PSD-95, whereas only ion flux dependent LTD occurs at more mature synapses, which have more PSD-95 that would block ion-flux independent LTD. These results may reconcile different findings regarding ion-flux independent LTD.

The effects of X-irradiation on developing neurons and their functions are unclear. We used primary cultures of mouse hippocampal neurons to investigate the effects of X-irradiation on cell death in developing neurons by analyzing caspase-3, MAP2 and DAPI-labeled cells, and the phenotypes and function of surviving neurons, by examining GAD67-positive cells as a GABAergic marker, and the synaptic markers synapsin 1, drebrin and PSD-95 through its maturation. One-day in vitro (DIV 1) cells were exposed to 0.5 Gy or 1 Gy of X-rays. A significant increase in the percentage of activated caspase-3, a decrease in the number of MAP2/DAPI-positive cells and change in the percentage of GAD67 positive neurons, compared with sham controls, were found 6 days after 1 Gy and 13 days after 0.5 Gy of X-rays. The expression of PSD-95 and drebrin, as well as drebrin clusters, in the remaining neurons was decreased at DIV 21, in both 0.5 Gy and on 1 Gy-irradiation there was a reduced number of dendritic intersection as well. Together, our findings show that 0.5 Gy and 1 Gy of X-irradiation at DIV 1 not only causes neuronal cell death but elicits an increase in the percentage of inhibitory neurons, changes in the dendrites and decrease in expression of important synaptic proteins in the surviving neurons at maturity 3 weeks after exposure.

Nanoscale distribution of proteins and their relative positioning within a defined subcellular region are key to their physiological functions. Thanks to the super-resolution imaging methods, especially single-molecule localization microscopy (SMLM), mapping the three-dimensional distribution of multiple proteins has been easier and more efficient than ever. Nevertheless, in spite of the many tools available for efficient localization detection and image rendering, it has been a challenge to quantitatively analyze the 3D distribution and relative positioning of proteins in these SMLM data. Here, using heterogeneously distributed synaptic proteins as examples, we describe in detail a series of analytical methods including detection of nanoscale density clusters, quantification of the trans-synaptic alignment between these protein densities, and automatic en face projection and averaging. These analyses were performed within customized Matlab routines and we make the full scripts available. The concepts behind these analytical methods and the scripts can be adapted for quantitative analysis of spatial organization of other macromolecular complexes.

Excessive drinking in adolescence continues to be a problem, and almost a quarter of young Canadians have reported consuming five or more alcoholic drinks in one occasion in recent surveys. The consequences of such drinking may be more pronounced when commenced in adolescence, given the ongoing brain development during this period of life. Here, we investigated the consequences of 3 weeks’ intermittent access to ethanol in mid-adolescence to early adulthood in rats, and the extent to which a stress history moderated the negative consequences of ethanol access. In experiment 1, male rats that underwent adolescent social instability stress (SS; daily 1 h isolation + return to unfamiliar cage partner every day from postnatal day [PND] 30–45) did not differ from control (CTL) rats in intake of 10% ethanol sweetened with 0.1% saccharin (access period; PND 47–66). Ethanol drinking reduced proteins relevant for synaptic plasticity (αCaMKII, βCaMKII, and PSD-95) in the dorsal hippocampus, and in CTL rats only in the prefrontal cortex (αCaMKII and PSD 95), attenuating the difference between CTL and SS rats in the water-drinking group. In experiment 2, ethanol also attenuated the difference between SS and CTL rats in a social interaction test by reducing social interaction in SS rats; CTL rats, however, had a higher intake of ethanol than did SS rats during the access period. Ethanol drinking reduced baseline and fear recall recovery concentrations of corticosterone relative to those exposed only to water, although there was no effect of either ethanol or stress history on fear conditioning. Ethanol drinking did not influence intake after 9 days of withdrawal; however, ethanol-naive SS rats drank more than did CTL rats when given a 24-h access in adulthood. These results reveal a complex relationship between stress history and ethanol intake in adolescence on outcomes in adulthood.

Prepulse inhibition (PPI) allows assessing schizophrenia-like sensorimotor gating deficits in rodents. Previous studies indicate that PPI is modulated by the medial prefrontal cortex (mPFC), which is in agreement with our findings showing that PPI differences in the Roman rats are associated with divergences in mPFC activity. Here, we explore whether differences in PPI and mPFC activity in male Roman rats can be explained by (i) differences in the activation (c-Fos) of inhibitory neurons (parvalbumin (PV) interneurons); and/or (ii) reduced excitatory drive (PSD-95) to PV interneurons. Our data show that low PPI in the Roman high-avoidance (RHA) rats is associated with reduced activation of PV interneurons. Moreover, the RHA rats exhibit decreased density of both PV interneurons and PSD-95 puncta on active PV interneurons. These findings point to reduced cortical inhibition as a candidate to explain the schizophrenia-like features observed in RHA rats and support the role of impaired cortical inhibition in schizophrenia.

Cytosolic PSD-95 interactor (cypin) is a multifunctional, guanine deaminase that plays a major role in shaping the morphology of the dendritic arbor of hippocampal and cortical neurons. Cypin catalyzes the Zn

Asparagine-linked glycosylation 13 (ALG13) is an X-linked gene that encodes a protein involved in the glycosylation of the N-terminus. ALG13 deficiency leads to ALG13-congenital disorders of glycosylation (ALG13-CDG), usually in females presenting with mental retardation and epilepsy. Cognitive function is an important function of the hippocampus, and forms the basis for learning, memory and social abilities. However, researchers have not yet investigated the effect of ALG13 on hippocampal cognitive function. In this study, the exploration, learning, memory and social abilities of ALG13 knockout (KO) female mice were decreased in behavioral experiments. Golgi staining demonstrated a decrease in the complexity of hippocampal neurons. Western blot and immunofluorescence staining of the synaptic plasticity factors postsynaptic density protein 95 (PSD95) and synaptophysin (SYP) displayed varying degrees of decline. In other words, the KO of ALG13 may have reduced the expression of PSD95 and SYP in the hippocampus of female mice. Moreover, it may have lowered the synaptic plasticity in various areas of the hippocampus, thus resulting in decreased dendrite length, complexity, and dendrite spine density, which affected the hippocampal function and reduced the cognitive function in female mice.

The critical role of AMPA receptor (AMPAR) trafficking in long-term potentiation (LTP) of excitatory synaptic transmission is now well established, but the underlying molecular mechanism is still uncertain. Recent research suggests that PSD-95 captures AMPARs via an interaction with the AMPAR auxiliary subunits-transmembrane AMPAR regulatory proteins (TARPs). To determine if such interaction is a core minimal component of the AMPAR trafficking and LTP mechanism, we engineered artificial binding partners, which individually were biochemically and functionally dead but which, when expressed together, rescue binding and both basal synaptic transmission and LTP. These findings establish the TARP/PSD-95 complex as an essential interaction underlying AMPAR trafficking and LTP.

