Literature context: lipore, Catalog #AB_1504; RRID:AB_2113602), rabbit anti-GluA2/3 (0.1 Î¼g/m
Astrocyte-derived factors can control synapse formation and functions, making astrocytes an attractive target for regulating neuronal circuits and associated behaviors. Abnormal astrocyte-neuronal interactions are also implicated in neurodevelopmental disorders and neurodegenerative diseases associated with impaired learning and memory. However, little is known about astrocyte-mediated mechanisms that regulate learning and memory. Here, we propose astrocytic ephrin-B1 as a regulator of synaptogenesis in adult hippocampus and mouse learning behaviors. We found that astrocyte-specific ablation of ephrin-B1 in male mice triggers an increase in the density of immature dendritic spines and excitatory synaptic sites in the adult CA1 hippocampus. However, the prevalence of immature dendritic spines is associated with decreased evoked postsynaptic firing responses in CA1 pyramidal neurons, suggesting impaired maturation of these newly formed and potentially silent synapses or increased excitatory drive on the inhibitory neurons resulting in the overall decreased postsynaptic firing. Nevertheless, astrocyte-specific ephrin-B1 knock-out male mice exhibit normal acquisition of fear memory but enhanced contextual fear memory recall. In contrast, overexpression of astrocytic ephrin-B1 in the adult CA1 hippocampus leads to the loss of dendritic spines, reduced excitatory input, and impaired contextual memory retention. Our results suggest that astrocytic ephrin-B1 may compete with neuronal ephrin-B1 and mediate excitatory synapse elimination through its interactions with neuronal EphB receptors. Indeed, a deletion of neuronal EphB receptors impairs the ability of astrocytes expressing functional ephrin-B1 to engulf synaptosomes in vitro Our findings demonstrate that astrocytic ephrin-B1 regulates long-term contextual memory by restricting new synapse formation in the adult hippocampus.SIGNIFICANCE STATEMENT These studies address a gap in our knowledge of astrocyte-mediated regulation of learning and memory by unveiling a new role for ephrin-B1 in astrocytes and elucidating new mechanisms by which astrocytes regulate learning. Our studies explore the mechanisms underlying astrocyte regulation of hippocampal circuit remodeling during learning using new genetic tools that target ephrin-B signaling in astrocytes in vivo On a subcellular level, astrocytic ephrin-B1 may compete with neuronal ephrin-B1 and trigger astrocyte-mediated elimination of EphB receptor-containing synapses. Given the role EphB receptors play in neurodevelopmental disorders and neurodegenerative diseases, these findings establish a foundation for future studies of astrocyte-mediated synaptogenesis in clinically relevant conditions that can help to guide the development of clinical applications for a variety of neurological disorders.
Literature context: olution, Millipore Cat# AB_1504 RRID:AB_2113602) for 72â€¯h rocking at 4â€¯Â°C. Labe
Neural networks that undergo acute insults display remarkable reorganization. This injury related plasticity is thought to permit recovery of function in the face of damage that cannot be reversed. Previously, an increase in the transmission strength at Schaffer collateral to CA1 pyramidal cell synapses was observed after long-term activity reduction in organotypic hippocampal slices. Here we report that, following acute preparation of adult rat hippocampal slices and surgical removal of area CA3, input to area CA1 was reduced and Schaffer collateral synapses underwent functional strengthening. This increase in synaptic strength was limited to Schaffer collateral inputs (no alteration to temporoammonic synapses) and acted to normalize postsynaptic discharge, supporting a homeostatic or compensatory response. Short-term plasticity was not altered, but an increase in immunohistochemical labeling of GluA1 subunits was observed in the stratum radiatum (but not stratum moleculare), suggesting increased numbers of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and a postsynaptic locus of expression. Combined, these data support the idea that, in response to the reduction in presynaptic activity caused by removal of area CA3, Schaffer collateral synapses undergo a relatively rapid increase in functional efficacy likely supported by insertion of more AMPARs, which maintains postsynaptic excitability in CA1 pyramidal neurons. This novel fast compensatory plasticity exhibits properties that would allow it to maintain optimal network activity levels in the hippocampus, a brain structure lauded for its ongoing experience-dependent malleability.
Literature context: llipore catalog number AB_1504; RRID:AB_2113602 dilution 1:1000 in I-Block
The onset of abnormal movements in DYT1 dystonia is between childhood and adolescence, although it is unclear why clinical manifestations appear during this developmental period. Plasticity at corticostriatal synapses is critically involved in motor memory. In the Tor1a+/Δgag DYT1 dystonia mouse model, long-term potentiation (LTP) appeared prematurely in a critical developmental window in striatal spiny neurons (SPNs), while long-term depression (LTD) was never recorded. Analysis of dendritic spines showed an increase of both spine width and mature mushroom spines in Tor1a+/Δgag neurons, paralleled by an enhanced AMPA receptor (AMPAR) accumulation. BDNF regulates AMPAR expression during development. Accordingly, both proBDNF and BDNF levels were significantly higher in Tor1a+/Δgag mice. Consistently, antagonism of BDNF rescued synaptic plasticity deficits and AMPA currents. Our findings demonstrate that early loss of functional and structural synaptic homeostasis represents a unique endophenotypic trait during striatal maturation, promoting the appearance of clinical manifestations in mutation carriers.