PSD-95 is a component and a building block of an excitatory synapse. PSD-95 is a specialized protein that is part of a "combination lock" system responsible for plastic events at the synapse, such as receptor expression, which consequently induces changes in the PSD structure and thus affects synaptic plasticity. The possible involvement of PSD-95 in antidepressant side effects related to cognitive function and psychosis will be considered. An attempt will be made to trace the sequence of events in the proposed mechanism leading to these disorders, focusing mainly on NMDA receptors. Understanding the mechanisms of action of compounds with antidepressant potential may facilitate the design of safer drugs.

Impulsivity is a multifaceted behavioral manifestation with implications in several neuropsychiatric disorders. Glutamate neurotransmission through the N-methyl-D-aspartate receptors (NMDARs) in the medial prefrontal cortex (mPFC), an important brain region in decision-making and goal-directed behaviors, plays a key role in motor impulsivity. We discovered that inherent motor impulsivity predicted responsiveness to D-cycloserine (DCS), a partial NMDAR agonist, which prompted the hypothesis that inherent motor impulsivity is associated with the pattern of expression of cortical NMDAR subunits (GluN1, GluN2A, GluN2B), specifically the protein levels and synaptosomal trafficking of the NMDAR subunits. Outbred male Sprague-Dawley rats were identified as high (HI) or low (LI) impulsive using the one-choice serial reaction time task. Following phenotypic identification, mPFC synaptosomal protein was extracted from HI and LI rats to assess the expression pattern of the NMDAR subunits. Synaptosomal trafficking and stabilization for the GluN2 subunits were investigated by co-immunoprecipitation for postsynaptic density 95 (PSD95) and synapse associated protein 102 (SAP102). HI rats had lower mPFC GluN1 and GluN2A, but higher GluN2B and pGluN2B synaptosomal protein expression versus LI rats. Further, higher GluN2B:PSD95 and GluN2B:SAP102 protein:protein interactions were detected in HI versus LI rats. Thus, the mPFC NMDAR subunit expression pattern and/or synaptosomal trafficking associates with high inherent motor impulsivity. Increased understanding of the complex regulation of NMDAR balance within the mPFC as it relates to inherent motor impulsivity may lead to a better understanding of risk factors for impulse-control disorders.

The aim of this study investigates whether β-asarone can improve cognition deficits in dizocilpine (MK-801) treated mice. Six-week-old male C57BL/6 mice were divided into four groups: control group (CON), MK-801-treated group (MK-801), MK-801 plus β-asarone group (MK-801+β-asa) and β-asarone group (β-asa). Behavioral tests, including sociability test, open field test (OPT) and Morris water-maze (MWM), were performed. Extracellular field excitatory postsynaptic potentials were recorded in the hippocampal dentate gyrus (DG) region. Western blot was employed to measure the expression of cognitive function-associated proteins and pro-inflammatory cytokines in the hippocampus. Immunofluorescence was performed to assess the microglial activation in the hippocampus DG region. The data show that social interactions and spatial learning and memory were impaired by MK-801. However, β-asarone significantly mitigated the impairments. Furthermore, it was found that MK-801 aggravated the hyperactivity and anxiety-like behavior, but β-asarone alleviated them. Moreover, β-asarone alleviated the impairments of hippocampal synaptic plasticity and enhanced the expression of hippocampal synaptophysin (SYP) and postsynaptic density protein 95 (PSD95) in MK-801-treated mice. In addition, it suppressed the expression of interleukin-6 (IL-6), interleukin-1β (IL-1β), inducible nitric oxide synthase (i-Nos) and cyclo-oxygenase-2 (COX-2) expression in MK-801-treated mice. The results suggest that β-asarone improved the impairment of cognition and synaptic plasticity possibly through modulating the excess release of pro-inflammatory cytokines and microglia activation in MK-801-treated mice.

Apolipoprotein E (apoE) colocalizes with amyloid-β (Aβ) in Alzheimer disease (AD) plaques and in synapses, and evidence suggests that direct interactions between apoE and Aβ are important for apoE's effects in AD. The present work examines the hypothesis that apoE receptors mediate uptake of apoE/Aβ complex into synaptic terminals. Western blot analysis shows multiple SDS-stable assemblies in synaptosomes from human AD cortex; apoE/Aβ complex was markedly increased in AD compared with aged control samples. Complex formation between apoE and Aβ was confirmed by coimmunoprecipitation experiments. The apoE receptors low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) were quantified in synaptosomes using flow cytometry, revealing up-regulation of LRP1 in early- and late-stage AD. Dual-labeling flow cytometry analysis of LRP1- and LDLR positives indicate most (approximately 65%) of LDLR and LRP1 is associated with postsynaptic density-95 (PSD-95)–positive synaptosomes, indicating that remaining LRP1 and LDLR receptors are exclusively presynaptic. Flow cytometry analysis of Nile red labeling revealed a reduction in cholesterol esters in AD synaptosomes. Dual-labeling experiments showed apoE and Aβ concentration into LDLR and LRP1-positive synaptosomes, along with free and esterified cholesterol. Synaptic Aβ was increased by apoE4 in control and AD samples. These results are consistent with uptake of apoE/Aβ complex and associated lipids into synaptic terminals, with subsequent Aβ clearance in control synapses and accumulation in AD synapses.

In the adult hippocampus new neurons are generated in the dentate gyrus from neural progenitor cells. Adult-born neurons integrate into the hippocampal circuitry and contribute to hippocampal function. PSD95 is a major postsynaptic scaffold protein that is crucial for morphological maturation and synaptic development of hippocampal neurons. Here we study the function of PSD95 in adult hippocampal neurogenesis by downregulating PSD95 expression in newborn cells using retroviral-mediated RNA interference. Retroviruses coding for a control shRNA or an shRNA targeting PSD95 (shPSD95) were stereotaxically injected into the dorsal dentate gyrus of 2-month-old C57BL/6 mice. PSD95 knockdown did not affect neuronal differentiation of newborn cells into neurons, or migration of newborn neurons into the granule cell layer. Morphological analysis revealed that newborn neurons expressing shPSD95 showed increased dendritic length and increased number of high-order dendrites. Concomitantly, dendrites from shPSD95-expressing newborn granule neurons showed a reduction in the density of dendritic spines. These results suggest that PSD95 is required for proper dendritic and spine maturation of adult-born neurons, but not for early stages of neurogenesis in the hippocampus.

Metabolic conditions during brain development may have long-term consequences on brain metabolism, thereby influencing the risk of neurodegenerative disease in later life. To ascertain the long-term consequences of omega-3 (ω3) fatty acid deficiency during brain development on oxidative fatty acid degradation in the brain and the development of Alzheimer-like pathology, wild-type (WT) female mice were fed diets that were either replete or deficient in ω3 fatty acids for 5 weeks. These females were then mated with hemizygous 5xFAD male transgenic (TG) mouse models of Alzheimer's disease, and the progeny were continued on diets that were either ω3-replete or ω3-deficient. When the progeny were 6 months of age, they received radiolabeled arachidonic acid (ARA) by intracerebroventricular injection. Five days after these injections, the brains were harvested and oxidative degradation of the radiolabeled ARA was characterized. Among the progeny of female mice on an ω3-replete diet, TG progeny had lower PSD-95 expression and higher oxidative ARA degradation than WT progeny. Progeny on an ω3-deficient diet, however, had no significant differences in PSD-95 expression between TG and WT mice, or in the extent of ARA degradation. In TG mice, an ω3-deficient diet reduced oxidative ARA degradation to a greater extent than in WT mice. The reductions in oxidative ARA degradation occurred even if the progeny of female mice on an ω3-deficient diet resumed an ω3-replete diet immediately on weaning. These results demonstrate that dietary ω3 fatty acid deficiency during development can cause long-term changes in the expression of a synaptic marker and long-term reductions in the rate of ARA degradation in the WT brain, which are not completely alleviated by an ω3-replete diet after weaning. The elimination of differences between TG and WT mice by an ω3-deficient diet suggests that mechanisms regulating PSD-95 expression and the oxidative degradation of ARA are related and that the timing of dietary ω3 intake during development may influence Alzheimer's disease-related pathological changes later in life.

Glutamate receptors play a crucial pathogenic role in brain damage induced by status epilepticus (SE). SE may initiate NMDAR-dependent excitotoxicity through the production of oxidative damage mediated by the activation of a ternary complex formed by the NMDA receptor, the post-synaptic density scaffolding protein 95 (PSD95) and the neuronal NO synthase (nNOS). The inhibition of the protein-protein-interaction (PPI) of the NMDAR-PSD95-nNOS complex is one of the most intriguing challenges recently developed to reduce neuronal death in both animal models and in patients with cerebral ischemia. We took advantage of this promising approach to verify whether early administration of a neuroprotective NMDAR-PSD95-nNOS PPI inhibitor preserves the brain from SE-induced damage in a model of acquired cortical dysplasia, the methylazoxymethanol (MAM)/pilocarpine rat. Pilocarpine-induced SE rapidly determined neurodegenerative changes mediated by a NMDAR-downstream neurotoxic pathway in MAM rats. We demonstrated that SE rapidly induces NMDAR activation, nNOS membrane translocation, PSD95-nNOS molecular interaction associated with neuronal and glial peroxynitrite accumulation in the neocortex of MAM-pilocarpine rats. These changes were paralleled by rapid c-fos overexpression and by progressive spectrin proteolysis, suggestive of calpain activity and irreversible cytoskeletal damage. Early administration of a cell-penetrating Tat-N-dimer peptide inhibitor of NMDAR-PSD95-nNOS PPI during SE significantly rescued the MAM-pilocarpine rats from SE-induced mortality, reduced the number of degenerating neurons, decreased neuronal c-fos activation, peroxynitrite formation and cytoskeletal degradation and prevented astrogliosis. Our findings suggest an overall neuroprotective effect of blocking PSD95-nNOS protein-protein-interaction against SE insult.

We combined translating ribosome affinity purification (TRAP) with in utero electroporation (IUE), called iTRAP to identify the molecular profile of specific neuronal populations during neonatal development without the need for viral approaches and FACS sorting. We electroporated a plasmid encoding EGFP-tagged ribosomal protein L10a at embryonic day (E) 14–15 to target layer 2–4 cortical neurons of the somatosensory cortex. At three postnatal (P) ages—P0, P7, and P14—when morphogenesis occurs and synapses are forming, TRAP and molecular profiling was performed from electroporated regions. We found that ribosome bound (Ribo)-mRNAs from ~7,300 genes were significantly altered over time and included classical neuronal genes known to decrease (e.g., Tbrl, Dcx) or increase (e.g., Eno2, Camk2a, Synl) as neurons mature. This approach led to the identification of specific developmental patterns for Ribo-mRNAs not previously reported to be developmentally regulated in neurons, providing rationale for future examination of their role in selective biological processes. These include upregulation of Lynxl, Nrnl, Cntnapl over time; downregulation of St8sia2 and Draxin; and bidirectional changes to Fkbplb. iTRAP is a versatile approach that allows researchers to easily assess the molecular profile of specific neuronal populations in selective brain regions under various conditions, including overexpression and knockdown of target genes, and in disease settings.

Analysis of affinity-purified PSD-95 complexes had previously identified a ‘hypothetical protein’, product of the gene FAM81A [ 1 ]. The present study examined the tissue and subcellular distribution of FAM81A protein and its expression levels during development. Comparison of different organs indicates selective expression of FAM81A protein in brain. FAM81A is expressed late in development, with a post-natal gradual increase in brain levels that parallels the expression of PSD-95. Comparison of subcellular fractions from adult brain shows that the distribution of FAM81A protein is similar to that of PSD-95, with a drastic enrichment in the postsynaptic density fraction. Immuno-electron microscopy of adult brain tissue reveals specific immunogold labeling for FAM81A protein at postsynaptic densities in the forebrain. The label for FAM81A protein is concentrated at the cytoplasmic edge of the electron-dense core of the postsynaptic density, with a mean distance of ∼33 nm from the postsynaptic membrane. These observations firmly establish FAM81A protein as a component of the postsynaptic density in the adult brain, suggesting a role in synaptic function.

Background Piper nigrum L. and Piper longum L. consist a classic formula in traditional Chinese Hui medicine and are widely used in treatment of stroke. To examine the therapeutic effect of neuron injury after apoplexy, we used a permanent middle cerebral artery occlusion model in rats to investigate the effects of dichloromethane fraction (DF) of Piper nigrum L. and Piper longum L. Materials and Methods After subjecting the rats to permanent middle cerebral artery occlusion, DF (100 and 200 mg/kg) were administered for 14 days. Neurological deficits and the degree of cerebral tissue injury was detected by 2,3,5-Triphenyltetrazolium Chloride Staining Hematoxylin and eosin staining and Nissl staining. Postsynaptic density protein 95 (PSD-95), synapsin-I (syn-I), and α-synuclein (α-syn) were stained by immunohistochemistry. PSD-95, Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMK II), phosphorylated CaMK II (p-CaMK II), CaM, N-methyl D-aspartate receptor subtype 2B (NR2B) expression were detected by Western blot. Meanwhile, phytochemical profile of DF was determined through ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Results DF alleviated neurological deficits and markedly prevented ischemia-induced cellular damage. Immunohistochemical micrographs revealed that PSD-95 and syn-I proteins increased, and α-syn presented reduced expression in brain samples from the sham group. Western blot analyses revealed that the model group exhibited a noticeable reduction in PSD-95, p-CaMK II, CaM, and NR2B. The DF-treated model group exhibited increased PSD-95, p-CaMK II, CaM, and NR2B. UPLC-Q-TOF/MS analysis revealed eight main components of DF, of which piperine accounted for the largest proportion.