Literature context: 1:100050, 60GluA1MilliporeAb150421136021:1000100GluA2MilliporeMab397211
A homozygous nonsense mutation in the cereblon (CRBN) gene results in autosomal recessive, nonsyndromic intellectual disability that is devoid of other phenotypic features, suggesting a critical role of CRBN in mediating learning and memory. In this study, we demonstrate that adult male Crbn knock-out (CrbnKO) mice exhibit deficits in hippocampal-dependent learning and memory tasks that are recapitulated by focal knock-out of Crbn in the adult dorsal hippocampus, with no changes in social or repetitive behavior. Cellular studies identify deficits in long-term potentiation at Schaffer collateral CA1 synapses. We further show that Crbn is robustly expressed in the mouse hippocampus and CrbnKO mice exhibit hyperphosphorylated levels of AMPKα (Thr172). Examination of processes downstream of AMP-activated protein kinase (AMPK) finds that CrbnKO mice have a selective impairment in mediators of the mTORC1 translation initiation pathway in parallel with lower protein levels of postsynaptic density glutamatergic proteins and higher levels of excitatory presynaptic markers in the hippocampus with no change in markers of the unfolded protein response or autophagy pathways. Acute pharmacological inhibition of AMPK activity in adult CrbnKO mice rescues learning and memory deficits and normalizes hippocampal mTORC1 activity and postsynaptic glutamatergic proteins without altering excitatory presynaptic markers. Thus, this study identifies that loss of Crbn results in learning, memory, and synaptic defects as a consequence of exaggerated AMPK activity, inhibition of mTORC1 signaling, and decreased glutamatergic synaptic proteins. Thus, CrbnKO mice serve as an ideal model of intellectual disability to further explore molecular mechanisms of learning and memory.SIGNIFICANCE STATEMENT Intellectual disability (ID) is one of the most common neurodevelopmental disorders. The cereblon (CRBN) gene has been linked to autosomal recessive, nonsyndromic ID, characterized by an intelligence quotient between 50 and 70 but devoid of other phenotypic features, making cereblon an ideal protein for the study of the fundamental aspects of learning and memory. Here, using the cereblon knock-out mouse model, we show that cereblon deficiency disrupts learning, memory, and synaptic function via AMP-activated protein kinase hyperactivity, downregulation of mTORC1, and dysregulation of excitatory synapses, with no changes in social or repetitive behaviors, consistent with findings in the human population. This establishes the cereblon knock-out mouse as a model of pure ID without the confounding behavioral phenotypes associated with other current models of ID.
Literature context: 0; Millipore, catalog #AB_1504, RRID:AB_2113602) antisera for 24 h at room temp
Exposure to cocaine-associated contextual cues contributes significantly to relapse. Extinction of these contextual associations, which involves a new form of learning, reduces cocaine-seeking behavior; however, the molecular mechanisms underlying this process remain largely unknown. We report that extinction, but not acquisition, of cocaine conditioned place preference (CPP) in male mice increased Cav1.2 L-type Ca2+ channel mRNA and protein in postsynaptic density (PSD) fractions of the hippocampus, a brain region involved in drug-context associations. Moreover, viral-mediated deletion of Cav1.2 in the dorsal hippocampus attenuated extinction of cocaine CPP. Molecular studies examining downstream Cav1.2 targets revealed that extinction recruited calcium/calmodulin (Ca2+/CaMK)-dependent protein kinase II (CaMKII) to the hippocampal PSD. This occurred in parallel with an increase in phosphorylation of the AMPA GluA1 receptor subunit at serine 831 (S831), a CaMKII site, along with an increase in total PSD GluA1. The necessity of S831 GluA1 was further demonstrated by the lack of extinction in S831A GluA1 phosphomutant mice. Of note hippocampal GluA1 levels remained unaltered at the PSD, but were reduced near the PSD and at perisynaptic sites of dendritic spines in extinction-resistant S831A mutant mice. Finally, conditional knock-out of Cav1.2 in dopamine D1 receptor (D1R)-expressing cells resulted in attenuation of cocaine CPP extinction and lack of extinction-dependent changes in hippocampal PSD CaMKII expression and S831 GluA1 phosphorylation. In summary, we demonstrate an essential role for the hippocampal Cav1.2/CaMKII/S831 GluA1 pathway in cocaine CPP extinction, with data supporting contribution of hippocampal D1R-expressing cells in this process. These findings demonstrate a novel role for Cav1.2 channels in extinction of contextual cocaine-associated memories.SIGNIFICANCE STATEMENT Continued drug-seeking behavior, a defining characteristic of cocaine addiction, can be precipitated by contextual cues, yet the molecular mechanisms required for extinction of these context-specific memories remain poorly understood. Here, we have uncovered a novel and selective role of the Cav1.2 L-type Ca2+ channel and its downstream signaling pathway in the hippocampus that mediate extinction of cocaine conditioned place preference (CPP). We additionally provide evidence that supports a role of Cav1.2 within dopamine D1 receptor-expressing cells of the hippocampus for extinction of cocaine CPP. Therefore, these findings reveal a previously unknown role of Cav1.2 channels within the hippocampus and in D1 receptor-expressing cells in extinction of cocaine-associated memories, providing a framework for further exploration of mechanisms underlying extinction of cocaine-seeking behavior.
Literature context: llipore CAT# AB_1504; RRID:AB_2113602 Mouse monoclonal anti-NeuN Mill
The generation of precise synaptic connections between developing neurons is critical to the formation of functional neural circuits. Astrocyte-secreted glypican 4 induces formation of active excitatory synapses by recruiting AMPA glutamate receptors to the postsynaptic cell surface. We now identify the molecular mechanism of how glypican 4 exerts its effect. Glypican 4 induces release of the AMPA receptor clustering factor neuronal pentraxin 1 from presynaptic terminals by signaling through presynaptic protein tyrosine phosphatase receptor δ. Pentraxin then accumulates AMPA receptors on the postsynaptic terminal forming functional synapses. Our findings reveal a signaling pathway that regulates synaptic activity during central nervous system development and demonstrates a role for astrocytes as organizers of active synaptic connections by coordinating both pre and post synaptic neurons. As mutations in glypicans are associated with neurological disorders, such as autism and schizophrenia, this signaling cascade offers new avenues to modulate synaptic function in disease.
Literature context: t#AB1504, RRID:AB_2113602), mouse an
OBJECTIVE: The roles of hippocampal AMPARs were investigated in irritable bowel syndrome (IBS)-like rats to clarify the central sensitization mechanisms. METHODS: IBS model was induced by neonatal maternal separation. The effects of AMPARs on visceral hypersensitivity were examined by the responses of abdominal muscle to colorectal distension after the bilateral intrahippocampal injections of CNQX (an AMPAR inhibitor). The expressions of hippocampal AMPARs (GluR1 and GluR2) were determined by Western blot. RESULTS: The IBS-like rats showed visceral hypersensitivity when compared with controls. Bilateral intrahippocampal injections of CNQX alleviated the visceral pain in IBS-like rats. The maximal effect appeared at the time point of 30 min, and the duration lasted for 90 min after CNQX application, under 40 and 60 mmHg CRD. The expressions of hippocampal GluR2 significantly increased in IBS-like rats when compared with controls (p < .05). However, the levels of hippocampal GluR1 had no significant differences in rats. Hippocampal LTP induced by HFS was significantly enhanced when compared with controls (p < .05). The expressions of GluR2 significantly increased in the control and IBS-like rats after 60 min LTP of recordings (p < .05), but not GluR1. CONCLUSION: Neonatal maternal separation enhances the expression of GluR2 and facilitates the LTP in the hippocampus, which could lead to the formation of visceral hypersensitivity when grown up.