Neuroligins (NLGs) are postsynaptic adhesion molecules known to play essential roles in synapse development and maturation, but their effects on synaptic plasticity at mature synapses remain unclear. In this study, we investigate the involvement of NLG1 in hippocampal long-term depression (LTD), a key form of long lasting synaptic plasticity, critical for memory formation and brain disorders, by using mice deficient in the expression of NLG1. We find that although NLG1 homozygous (NLG1-/-) mice show no impairments in either NMDA receptor- (NMDAR-LTD) or metabotropic glutamate receptor-dependent LTD (mGluR-LTD), the heterozygous (NLG1+/-) mice are significantly altered in both forms of LTD characterized by the absence of NMDAR-LTD but enhanced mGluR-LTD. Accordingly, the NLG1+/-, but not the NLG1-/- mice are altered in synaptic proteins, including PSD95, GluA2 and phosphorylated GluA1 at serine 845, all of which are involved in the expression of LTD. The NLG1+/- mice also exhibit autistic-like behaviors including increased grooming and impaired recognition memory. We further show that the expression of NLG3, a close family member of NLG1, is elevated in the NLG1-/-, but not in NLG1+/- mice, suggesting that the lack of LTD deficits in the NLG1-/- mice might be due to the increased NLG3. Our results reveal a gene dosage dependent role for NLG1 in the regulation of LTD and suggest that moderate changes in NLG1 protein level may be sufficient to cause synaptic and behavior deficits in brain disorders where copy number variants and hemizygosity of gene mutations are common.

Mutation or disruption of the Shank/ProSAP family of genes is a high risk factor for autism spectrum disorders (ASDs) and intellectual disability. N-methyl-D-aspartate glutamate receptor (NMDAR) dysfunction contributes to the development of autism-like behaviors. However, the molecular mechanism of Shank-mediated NMDAR modulation is still not clear. Here, we show that the scaffold protein plenty of SH3s (POSH) directly interacts with two other scaffold proteins, PSD95 and SHANK2/3, at excitatory synapses. In POSH conditional knockout (cKO) mice, normal synaptic clustering of NMDAR/PSD-95/SHANK complex is disrupted, accompanied by abnormal dendritic spine development and glutamatergic transmission in hippocampal neurons. POSH cKO mice display profound autism-like behaviors, including impairments in social interactions, social communication, repetitive behaviors, and deficits in learning and memory. Thus, POSH clusters at the postsynaptic density (PSD) with PSD-95 and SHANK2/3 and plays important roles in the signaling mechanisms of the NMDAR/PSD-95/POSH/SHANK complex as well as in spine development and brain function.

Cocaine use disorder is a serious, chronic and relapsing disease of the nervous system, for which effective treatments do not yet exist. Recently, the role of the N-methyl-d-aspartate (NMDA) receptor subunit GluN2B has been highlighted in cocaine abstinence followed by extinction training. Since the GluN2B subunit is stabilized at synaptic level by the interaction with its scaffolding protein PSD95, in this study we aimed at investigating efficacy of Tat-NR2B9c peptide, a PSD95 inhibitor, which disrupts the interaction of PSD95 with GluN2B, in the attenuation of cocaine seeking-behavior or cue-induced reinstatement. We found that Tat-NR2B9c, administered intravenously, attenuated the reinstatement of active lever presses induced by a priming dose of cocaine or by drug-associated conditioned stimuli. At the same time, the GluN2B/PSD95 complex levels were decreased in the ventral hippocampus of rats that previously self-administered cocaine injected with Tat-NR2B9c during cocaine- or cue-induced reinstatement. In conclusion, we here provide the first evidence showing that the disruption of the GluN2B/PSD95 complexes during cocaine abstinence followed by extinction training may represent a useful strategy to reduce reinstatement of cocaine-seeking behavior.

The metabotropic glutamate 2/3 (mGlu2/3) receptor antagonists are reported to produce ketamine-like rapid-acting and sustained antidepressant-like effects in rodents. In this study, we compared the effects of single administration of the new mGlu2/3 receptor antagonist TP0178894 and the selective serotonin reuptake inhibitor (SSRI) escitalopram in the chronic social defeat stress (CSDS) model of depression, a model which has been shown to be resistant to treatment with a single dose of SSRI. In the tail suspension test and forced swimming test, high dose (3.0 mg/kg) of TP0178894 significantly attenuated the increased immobility time of these tests in CSDS susceptible mice, compared with vehicle-treated mice. In contrast, low doses (0.3 and 1.0 mg/kg) of TP0178894 and escitalopram (10 mg/kg) did not alter the increased immobility time of these two tests. In the sucrose preference test, TP0178894 (3.0 mg/kg) significantly improved the reduced sucrose preference of CSDS susceptible mice, three and seven days after a single dose. In addition, Western blot analyses showed that TP0178894 (3.0 mg/kg), but not low doses of TP0178894 and escitalopram, significantly attenuated the reduced expression of synaptic proteins [α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (GluA1) and postsynaptic density protein 95 (PSD-95)] in the prefrontal cortex from CSDS susceptible mice. This study suggests that TP0178894 shows rapid-acting and sustained antidepressant-like effects in CSDS model, as ketamine does.

MiR-335-3p, a neuron-enriched microRNA, has been reported to be involved in aging and age-related neurological diseases. However, the role of miR-335-3p in cholesterol metabolism of astrocytes, and whether it affects neuronal functions, particularly during aging process, largely remains unknown. In this study, we uncover that miR-335-3p is significantly increased in aged cultured astrocytes and aged hippocampal brains, accompanied by decreased cellular cholesterol and diminished expression of HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase) and 3-hydroxy-3-methylglutaryl-CoA synthase-1 (HMGCS1), both step-limiting enzymes in cholesterol synthesis pathway. We also demonstrate that miR-335-3p suppresses HMGCS1 post-transcriptionally by directly binding to its 3'UTR, and HMGCR through binding mediated by SFRS2. More importantly, aged mice with miR-335-3p deficiency in hippocampal brains exhibit improved learning and memory, accompanied by enhanced levels of postsynaptic density protein 95 (PSD95). We further reveal that the level change of PSD95 is resulted from altered cholesterol metabolism. Our findings provide a novel insight into the regulatory role of miR-335-3p in cholesterol metabolism in astrocytes, and consequently cognitive functions during aging.

Neuroligin 2 is a synaptic cell adhesion molecule that is mainly located in inhibitory synapses and is crucial in the regulation of synapse function through protein-protein interactions. However, researchers have not clearly determined whether neuroligin 2 is involved in the development of postoperative pain. In the current study, Western blot, immunofluorescence staining and co-immunoprecipitation were used to examine the critical role of neuroligin 2 in postoperative pain hypersensitivity. A small interfering ribonucleic acid (siRNA)-targeting neuroligin 2 was used to inhibit neuroligin 2 expression. Our data found that plantar incision induced postoperative pain hypersensitivity, which was characterized by paw withdrawal threshold and cumulative pain score. The upregulation of neuroligin 2 and GluR1 expression in the postsynaptic membranes of ipsilateral spinal dorsal horn was observed at 3 h and 1 day after plantar incision. Additionally, at 3 h after plantar incision, the amount of PSD-95 that was co-immunoprecipitated with neuroligin 2 antibody was significantly increased in the ipsilateral dorsal horn, as compared to that of the control group. Intrathecal pretreatment of siRNA-targeting neuroligin 2 to reduce the neuroligin 2 expression in the spinal cord significantly inhibited the pain hypersensitivity and reduced the synaptic targeting of GluR1 in ipsilateral dorsal horns. Our study indicates that the incision-induced interaction between neuroligin 2 and PSD-95 and subsequent synaptic targeting of GluR1 in ipsilateral dorsal horns contribute to postoperative pain hypersensitivity.