Literature context: illipore (RRID:AB_2113602)], and Glu
Modification of the strength of excitatory synaptic connections is a fundamental mechanism by which neural circuits are refined during development and learning. Synapse Differentiation Induced Gene 1 (SynDIG1) has been shown to play a key role in regulating synaptic strength in vitro. Here, we investigated the role of SynDIG1 in vivo in mice with a disruption of the SynDIG1 gene rather than use an alternate loxP-flanked conditional mutant that we find retains a partial protein product. The gene-trap insertion with a reporter cassette mutant mice shows that the SynDIG1 promoter is active during embryogenesis in the retina with some activity in the brain, and postnatally in the mouse hippocampus, cortex, hindbrain, and spinal cord. Ultrastructural analysis of the hippocampal CA1 region shows a decrease in the average PSD length of synapses and a decrease in the number of synapses with a mature phenotype. Intriguingly, the total synapse number appears to be increased in SynDIG1 mutant mice. Electrophysiological analyses show a decrease in AMPA and NMDA receptor function in SynDIG1-deficient hippocampal neurons. Glutamate stimulation of individual dendritic spines in hippocampal slices from SynDIG1-deficient mice reveals increased short-term structural plasticity. Notably, the overall levels of PSD-95 or glutamate receptors enriched in postsynaptic biochemical fractions remain unaltered; however, activity-dependent synapse development is strongly compromised upon the loss of SynDIG1, supporting its importance for excitatory synapse maturation. Together, these data are consistent with a model in which SynDIG1 regulates the maturation of excitatory synapse structure and function in the mouse hippocampus in vivo.
Literature context: # AB_1504 RRID:AB_2113602 Guinea pig anti-vGLUT1 Millipor
Hippocampal CA3 neurons form synapses with CA1 neurons in two layers, stratum oriens (SO) and stratum radiatum (SR). Each layer develops unique synaptic properties but molecular mechanisms that mediate these differences are unknown. Here, we show that SO synapses normally have significantly more mushroom spines and higher-magnitude long-term potentiation (LTP) than SR synapses. Further, we discovered that these differences require the Type II classic cadherins, cadherins-6, -9, and -10. Though cadherins typically function via trans-cellular homophilic interactions, our results suggest presynaptic cadherin-9 binds postsynaptic cadherins-6 and -10 to regulate mushroom spine density and high-magnitude LTP in the SO layer. Loss of these cadherins has no effect on the lower-magnitude LTP typically observed in the SR layer, demonstrating that cadherins-6, -9, and -10 are gatekeepers for high-magnitude LTP. Thus, Type II cadherins may uniquely contribute to the specificity and strength of synaptic changes associated with learning and memory.
Literature context: _1504 RRID:AB_2113602 PSD-95 (1:1000) NeuroMab Cat# 7
Most disease variants lie within noncoding genomic regions, making their functional interpretation challenging. Because chromatin openness strongly influences transcriptional activity, we hypothesized that cell-type-specific open chromatin regions (OCRs) might highlight disease-relevant noncoding sequences. To investigate, we mapped global OCRs in neurons differentiating from hiPSCs, a cellular model for studying neurodevelopmental disorders such as schizophrenia (SZ). We found that the OCRs are highly dynamic and can stratify GWAS-implicated SZ risk variants. Of the more than 3,500 SZ-associated variants analyzed, we prioritized ∼100 putatively functional ones located in neuronal OCRs, including rs1198588, at a leading risk locus flanking MIR137. Excitatory neurons derived from hiPSCs with CRISPR/Cas9-edited rs1198588 or a rare proximally located SZ risk variant showed altered MIR137 expression, dendrite arborization, and synapse maturation. Our study shows that noncoding disease variants in OCRs can affect neurodevelopment, and that analysis of open chromatin regions can help prioritize functionally relevant noncoding variants identified by GWAS.
Literature context: antibody (RRID:AB_2113602) and anti-
Homocysteine, a metabolite of the methionine cycle has been reported to play a role in neurotoxicity through activation of N-methyl-d-aspartate receptors (NMDAR)-mediated signaling pathway. The proposed mechanisms associated with homocysteine-NMDAR-induced neurotoxicity involve a unique signaling pathway that triggers a crosstalk between extracellular signal-regulated kinase (ERK) and p38 MAPKs, where activation of p38 MAPK is downstream of and dependent on ERK MAPK. However, the molecular basis of the ERK MAPK-mediated p38 MAPK activation is not understood. This study investigates whether α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) play a role in facilitating the ERK MAPK-mediated p38 MAPK activation. Using surface biotinylation and immunoblotting approaches we show that treatment with homocysteine leads to a decrease in surface expression of GluA2-AMPAR subunit in neurons, but have no effect on the surface expression of GluA1-AMPAR subunit. Inhibition of NMDAR activation with D-AP5 or ERK MAPK phosphorylation with PD98059 attenuates homocysteine-induced decrease in surface expression of GluA2-AMPAR subunit. The decrease in surface expression of GluA2-AMPAR subunit is associated with p38 MAPK phosphorylation, which is inhibited by 1-napthyl acetyl spermine trihydrochloride (NASPM), a selective antagonist of GluA2-lacking Ca2+ -permeable AMPARs. These results suggest that homocysteine-NMDAR-mediated ERK MAPK phosphorylation leads to a decrease in surface expression of GluA2-AMPAR subunit resulting in Ca2+ influx through the GluA2-lacking Ca2+ -permeable AMPARs and p38 MAPK phosphorylation. Cell death assays further show that inhibition of AMPAR activity with 2,3-dioxo-6-nitro-1,2,3,4,tetrahydrobenzoquinoxaline-7-sulfonamide (NBQX)/6-cyano-7-nitroquinoxaline-2,3, -dione (CNQX) or GluA2-lacking Ca2+ -permeable AMPAR activity with NASPM attenuates homocysteine-induced neurotoxicity. We have identified an important mechanism involved in homocysteine-induced neurotoxicity that highlights the intermediary role of GluA2-lacking Ca2+ -permeable AMPARs in the crosstalk between ERK and p38 MAPKs.