Maternal sleep deprivation (MSD) has been suggested to be associated with increased frequency of neurodevelopmental disorders in offspring in both humans and animal models. However, the underlying cellular and molecular mechanism is still unclear. We have recently reported that MSD at different stages of pregnancy impairs the emotional and cognitive functions, and suppresses hippocampal CA1 long-term potentiation (LTP) in the offspring rats. Here, we report that the MSD induced LTP impairment at the CA1 hippocampus of the offspring rats is associated with increased long-term depression (LTD) and reduced expression of postsynaptic GluA2-containing α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptors (AMPARs). Importantly, we found that inhibition of AMPAR endocytosis by a synthetic peptide Tat-GluA23Y (3 μmol/kg, i.p.) not only increased level of AMPARs and reduced LTD, but also restored LTP. Moreover, treatment with Tat-GluA23Y peptide markedly alleviated the MSD-induced impairments of spatial learning and memory; and decreased depressive- and anxiety-like behaviors in the offspring. Together, our findings suggest that the MSD-induced postsynaptic AMPAR endocytosis causally contributes to the impairments of hippocampal synaptic plasticity, thereby disrupting the emotional and cognitive functions in the offspring.

Our previous study has reported that maternal separation (MS) results in abnormal behaviors such as depressive-like and anxious-like behaviors in adult rats. However, the underlying mechanism remains unclear. In this study, we focused on the relationship between MS and autophagy in an effort to explain potential mechanisms. Pups experienced MS from postnatal days 7 to 21 and were randomly divided into the control (CON) group and MS group. Data showed that MS induced depressive-like and anxious-like behaviors in adult rats. Compared with the CON group, the glutamate level was obviously increased in the hippocampus (HP) and prefrontal cortex (PFC) of rats in the MS group. Interestingly, both beclin-1 expression and ratio of LC3II/LC3I were decreased in the HP, but they were increased in the PFC. Furthermore, N-methyl-d-aspartate receptor subunit 2B (NR2B) expression was significantly increased in the HP and PFC. Additionally, postsynaptic density-95 was increased in the HP; however, it was unchanged in the PFC. Moreover, the expression of synaptophysin was obviously decreased in both the HP and PFC. In conclusion, these findings suggest that MS induces different autophagic responses in the HP and PFC (i.e., the inhibition of autophagy in the HP and the activation of autophagy in the PFC), which can be related to the NR2B signaling pathway.

Anxiety disorders are a category of mental disorders characterized by feelings of anxiety, stress, and fear attached to various sources. However, their pathogenesis is complicated and has not been fully elucidated. The amygdala is a vital brain region that regulates anxiety and mental disorders. Cytoplasmic polyadenylation element binding protein 1 (CPEB1) mediates the extension of the mRNA polyadenylation tail and facilitates the translation of target RNA. CPEB1 is closely related to neuronal diseases, such as Fragile X Syndrome, learning and memory disorders, and chronic pain. In this study, the role of CPEB1 in anxiety development was determined in a pain-mediated anxiety mouse model. The anxiety model was established in mice by injecting with Complete Freund's Adjuvant (CFA) into the hindpaw. CFA injection then led to anxiety-like behaviors and increased the CPEB1 levels in the mouse basolateral amygdala (BLA). CPEB1 enhancement facilitated the translation of GluA1, GluN2A, GluN2B, PSD95, and GABA receptors, which disturbed the E/I balance in the BLA as shown by enhanced excitatory presynaptic release and reduced inhibitory presynaptic release. CPEB1 knockdown with AAV-CPEB1-shRNA alleviated the anxiety-like behaviors but not the pain-like behaviors by enhancing inhibitory transmission in the BLA of model mice. The data suggest that CPEB1 participates in anxiety development by regulating excitatory/inhibitory synaptic transmission in the BLA.

Ketamine is a non-competitive antagonist of N-methyl-D-aspartate receptors (NMDARs). Growing evidence suggests that a single dose of ketamine produces a series of rapid and remarkable antidepressant properties. However, the mechanisms remain unclear. In our study, the antidepressant properties of a single dose of ketamine (10 mg/kg, i.p.) in mice exposed to chronic unpredictable stress (CUS) were assessed using the open-field test (OFT) and the forced swimming test (FST). Early growth response 1 (Egr-1) and postsynaptic density protein 95 (PSD-95) mRNA and protein expression levels were examined using qRT-PCR and western blot, respectively. Dendritic spine density in the CA1 region of the hippocampus was detected by Golgi staining. AMPAR currents in hippocampal slices were measured by electrophysiology. Our study showed that CUS induced a significant depression-like behavior accompanied by an upregulation of Egr-1 and downregulations of PSD-95, spine density, and AMPAR currents in the hippocampus, and a single dose of ketamine rapidly restored these changes. Interestingly, a single dose of Ro-25-6981 (an GluN2B antagonist, 10 mg/kg, i.p.) or Egr-1 siRNA, but not NVP AAM077 (an GluN2A antagonist, 10 mg/kg, i.p.), could produce the same antidepressant effects as ketamine. These data demonstrate that ketamine may produce its rapid antidepressant effects by downregulating the expression of Egr-1 via blocking GluN2B in the hippocampus.

Discs large (DLG) MAGUKs are abundantly expressed in glutamatergic synapses, crucial for synaptic transmission, and plasticity by anchoring various postsynaptic components including glutamate receptors, downstream scaffold proteins and signaling enzymes. Different DLG members have shared structures and functions, but also contain unique features. How DLG family proteins function individually and cooperatively is largely unknown. Here, we report that PSD-95 PDZ3 directly couples with SH3-GK tandem in a PDZ ligand binding-dependent manner, and the coupling can promote PSD-95 dimerization and multimerization. Aided by sortase-mediated protein ligation and selectively labeling, we elucidated the PDZ3/SH3-GK conformational coupling mechanism using NMR spectroscopy. We further demonstrated that PSD-93, but not SAP102, can also undergo PDZ3 ligand binding-induced conformational coupling with SH3-GK and form homo-oligomers. Interestingly, PSD-95 and PSD-93 can also form ligand binding-induced hetero-oligomers, suggesting a cooperative assembly mechanism for the mega-N-methyl-d-aspartate receptor synaptic signaling complex. Finally, we provide evidence showing that ligand binding-induced conformational coupling between PDZ and SH3-GK is a common feature for other MAGUKs including CASK and PALS1.