Literature context: ore, Cat# AB_1504, RRID:AB_2113602), GluA2 (1:1000, EMD Millipore,
Inhibition of monoacylglycerol lipase (MAGL), the primary enzyme that hydrolyzes the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain, produces profound anti-inflammatory and neuroprotective effects and improves synaptic and cognitive functions in animal models of Alzheimer's disease (AD). However, the molecular mechanisms underlying the beneficial effects produced by inhibition of 2-AG metabolism are still not clear. The cannabinoid receptor type 2 (CB2R) has been thought to be a therapeutic target for AD. Here, we provide evidence, however, that CB2R does not play a role in ameliorating AD neuropathology produced by inactivation of MAGL in 5XFAD APP transgenic mice, an animal model of AD. We observed that expression of APP and β-secretase as well as production of total Aβ and Aβ42 were significantly reduced in APP transgenic mice lacking CB2R (TG-CB2-KO) treated with JZL184, a selective and potent inhibitor for MAGL. Inactivation of MAGL also alleviated neuroinflammation and neurodegeneration in TG-CB2-KO mice. Importantly, TG-CB2-KO mice treated with JZL184 still exhibited improvements in spatial learning and memory. In addition, MAGL inhibition prevented deterioration in expression of important synaptic proteins in TG-CB2-KO mice. Our results suggest that CB2R is not required in ameliorating neuropathology and preventing cognitive decline by inhibition of 2-AG metabolism in AD model animals.
Literature context: # AB1504, RRID:AB_2113602), GluA2 (1
The formation of functional synapses requires coordinated assembly of presynaptic transmitter release machinery and postsynaptic trafficking of functional receptors and scaffolds. Here, we demonstrate a critical role of presynaptic cadherin/catenin cell adhesion complexes in stabilizing functional synapses and spines in the developing neocortex. Importantly, presynaptic expression of stabilized β-catenin in either layer (L) 4 excitatory neurons or L2/3 pyramidal neurons significantly upregulated excitatory synaptic transmission and dendritic spine density in L2/3 pyramidal neurons, while its sparse postsynaptic expression in L2/3 neurons had no such effects. In addition, presynaptic β-catenin expression enhanced release probability of glutamatergic synapses. Newly identified β-catenin-interacting protein p140Cap is required in the presynaptic locus for mediating these effects. Together, our results demonstrate that cadherin/catenin complexes stabilize functional synapses and spines through anterograde signaling in the neocortex and provide important molecular evidence for a driving role of presynaptic components in spinogenesis in the neocortex.
Literature context: 1:500 dilutionAnti-GluR1, RRID: AB_2213602KLH-conjugated linear peptide co
Dopamine- and tyrosine hydroxylase-immunopositive cells (TH cells) modulate visually driven signals as they flow through retinal photoreceptor, bipolar, and ganglion cells. Previous studies suggested that TH cells release dopamine from varicose axons arborizing in the inner and outer plexiform layers after glutamatergic synapses depolarize TH cell dendrites in the inner plexiform layer and these depolarizations propagate to the varicosities. Although it has been proposed that these excitatory synapses are formed onto appendages resembling dendritic spines, spines have not been found on TH cells of most species examined to date or on TH cell somata that release dopamine when exposed to glutamate receptor agonists. By use of protocols that preserve proximal retinal neuron morphology, we have examined the shape, distribution, and synapse-related immunoreactivity of adult rat TH cells. We report here that TH cell somata, tapering and varicose inner plexiform layer neurites, and varicose outer plexiform layer neurites all bear spines, that some of these spines are immunopositive for glutamate receptor and postsynaptic density proteins (viz., GluR1, GluR4, NR1, PSD-95, and PSD-93), that TH cell somata and tapering neurites are also immunopositive for a γ-aminobutyric acid (GABA) receptor subunit (GABAA Rα1 ), and that a synaptic ribbon-specific protein (RIBEYE) is found adjacent to some colocalizations of GluR1 and TH in the inner plexiform layer. These results identify previously undescribed sites at which glutamatergic and GABAergic inputs may stimulate and inhibit dopamine release, especially at somata and along varicose neurites that emerge from these somata and arborize in various levels of the retina. J. Comp. Neurol. 525:1707-1730, 2017. © 2016 Wiley Periodicals, Inc.
Literature context: re AB1504 RRID:AB_2113602 GluA2 Mous
Deeper exploration of the brain's vast synaptic networks will require new tools for high-throughput structural and molecular profiling of the diverse populations of synapses that compose those networks. Fluorescence microscopy (FM) and electron microscopy (EM) offer complementary advantages and disadvantages for single-synapse analysis. FM combines exquisite molecular discrimination capacities with high speed and low cost, but rigorous discrimination between synaptic and non-synaptic fluorescence signals is challenging. In contrast, EM remains the gold standard for reliable identification of a synapse, but offers only limited molecular discrimination and is slow and costly. To develop and test single-synapse image analysis methods, we have used datasets from conjugate array tomography (cAT), which provides voxel-conjugate FM and EM (annotated) images of the same individual synapses. We report a novel unsupervised probabilistic method for detection of synapses from multiplex FM (muxFM) image data, and evaluate this method both by comparison to EM gold standard annotated data and by examining its capacity to reproduce known important features of cortical synapse distributions. The proposed probabilistic model-based synapse detector accepts molecular-morphological synapse models as user queries, and delivers a volumetric map of the probability that each voxel represents part of a synapse. Taking human annotation of cAT EM data as ground truth, we show that our algorithm detects synapses from muxFM data alone as successfully as human annotators seeing only the muxFM data, and accurately reproduces known architectural features of cortical synapse distributions. This approach opens the door to data-driven discovery of new synapse types and their density. We suggest that our probabilistic synapse detector will also be useful for analysis of standard confocal and super-resolution FM images, where EM cross-validation is not practical.