Little is known about the consequences of altered endocannabinoid signalling in adolescence. We hypothesized that CB1 receptor antagonism (AM251, 1 mg/kg) and stress exposures (1 h confinement stress) in adolescence (daily, postnatal days 30–44) would interact to increase neuroendocrine stress responses and anxiety when investigated a minimum of 24 h after drug and stress treatments; these treatment effects were independent of each other. Changes in homecage behaviour and in weight gain confirmed that both males and females were sensitive to the treatments. Nevertheless, in males, repeated AM251 administration was without effect on any of the measures investigated in days post-treatment. Males had reduced corticosterone release to the repeated stress and had increased GAD67 expression in the ventral hippocampus under baseline conditions. In females, AM251 also reduced weight gain and increased stereotypic behaviours in the homecage; these same females showed increased sociality, reduced CB1 receptor expression in the dorsal hippocampus, and increased GAD67 expression in the prefrontal cortex. Further, females exposed to repeated stress had enhanced recovery to baseline corticosterone concentrations after stress. The inclusion of a non-injected comparison group also revealed stress of injection effects in both sexes that otherwise would have been masked. Together, the findings demonstrate effects of CB1 receptor antagonism and stress that were more evident in females than males, suggesting that females may be more vulnerable to the consequences of disrupted endocannabinoid signalling during adolescence.

The most common cause of dementia is Alzheimer's disease. The etiology of the disease is unknown, although considerable evidence suggests a critical role for the soluble oligomers of amyloid beta peptide (Aβ). Because Aβ increases the expression of purinergic receptors (P2XRs) in vitro and in vivo, we studied the functional correlation between long-term exposure to Aβ and the ability of P2XRs to modulate network synaptic tone. We used electrophysiological recordings and Ca2+ microfluorimetry to assess the effects of chronic exposure (24 h) to Aβ oligomers (0.5 μM) together with known inhibitors of P2XRs, such as PPADS and apyrase on synaptic function. Changes in the expression of P2XR were quantified using RT-qPCR. We observed changes in the expression of P2X1R, P2X7R and an increase in P2X2R; and also in protein levels in PC12 cells (143%) and hippocampal neurons (120%) with Aβ. In parallel, the reduction on the frequency and amplitude of mEPSCs (72% and 35%, respectively) were prevented by P2XR inhibition using a low PPADS concentration. Additionally, the current amplitude and intracellular Ca2+ signals evoked by extracellular ATP were increased (70% and 75%, respectively), suggesting an over activation of purinergic neurotransmission in cells pre-treated with Aβ. Taken together, our findings suggest that Aβ disrupts the main components of synaptic transmission at both pre- and post-synaptic sites, and induces changes in the expression of key P2XRs, especially P2X2R; changing the neuromodulator function of the purinergic tone that could involve the P2X2R as a key factor for cytotoxic mechanisms. These results identify novel targets for the treatment of dementia and other diseases characterized by increased purinergic transmission.

Background In the present study, we aimed to investigate the effects of probucol on aging-related hippocampus-dependent cognitive impairment and explore the potential mechanisms. Methods D -galactose (100 mg/kg, once daily for 6 weeks) was subcutaneously injected to induce aging in mice. Then the mice were administered with probucol or vehicle once a day for 2 weeks. The hippocampus-related cognition was evaluated with Morris water maze test, novel object recognition test, and contextual fear conditioning test. Moreover, synaptic plasticity was assessed, and RNA-sequencing was applied to further explore the molecular mechanisms. Results Aging mice induced by D -galactose showed conspicuous learning and memory impairment, which was significantly ameliorated by probucol. Meanwhile, probucol enhanced the spine density and dendritic branches, improved long-term potentiation, and increased the expression of PSD95 of aging mice. Probucol regulated 70 differentially expressed genes compared to D -galactose group, of which 38 genes were upregulated and 32 genes were downregulated. At last, RNA-sequencing results were verified by quantitative reverse transcription-polymerase chain reaction. Conclusions Probucol improved learning and memory in aging mice through enhancing synaptic plasticity and regulating gene expression, indicating the potential application of probucol to prevent and treat aging-related disorders.

As an N-methyl- D -aspartate (NMDA) receptor inhibitor, ketamine has become a popular recreational substance and currently is used to address treatment-resistant depression. Since heavy ketamine use is associated with persisting psychosis, cognitive impairments, and neuronal damage, the safety of ketamine treatment for depression should be concerned. The nutrient supplement betaine has been shown to counteract the acute ketamine-induced psychotomimetic effects and cognitive dysfunction through modulating NMDA receptors. This study aimed to determine whether the adjunctive or subsequent betaine treatment would improve the enduring behavioral disturbances and hippocampal synaptic abnormality induced by repeated ketamine exposure. Mice received ketamine twice daily for 14 days, either combined with betaine co-treatment or subsequent betaine post-treatment for 7 days. Thereafter, three-chamber social approach test, reciprocal social interaction, novel location/object recognition test, forced swimming test, and head-twitch response induced by serotonergic hallucinogen were monitored. Data showed that the enduring behavioral abnormalities after repeated ketamine exposure, including disrupted social behaviors, recognition memory impairments, and increased depression-like and hallucinogen-induced head-twitch responses, were remarkably improved by betaine co-treatment or post-treatment. Consistently, betaine protected and reversed the reduced hippocampal synaptic activity, such as decreases in field excitatory post-synaptic potentiation (fEPSP), long-term potentiation (LTP), and PSD-95 levels, after repeated ketamine treatment. These results demonstrated that both co-treatment and post-treatment with betaine could effectively prevent and reverse the adverse behavioral manifestations and hippocampal synaptic plasticity after repeated ketamine use, suggesting that betaine can be used as a novel adjunct therapy with ketamine for treatment-resistant depression and provide benefits for ketamine use disorders.

SUMMARY Cortical function relies on the balanced activation of excitatory and inhibitory neurons. However, little is known about the organization and dynamics of shaft excitatory synapses onto cortical inhibitory interneurons. Here, we use the excitatory postsynaptic marker PSD-95, fluorescently labeled at endogenous levels, as a proxy for excitatory synapses onto layer 2/3 pyramidal neurons and parvalbumin-positive (PV+) interneurons in the barrel cortex of adult mice. Longitudinal in vivo imaging under baseline conditions reveals that, although synaptic weights in both neuronal types are log-normally distributed, synapses onto PV+ neurons are less heterogeneous and more stable. Markov model analyses suggest that the synaptic weight distribution is set intrinsically by ongoing cell-type-specific dynamics, and substantial changes are due to accumulated gradual changes. Synaptic weight dynamics are multiplicative, i.e., changes scale with weights, although PV+ synapses also exhibit an additive component. These results reveal that cell-type-specific processes govern cortical synaptic strengths and dynamics., Graphical Abstract, In brief Melander et al. use a genetic strategy to visualize excitatory neuronal connections that cannot be inferred from morphology, and they monitor how the connections change over weeks in mice. They find distinct characteristics between synapses onto cells that “suppress” brain activity and those onto cells that “excite” brain activity.

In view of postsynaptic density 95kDA (PSD95) tethers neuronal NO synthase (nNOS) to N-methyl- d -aspartate receptor (NMDAR), the PSD95-nNOS complex represents a therapeutic target of neuropathic pain. This study therefore sought to explore the ability of PCC-0105002, a novel PSD95-nNOS small molecule inhibitor, to alter pain sensitivity in rodent neuropathic pain models. Firstly, the IC50 of PCC-0105002 for PSD95 and NOS1 binding activity was determined using an Alpha Screen assay kit. Then, we examined the effects of PCC-0105002 in the mouse formalin test and in the rat spinal nerve ligation (SNL) model, and explored the ability of PCC-0105002 to mediate analgesia and to effect motor coordination in a rota-rod test. Moreover, the mechanisms whereby PCC-0105002 mediates analgesia was explored via western blotting, Golgi staining, and co-immunoprecipitation experiments in dorsal horn. The outcomes indicated that PCC-0105002 exhibited dose-dependent attenuation of phase II pain-associated behaviors in the formalin test. The result indicated that PCC-0105002 disrupted the PSD95-nNOS interaction with IC50 of 1.408 μM. In the SNL model, PCC-0105002 suppressed mechanical allodynia, thermal hyperalgesia, and abnormal dorsal horn wide dynamic range neuron discharge. PCC-0105002 mediated an analgesic effect comparable to that of MK-801, while it was better able to enhance motor coordination as compared with MK-801. Moreover, PCC-0105002 altered signaling downstream of NMDAR and thus functionally and structurally attenuating synaptic plasticity through respective regulation of the NR2B/GluR1/CaMKIIα and Rac1/RhoA pathways. These findings suggest that the novel PSD95-nNOS inhibitor PCC-0105002 is an effective agent for alleviating neuropathic pain, and that it produces fewer motor coordination-associated side effects than do NMDAR antagonists.

Using marijuana has become popular and is allowed for medical purposes in some countries. The effect of marijuana on Parkinson's disease is controversial and Medical marijuana may benefit for motor and non-motor symptoms of patients with Parkinson's disease. No research has been conducted to fully prove the benefits, risks, and uses of marijuana as a treatment for patients with Parkinson's disease. In the present study, several different approaches, including behavioral measures and the western blot method for protein level assay, were used to investigate whether exposure to marijuana affects the motor and synaptic plasticity impairment induced by 6-OHDA. Marijuana consumption significantly decreased apomorphine-induced contralateral rotation, beam travel time, beam freeze time, and catalepsy time, but significantly increased latency to fall in the rotarod test, balance time, and protein level of PSD-95 and dopamine receptor D1 in the 6-OHDA + marijuana group. These results suggest that marijuana may be helpful for motor disorders and synaptic changes in patients with Parkinson's disease.

Clinical and preclinical studies report that chronic stress induces behavioral deficits as well as volumetric and synaptic alterations in corticolimbic brain regions including the anterior cingulate cortex (ACC), amygdala (AMY), nucleus accumbens (NAc) and hippocampus (HPC). Here, we aimed to investigate the volumetric changes associated with chronic restraint stress (CRS) and link these changes to the CRS-induced behavioral and synaptic deficits. We first confirmed that CRS increases behavioral emotionality, defined as collective scoring of anxiety- and anhedonia-like behaviors. We then demonstrated that CRS induced a reduction of total brain volume which negatively correlated with behavioral emotionality. Region-specific analysis identified that only the ACC showed significant decrease in volume following CRS (p 0.05). Reduced ACC correlated with increased behavioral emotionality (r = -0.56; p = 0.0003). Although not significantly altered by CRS, AMY and NAc (but not the HPC) volumes were negatively correlated with behavioral emotionality. Finally, using structural covariance network analysis to assess shared volumetric variances between the corticolimbic brain regions and associated structures, we found a progressive decreased ACC degree and increased AMY degree following CRS. At the cellular level, reduced ACC volume correlated with decreased PSD95 (but not VGLUT1) puncta density (r = 0.35, p 0.05), which also correlated with increased behavioral emotionality (r = -0.44, p 0.01), suggesting that altered synaptic strength is an underlying substrate of CRS volumetric and behavioral effects. Our results demonstrate that CRS effects on ACC volume and synaptic density are linked to behavioral emotionality and highlight key ACC structural and morphological alterations relevant to stress-related illnesses including mood and anxiety disorders.

Granule cells in the dentate gyrus regenerate constantly in adult hippocampus and then integrate into neural circuits in the hippocampus thereby providing the neural basis for learning and memory. Promoting the neurogenesis in the hippocampus facilitates learning and memory such as spatial learning, object identification, and extinction learning. The interaction between neuronal nitric oxide synthase (nNOS) and postsynaptic density protein-95 (PSD-95) is reported to negatively regulate neurogenesis in brain, so we hypothesized that disrupting this interaction might facilitate the neurogenesis in the dentate gyrus (DG) and thus enhance the extinction memory retrieval of fear learning. We found that uncoupling the nNOS-PSD-95 complex in remote contextual fear condition promoted both neuronal proliferation and survival in the DG, contributing to an enhanced retrieval of the extinction memory. Moreover, the nNOS-PSD-95 uncoupling-induced neurogenesis may be mediated by the extracellular signal-regulated kinase (ERK) as the phosphorylation level of ERK1/2 was increased after uncoupling. These findings suggest that the nNOS-PSD-95 complex may serve as a novel target for the treatment of post-traumatic stress disorder (PTSD).

Insulin-like growth factor-II (IGF-II) is a naturally occurring hormone that exerts neurotrophic and neuroprotective properties in a wide range of neurodegenerative diseases and ageing. Accumulating evidence suggests that the effects of IGF-II in the brain may be explained by its binding to the specific transmembrane receptor, IGFII/M6P receptor (IGF-IIR). However, relatively little is known regarding the role of IGF-II through IGF-IIR in neuroprotection. Here, using adult cortical neuronal cultures, we investigated whether IGF-II exhibits long-term antioxidant effects and neuroprotection at the synaptic level after oxidative damage induced by high and transient levels of corticosterone (CORT). Furthermore, the involvement of the IGF-IIR was also studied to elucidate its role in the neuroprotective actions of IGF-II. We found that neurons treated with IGF-II after CORT incubation showed reduced oxidative stress damage and recovered antioxidant status (normalized total antioxidant status, lipid hydroperoxides and NAD(P) H:quinone oxidoreductase activity). Similar results were obtained when mitochondria function was analysed (cytochrome c oxidase activity, mitochondrial membrane potential and subcellular mitochondrial distribution). Furthermore, neuronal impairment and degeneration were also assessed (synaptophysin and PSD-95 expression, presynaptic function and FluoroJade B® stain). IGF-II was also able to recover the long-lasting neuronal cell damage. Finally, the effects of IGF-II were not blocked by an IGF-IR antagonist, suggesting the involvement of IGF-IIR. Altogether these results suggest that, in or model, IGF-II through IGF-IIR is able to revert the oxidative damage induced by CORT. In accordance with the neuroprotective role of the IGF-II/IGF-IIR reported in our study, pharmacotherapy approaches targeting this pathway may be useful for the treatment of diseases associated with cognitive deficits (i.e., neurodegenerative disorders, depression, etc.)., Graphical abstract fx1, Highlights • First evidence that IGF-II reverts oxidative synaptic damage produced by corticoids. • IGF-II recovers mitochondrial function in synapses after oxidative damage. • IGF-II restores mitochondrial distribution in neurons after oxidative damage. • Evidence of the involvement of IGF-II receptor in the recovery of synaptic function. • IGF-II reverts neurodegeneration induced by oxidative damage produced by corticoids.