Literature context: t#AB1504, RRID:AB_2113602 GABAar Mil
Synaptic excitation mediates a broad spectrum of structural changes in neural circuits across the brain. Here, we examine the morphologies, wiring, and architectures of single synapses of projection neurons in the murine hippocampus that developed in virtually complete absence of vesicular glutamate release. While these neurons had smaller dendritic trees and/or formed fewer contacts in specific hippocampal subfields, their stereotyped connectivity was largely preserved. Furthermore, loss of release did not disrupt the morphogenesis of presynaptic terminals and dendritic spines, suggesting that glutamatergic neurotransmission is unnecessary for synapse assembly and maintenance. These results underscore the instructive role of intrinsic mechanisms in synapse formation.
Literature context: t# AB1504 RRID:AB_2113602); mouse an
Neuronal activity is regulated in a homeostatic manner through changes in inhibitory GABA and excitatory glutamate (Glu) AMPA (A) receptors (GluARs). Using immunofluorescent staining, we examined whether calcium/calmodulin-dependent protein kinase IIα (CaMKIIα)-labeled (+) excitatory neurons in the barrel cortex undergo such homeostatic regulation following enforced waking with associated cortical activation during the day when mice normally sleep the majority of the time. Sleep deprived mice were prevented from falling asleep by unilateral whisker stimulation and sleep recovery (SR) mice allowed to sleep freely following deprivation. In parallel with changes in c-Fos reflecting changes in activity, (β2-3 subunits of) GABAA Rs were increased on the membrane of CaMKIIα+ neurons with enforced waking and returned to baseline levels with SR in barrel cortex on sides both contra- and ipsilateral to the whisker stimulation. The GABAAR increase was correlated with increased gamma electroencephalographic (EEG) activity across conditions. On the other hand, (GluA1 subunits of) AMPA Rs were progressively removed from the membrane of CaMKIIα+ neurons by (Rab5+) early endosomes during enforced waking and returned to the membrane by (Rab11+) recycling endosomes during SR. The internalization of the GluA1Rs paralleled the expression of Arc, which mediates homeostatic regulation of AMPA receptors through an endocytic pathway. The reciprocal changes in GluA1Rs relative to GABAARs suggest homeostatic down-scaling during enforced waking and sensory stimulation and restorative up-scaling during recovery sleep. Such homeostatic changes with sleep-wake states and their associated cortical activities could stabilize excitability and activity in excitatory cortical neurons.
Literature context: illipore; RRID:AB_2113602) diluted 1
Cognitive impairments, uncontrolled drinking, and neuropathological cortical changes characterize alcohol use disorder. Dysfunction of the orbitofrontal cortex (OFC), a critical cortical subregion that controls learning, decision-making, and prediction of reward outcomes, contributes to executive cognitive function deficits in alcoholic individuals. Electrophysiological and quantitative synaptomics techniques were used to test the hypothesis that heavy drinking produces neuroadaptations in the macaque OFC. Integrative bioinformatics and reverse genetic approaches were used to identify and validate synaptic proteins with novel links to heavy drinking in BXD mice. In drinking monkeys, evoked firing of OFC pyramidal neurons was reduced, whereas the amplitude and frequency of postsynaptic currents were enhanced compared with controls. Bath application of alcohol reduced evoked firing in neurons from control monkeys, but not drinking monkeys. Profiling of the OFC synaptome identified alcohol-sensitive proteins that control glutamate release (e.g., SV2A, synaptogyrin-1) and postsynaptic signaling (e.g., GluA1, PRRT2) with no changes in synaptic GABAergic proteins. Western blot analysis confirmed the increase in GluA1 expression in drinking monkeys. An exploratory analysis of the OFC synaptome found cross-species genetic links to alcohol intake in discrete proteins (e.g., C2CD2L, DIRAS2) that discriminated between low- and heavy-drinking monkeys. Validation studies revealed that BXD mouse strains with the D allele at the C2cd2l interval drank less alcohol than B allele strains. Thus, by profiling of the OFC synaptome, we identified changes in proteins controlling glutamate release and postsynaptic signaling and discovered several proteins related to heavy drinking that have potential as novel targets for treating alcohol use disorder.SIGNIFICANCE STATEMENT Clinical research identified cognitive deficits in alcoholic individuals as a risk factor for relapse, and alcoholic individuals display deficits on cognitive tasks that are dependent upon the orbitofrontal cortex (OFC). To identify neurobiological mechanisms that underpin OFC dysfunction, this study used electrophysiology and integrative synaptomics in a translational nonhuman primate model of heavy alcohol consumption. We found adaptations in synaptic proteins that control glutamatergic signaling in chronically drinking monkeys. Our functional genomic exploratory analyses identified proteins with genetic links to alcohol and cocaine intake across mice, monkeys, and humans. Future work is necessary to determine whether targeting these novel targets reduces excessive and harmful levels of alcohol drinking.
Literature context: t# AB1504 RRID:AB_2113602; HA 1:1000
The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) are major excitatory receptors that mediate fast neurotransmission in the mammalian brain. The surface expression of functional AMPARs is crucial for synaptic transmission and plasticity. AMPAR auxiliary subunits control the biosynthesis, membrane trafficking, and synaptic targeting of AMPARs. Our previous report showed that α/β-hydrolase domain-containing 6 (ABHD6), an auxiliary subunit for AMPARs, suppresses the membrane delivery and function of GluA1-containing receptors in both heterologous cells and neurons. However, it remained unclear whether ABHD6 affects the membrane trafficking of glutamate receptor subunits, GluA2 and GluA3. Here, we examine the effects of ABHD6 overexpression in HEK293T cells expressing GluA1, GluA2, GluA3, and stargazin, either alone or in combination. The results show that ABHD6 suppresses the glutamate-induced currents and the membrane expression of AMPARs when expressing GluA2 or GluA3 in the HEK293T cells. We generated a series of GluA2 and GluA3 C-terminal deletion constructs and confirm that the C-terminus of GluAs is required for ABHD6's inhibitory effects on glutamate-induced currents and surface expression of GluAs. Meanwhile, our pull-down experiments reveal that ABHD6 binds to GluA1-3, and deletion of the C-terminal domain of GluAs abolishes this binding. These findings demonstrate that ABHD6 inhibits the AMPAR-mediated currents and its surface expression, independent of the type of AMPAR subunits, and this inhibitor's effects are mediated through the binding with the GluAs C-terminal regions.