Converging evidence indicates that abnormal glutamatergic and synaptic systems may be associated with the pathophysiology of depression. Over-activation of corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN) plays a key role in the hypothalamic-pituitary-adrenal (HPA) axis hyperactivity and is a prominent feature in depression. In this study, we examined the effect of chronic unpredictable stress (CUS) in rats on the expression of AMPA subunit GluR1 protein in the hypothalamic PVN as well as on the expression of GluR1 and several synapse-related proteins in the hypothalamus. We found that CUS in rats induced CRH over-expression accompanied by significant GluR1 up-regulation in the hypothalamic PVN and GluR1 co-localized with CRH in the PVN area. The expression of GluR1 and three synapse-related proteins (PSD95, SYP and MAP2) in the hypothalamus were also increased at both mRNA and protein levels in CUS rats. Correlation analysis showed that the levels of the above proteins are directly correlated with depression-like behaviors of rats in sucrose preference test, open field test and forced swim test. These findings suggest that the augmented glutamatergic and synaptic signaling might be a potential mechanism underlying CRH over-activation in the hypothalamic PVN and contribute to CUS-induced depression-like behaviors in rats.

Background: Premature infants are deprived of prenatal accumulation of brain docosahexaenoic acid [DHA (22:6n-3)], an omega-3 fatty acid [ω-3 FA (n-3 FA)] important for proper development of cognitive function. The resulting brain DHA deficit can be reversed by ω-3 FA supplementation.Objective: The objective was to test whether there is a critical period for providing ω-3 FA to correct cognitive deficits caused by developmental ω-3 FA deprivation in mice.Methods: Twelve timed-pregnant mice [embryonic day 14 (E14), C57/BL6NCr] were fed an ω-3 FA-deficient diet containing 0.04% α-linolenic acid [ALA (18:3n-3)], and their offspring were fed the same deficient diet (Def group) or changed to an ω-3 FA-adequate diet containing 3.1% ALA at 3 wk, 2 mo, or 4 mo of age. In parallel, 3 E14 pregnant mice were fed the adequate diet and their offspring were fed the same diet (Adeq group) throughout the experiment. Brain FA composition, learning and memory, and hippocampal synaptic protein expression were evaluated at 6 mo by gas chromatography, the Morris water maze test, and western blot analysis, respectively.Results: Maternal dietary ω-3 FA deprivation decreased DHA by >50% in the brain of their offspring at 3 wk of age. The Def group showed significantly worse learning and memory at 6 mo than those groups fed the adequate diet. These pups also had decreased hippocampal expression of postsynaptic density protein 95 (43% of Adeq group), Homer protein homolog 1 (21% of Adeq group), and synaptosome-associated protein of 25 kDa (64% of Adeq group). Changing mice to the adequate diet at 3 wk, 2 mo, or 4 mo of age restored brain DHA to the age-matched adequate concentration. However, deficits in hippocampal synaptic protein expression and spatial learning and memory were normalized only when the diet was changed at 3 wk.Conclusion: Developmental deprivation of brain DHA by dietary ω-3 FA depletion in mice may have a lasting impact on cognitive function if not corrected at an early age.

Postsynaptic density 95 (PSD-95) is a major synaptic scaffolding protein that plays a key role in bidirectional synaptic plasticity, which is a process important for learning and memory. It is known that PSD-95 shows increased dynamics upon induction of plasticity. However, the underlying structural and functional changes in PSD-95 that mediate its role in plasticity remain unclear. Here we show that phosphorylation of PSD-95 at Ser-561 in its guanylate kinase (GK) domain, which is mediated by the partitioning-defective 1 (Par1) kinases, regulates a conformational switch and is important for bidirectional plasticity. Using a fluorescence resonance energy transfer (FRET) biosensor, we show that a phosphomimetic mutation of Ser-561 promotes an intramolecular interaction between GK and the nearby Src homology 3 (SH3) domain, leading to a closed conformation, whereas a non-phosphorylatable S561A mutation or inhibition of Par1 kinase activity decreases SH3-GK interaction, causing PSD-95 to adopt an open conformation. In addition, S561A mutation facilitates the interaction between PSD-95 and its binding partners. Fluorescence recovery after photobleaching imaging reveals that the S561A mutant shows increased stability, whereas the phosphomimetic S561D mutation increases PSD-95 dynamics at the synapse. Moreover, molecular replacement of endogenous PSD-95 with the S561A mutant blocks dendritic spine structural plasticity during chemical long-term potentiation and long-term depression. Endogenous Ser-561 phosphorylation is induced by synaptic NMDA receptor activation, and the SH3-GK domains exhibit a Ser-561 phosphorylation-dependent switch to a closed conformation during synaptic plasticity. Our results provide novel mechanistic insight into the regulation of PSD-95 in dendritic spine structural plasticity through phosphorylation-mediated regulation of protein dynamics and conformation.

We recently demonstrated that prenatal exposure to valproic acid (VPA) at embryonic day 12.5 causes autism spectrum disorder (ASD)-like phenotypes such as hypolocomotion, anxiety-like behavior, social deficits and cognitive impairment in mice and that it decreases dendritic spine density in the hippocampal CA1 region. Previous studies show that some abnormal behaviors are improved by environmental enrichment in ASD rodent models, but it is not known whether environmental enrichment improves cognitive impairment. In the present study, we examined the effects of early environmental enrichment on behavioral abnormalities and neuromorphological changes in prenatal VPA-treated mice. We also examined the role of dendritic spine formation and synaptic protein expression in the hippocampus. Mice were housed for 4 weeks from 4 weeks of age under either a standard or enriched environment. Enriched housing was found to increase hippocampal brain-derived neurotrophic factor mRNA levels in both control and VPA-exposed mice. Furthermore, in VPA-treated mice, the environmental enrichment improved anxiety-like behavior, social deficits and cognitive impairment, but not hypolocomotion. Prenatal VPA treatment caused loss of dendritic spines in the hippocampal CA1 region and decreases in mRNA levels of postsynaptic density protein-95 and SH3 and multiple ankyrin repeat domains 2 in the hippocampus. These hippocampal changes were improved by the enriched housing. These findings suggest that the environmental enrichment improved most ASD-like behaviors including cognitive impairment in the VPA-treated mice by enhancing dendritic spine function.