Literature context: . AB1504, RRID:AB_2113602) antibody
The modulation of AMPA receptor (AMPAR) content at synapses is thought to be an underlying molecular mechanism of memory and learning. AMPAR content at synapses is highly plastic and is regulated by numerous AMPAR accessory transmembrane proteins such as TARPs, cornichons, and CKAMPs. SynDIG (synapse differentiation-induced gene) defines a family of four genes (SynDIG1-4) expressed in distinct and overlapping patterns in the brain. SynDIG1 was previously identified as a novel transmembrane AMPAR-associated protein that regulates synaptic strength. The related protein SynDIG4 [also known as Prrt1 (proline-rich transmembrane protein 1)] has recently been identified as a component of AMPAR complexes. In this study, we show that SynDIG1 and SynDIG4 have distinct yet overlapping patterns of expression in the central nervous system, with SynDIG4 having especially prominent expression in the hippocampus and particularly within CA1. In contrast to SynDIG1 and other traditional AMPAR auxiliary subunits, SynDIG4 is de-enriched at the postsynaptic density and colocalizes with extrasynaptic GluA1 puncta in primary dissociated neuron culture. These results indicate that, although SynDIG4 shares sequence similarity with SynDIG1, it might act through a unique mechanism as an auxiliary factor for extrasynaptic GluA1-containing AMPARs. J. Comp. Neurol. 524:2266-2280, 2016. © 2015 Wiley Periodicals, Inc.
Literature context: g #AB1504 RRID:AB_2113602), GluA2 (1
Abnormal accumulation of β-amyloid (Aβ) is the major neuropathological hallmark of Alzheimer's disease (AD). However, the mechanisms underlying aberrant Aβ formation in AD remain unclear. We showed previously that inhibition of monoacylglycerol lipase (MAGL), the primary enzyme that metabolizes the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain, robustly reduces Aβ by inhibiting β-site amyloid precursor protein cleaving enzyme 1 (BACE1), a key enzyme responsible for Aβ formation. However, the molecular mechanisms responsible for suppression of BACE1 by inhibition of 2-AG metabolism are largely unknown. We demonstrate here that expression of the noncoding small RNA miR-188-3p that targets BACE1 was significantly downregulated both in the brains of AD humans and APP transgenic (TG) mice, a mouse model of AD. The downregulated miR-188-3p expression was restored by MAGL inhibition. Overexpression of miR-188-3p in the hippocampus reduced BACE1, Aβ, and neuroinflammation and prevented deteriorations in hippocampal basal synaptic transmission, long-term potentiation, spatial learning, and memory in TG mice. 2-AG-induced suppression of BACE1 was prevented by miR-188-3p loss of function. Moreover, miR-188-3p expression was upregulated by 2-AG or peroxisome proliferator-activated receptor-γ (PPARγ) agonists and suppressed by PPARγ antagonism or NF-κB activation. Reducing Aβ and neuroinflammation by MAGL inhibition was occluded by PPARγ antagonism. In addition, BACE1 suppression by 2-AG and PPARγ activation was eliminated by knockdown of NF-κB. Our study provides a novel molecular mechanism underlying improved synaptic and cognitive function in TG mice by 2-AG signaling, which upregulates miR-188-3p expression through PPARγ and NF-κB signaling pathway, resulting in suppressions of BACE1 expression and Aβ formation.
Autoimmune forms of encephalitis have been associated with autoantibodies against synaptic cell surface antigens such as NMDA- and AMPA-type glutamate receptors, GABA(B) receptor, and LGI1. However, it remains unclear how many synaptic autoantigens are yet to be defined. Using immunoproteomics, we identified autoantibodies against the GABA(A) receptor in human sera from two patients diagnosed with encephalitis who presented with cognitive impairment and multifocal brain MRI abnormalities. Both patients had antibodies directed against the extracellular epitope of the β3 subunit of the GABA(A) receptor. The β3-subunit-containing GABA(A) receptor was a major target of the patients' serum antibodies in rat hippocampal neurons because the serum reactivity to the neuronal surface was greatly decreased by 80% when the β3 subunit was knocked down. Our developed multiplex ELISA testing showed that both patients had similar levels of GABA(A) receptor antibodies, one patient also had a low level of LGI1 antibodies, and the other also had CASPR2 antibodies. Application of the patients' serum at the time of symptom presentation of encephalitis to rat hippocampal neuron cultures specifically decreased both synaptic and surface GABA(A) receptors. Furthermore, treatment of neurons with the patients' serum selectively reduced miniature IPSC amplitude and frequency without affecting miniature EPSCs. These results strongly suggest that the patients' GABA(A) receptor antibodies play a central role in the patients' symptoms. Therefore, this study establishes anti-GABA(A) receptor encephalitis and expands the pathogenic roles of GABA(A) receptor autoantibodies.
Literature context: illipore, catalog #AB1504, RIID:AB_2113602), GluN1 (BD Biosciences, catalo
Elevation of inflammatory cytokines in the striatum precedes symptoms in a number of motor dysfunctions, but it is unclear whether this is part of the disease process or an adaptive response to the pathology. In pyramidal cells, TNFα drives the insertion of AMPA-type glutamate receptors into synapses, and contributes to the homeostatic regulation of circuit activity in the developing neocortex. Here we demonstrate that in the mouse dorsolateral striatum, TNFα drives the internalization of AMPARs and reduces corticostriatal synaptic strength, dephosphorylates DARPP-32 and GluA1, and results in a preferential removal of Ca(2+)-permeable AMPARs. Striatal TNFα signaling appears to be adaptive in nature, as TNFα is upregulated in response to the prolonged blockade of D2 dopamine receptors and is necessary to reduce the expression of extrapyramidal symptoms induced by chronic haloperidol treatment. These data indicate that TNFα is a regulator of glutamatergic synaptic strength in the adult striatum in a manner distinct from its regulation of synapses on pyramidal cells and mediates an adaptive response during pathological conditions.
Cannabinoid receptor type 1 (CB1R) participates in developmental processes in the central nervous system (CNS). The rodent retina represents an interesting and valuable model for studying CNS development, because it contains well-identified cell types with clearly established and distinct developmental timelines. Very little is known about the distribution or function of CB1R in the developing retina. In this study, we investigated the expression pattern of CB1R in the rat retina during all stages of postnatal development. Western blots were performed on retinal tissue at different time points between P1 and adulthood. In order to identify the cells expressing the receptor and the age at which this expression started, immunohistochemical co-staining was carried out for CB1R and markers of the different cell types comprising the retina. CB1R was already present at P1 in various cell types, i.e., ganglion, amacrine, horizontal, and mitotic cells. In the course of development, it appeared in cone photoreceptors and bipolar cells. For some cell types (bipolar, Müller, and some amacrine cells), CB1R was transiently expressed, suggesting a potential role of this receptor in developmental processes, such as migration, morphological changes, sub-identity acquisition, and patterned retinal spontaneous activity. Our results also indicated that CB1R is largely expressed in the adult retina (cone photoreceptors and horizontal, most amacrine, and retinal ganglion cells), and may therefore contribute to retinal functions. Overall these results indicate that, as shown in other structures of the brain, CB1R could play an instrumental role in the development and function of the retina.
Although glutamate receptor 1 (GluR1)-containing α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (GluR1-AMPARs) are implicated in synaptic plasticity, it has yet to be demonstrated whether endogenous GluR1-AMPARs undergo activity-dependent trafficking in vivo to synapses to support short-term memory (STM) formation. The paradigm of pavlovian fear conditioning (FC) can be used to address this question, because a discrete region-the lateral amygdala (LA)-has been shown unambiguously to be necessary for the formation of the associative memory between a neutral stimulus (tone [CS]) and a noxious stimulus (foot shock [US]). Acquisition of STM for FC can occur even in the presence of protein synthesis inhibitors, indicating that redistribution of pre-existing molecules to synaptic junctions underlies STM. We employed electron microscopic immunocytochemistry to evaluate alterations in the distribution of endogenous AMPAR subunits at LA synapses during the STM phase of FC. Rats were sacrificed 40 minutes following three CS-US pairings. In the LA of paired animals, relative to naïve animals, the proportion of GluR1-AMPAR-labeled synapses increased 99% at spines and 167% in shafts. In the LA of unpaired rats, for which the CS was never associated with the US, GluR1 immunoreactivity decreased 84% at excitatory shaft synapses. GluR2/3 immunoreactivity at excitatory synapses did not change detectably following paired or unpaired conditioning. Thus, the early phase of FC involves rapid redistribution specifically of the GluR1-AMPARs to the postsynaptic membranes in the LA, together with the rapid translocation of GluR1-AMPARs from remote sites into the spine head cytoplasm, yielding behavior changes that are specific to stimulus contingencies.
Down syndrome (DS) is a neurological disorder causing impaired learning and memory. Partial trisomy 16 mice (Ts65Dn) are a genetic model for DS. Previously, we demonstrated widespread alterations of pre- and postsynaptic elements and physiological abnormalities in Ts65Dn mice. The average diameter of presynaptic boutons and spines in the neocortex and hippocampus was enlarged. Failed induction of long-term potentiation (LTP) due to excessive inhibition was observed. In this paper we investigate the morphological substrate for excessive inhibition in Ts65Dn. We used electron microscopy (EM) to characterize synapses, confocal microscopy to analyze colocalization of the general marker for synaptic vesicle protein with specific protein markers for inhibitory and excitatory synapses, and densitometry to characterize the distribution of the receptor and several proteins essential for synaptic clustering of neurotransmitter receptors. EM analysis of synapses in the Ts65Dn vs. 2N showed that synaptic opposition lengths were significantly greater for symmetric synapses (approximately 18%), but not for asymmetric ones. Overall, a significant increase in colocalization coefficients of glutamic acid decarboxylase (GAD)65/p38 immunoreactivity (IR) (approximately 27%) and vesicular GABA transporter (VGAT)/p38 IR (approximately 41%) was found, but not in vesicular glutamate transporter 1 (VGLUT1)/p38 IR. A significant overall decrease of IR in the hippocampus of Ts65Dn mice compared with 2N mice for glutamate receptor 2 (GluR2; approximately 13%) and anti-gamma-aminobutyric acid (GABA)(A) receptor beta2/3 subunit (approximately 20%) was also found. The study of proteins essential for synaptic clustering of receptors revealed a significant increase in puncta size for neuroligin 2 (approximately 13%) and GABA(A) receptor-associated protein (GABARAP; approximately 13%), but not for neuroligin 1 and gephyrin. The results demonstrate a significant alteration of inhibitory synapses in the fascia dentata of Ts65Dn mice.
Prolonged benzodiazepine treatment leads to tolerance and increases the risk of dependence. Flurazepam (FZP) withdrawal is associated with increased anxiety correlated with increased alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor (AMPAR)-mediated synaptic function and AMPAR binding in CA1 pyramidal neurons. Enhanced AMPAR synaptic strength is also associated with a shift toward inward rectification of synaptic currents and increased expression of GluR1, but not GluR2, subunits, suggesting augmented membrane incorporation of GluR1-containing, GluR2-lacking AMPARs. To test this hypothesis, the postsynaptic incorporation of GluR1 and GluR2 subunits in CA1 neurons after FZP withdrawal was examined by postembedding immunogold quantitative electron microscopy. The percentage of GluR1 positively labeled stratum radiatum (SR) synapses was significantly increased in FZP-withdrawn rats (88.2% +/- 2.2%) compared with controls (74.4% +/- 1.9%). In addition, GluR1 immunogold density was significantly increased by 30% in SR synapses in CA1 neurons from FZP-withdrawn rats compared with control rats (FZP: 14.1 +/- 0.3 gold particles/mum; CON: 10.8 +/- 0.4 gold particles/mum). In contrast, GluR2 immunogold density was not significantly different between groups. Taken together with recent functional data from our laboratory, the current study suggests that the enhanced glutamatergic strength at CA1 neuron synapses during benzodiazepine withdrawal is mediated by increased incorporation of GluR1-containing AMPARs. Mechanisms underlying synaptic plasticity in this model of drug dependence are therefore fundamentally similar to those that operate during activity-dependent plasticity.
Dopaminergic (DA) neurons of mouse and rat retinas are of the interplexiform subtype (DA-IPC), i.e., they send processes distally toward the outer retina, exhibiting numerous varicosities along their course. The primary question we addressed was whether distally located DA-IPC varicosities, identified by tyrosine hydroxylase (TH) immunoreactivity, had the characteristic presynaptic proteins associated with calcium-dependent vesicular release of neurotransmitter. We found that TH immunoreactive varicosities in the outer retina possessed vesicular monoamine transporter 2 and vesicular GABA transporter, but they lacked immunostaining for any of nine subtypes of voltage-dependent calcium channel. Immunoreactivity for other channels that may permit calcium influx such as certain ionotropic glutamate receptors and canonical transient receptor potential channels (TRPCs) was similarly absent, although DA-IPC varicosities did show ryanodine receptor immunoreactivity, indicating the presence of intracellular calcium stores. The synaptic vesicle proteins sv2a and sv2b and certain other proteins associated with the presynaptic membrane were absent from DA-IPC varicosities, but the vesicular SNARE protein, vamp2, was present in a fraction of those varicosities. We identified a presumed second class of IPC that is GABAergic but not dopaminergic. Outer retinal varicosities of this putative GABAergic IPC did colocalize synaptic vesicle protein 2a, suggesting they possessed a conventional vesicular release mechanism.
We examined the expression profile of subunits of ionotropic glutamate receptors [N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA)] during postnatal development of connectivity in the rat vestibular nucleus. Vestibular nuclear neurons were functionally activated by constant velocity off-vertical axis rotation, a strategy to stimulate otolith organs in the inner ear. These neurons indicated Fos expression as a result. By immunodetection for Fos, otolith-related neurons that expressed NMDA/AMPA receptor subunits were identified as early as P7, and these neurons were found to increase progressively up to adulthood. Although there was developmental invariance in the percentage of Fos-immunoreactive neurons expressing the NR1, NR2A, GluR1, or GluR2/3 subunits, those expressing the NR2B subunit decreased from P14 onward, and those expressing the GluR4 subunit decreased in adults. These double-immunohistochemical data were corroborated by combined immuno-/hybridization histochemical data obtained from Fos-immunoreactive neurons expressing NR2B mRNA or GluR4 mRNA. The staining of both NR2B and GluR4 in the cytoplasm of these neurons decreased upon maturation. The percentage of Fos-immunoreactive neurons expressing the other ionotropic glutamate receptor subunits (viz. NR1, NR2A, GluR1, and GluR2/3) remained relatively constant throughout postnatal maturation. Triple immunofluorescence further demonstrated coexpression of NR1 and NR2 subunits in Fos-immunoreactive neurons. Coexpression of NR1 subunit with each of the GluR subunits was also observed among the Fos-immunoreactive neurons. Taken together, the different expression profiles of ionotropic glutamate receptor subunits constitute the histological basis for glutamatergic neurotransmission in the maturation of central vestibular connectivity for the coding of gravity-related horizontal head movements.
In adult cats, damage to the extrastriate visual cortex on the banks of the lateral suprasylvian (LS) sulcus causes severe deficits in motion perception that can recover as a result of intensive direction discrimination training. The fact that recovery is restricted to trained visual field locations suggests that the neural circuitry of early visual cortical areas, with their tighter retinotopy, may play an important role in attaining perceptual improvements after damage to higher level visual cortex. The present study tests this hypothesis by comparing the manner in which excitatory and inhibitory components of the supragranular circuitry in an early visual cortical area (area 18) are affected by LS lesions and postlesion training. First, the proportion of LS-projecting pyramidal cells as well as calbindin- and parvalbumin-positive interneurons expressing each of the four AMPA receptor subunits was estimated in layers II and III of area 18 in intact animals. The degree to which LS lesions and visual retraining altered these expression patterns was then assessed. Both LS-projecting pyramidal cells and inhibitory interneurons exhibited long-term, differential reductions in the expression of glutamate receptor (GluR)1, -2, -2/3, and -4 following LS lesions. Intensive visual training post lesion restored normal AMPAR subunit expression in all three cell-types examined. Furthermore, for LS-projecting and calbindin-positive neurons, this restoration occurred only in portions of the ipsi-lesional area 18 representing trained visual field locations. This supports our hypothesis that stimulation of early visual cortical areas-in this case, area 18-by training is an important factor in restoring visual perception after permanent damage to LS cortex.
This is the first part of a two-part study to investigate the cellular distribution and temporal regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) subunits in the developing white matter and cortex in rat (part I) and human (part II). Western blot and immunocytochemistry were used to evaluate the differential expression of AMPAR subunits on glial and neuronal subtypes during the first 3 postnatal weeks in the Long Evans and Sprague Dawley rat strains. In Long Evans rats during the first postnatal week, GluR2-lacking AMPARs were expressed predominantly on white matter cells, including radial glia, premyelinating oligodendrocytes, and subplate neurons, whereas, during the second postnatal week, these AMPARs were highly expressed on cortical neurons, coincident with decreased expression on white matter cells. Immunocytochemical analysis revealed that cell-specific developmental changes in AMPAR expression occurred 2-3 days earlier by chronological age in Sprague Dawley rats compared with Long Evans rats, despite overall similar temporal sequencing. In both white and gray matter, the periods of high GluR2 deficiency correspond to those of regional susceptibility to hypoxic/ischemic injury in each of the two rat strains, supporting prior studies suggesting a critical role for Ca2+-permeable AMPARs in excitotoxic cellular injury and epileptogenesis. The developmental regulation of these receptor subunits strongly suggests that Ca2+ influx through GluR2-lacking AMPARs may play an important role in neuronal and glial development and injury in the immature brain. Moreover, as demonstrated in part II, there are striking similarities between rat and human in the regional and temporal maturational regulation of neuronal and glial AMPAR expression.