DNA methylation of Vesicular Glutamate Transporters in the mesocorticolimbic brain following early-life stress and adult ethanol exposure-an explorative study.
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ABSTRACT:
SUBMITTER: Vrettou M
PROVIDER: S-EPMC8319394 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Alcohol use disorder (AUD) is a chronic mental illness in which patients often achieve protracted periods of abstinence prior to relapse. Epigenetic mechanisms may provide an explanation for the persisting gene expression changes that can be observed even after long periods of abstinence and may contribute to relapse. In this study, we examined two histone modifications, histone 3 lysine 4 tri-methylation (H3K4me3) and histone 3 lysine 27 tri-methylation (H3K27me3), in the prefrontal cortex of Withdrawal Seizure Resistant (WSR) mice 21 days after 72 h of ethanol vapor exposure. These histone modifications were selected because they are associated with active promoters (H3K4me3) and repressed gene expression in a euchromatic environment (H3K27me3). We performed a genome-wide analysis to identify differences in H3K4me3 and H3K27me3 levels in post-ethanol exposure vs. control mice by ChIP-seq. We detected a global reduction in H3K4me3 peaks and increase in H3K27me3 peaks in post-ethanol exposure mice compared to controls, these changes are consistent with persistent reductions in gene expression. Pathway analysis of genes displaying changes in H3K4me3 and H3K27me3 revealed enrichment for genes involved in proteoglycan and calcium signaling pathways, respectively. Microarray analysis of 7,683 genes and qPCR analysis identified eight genes displaying concordant regulation of gene expression and H3K4me3/H3K27me3. We also compared changes in H3K4me3 and/or H3K27me3 from our study with changes in gene expression in response to ethanol from published literature and we found that the expression of 52% of the genes with altered H3K4me3 binding and 40% of genes with H3K27me3 differences are altered by ethanol exposure. The chromatin changes associated with the 21-day post-exposure period suggest that this period is a unique state in the addiction cycle that differs from ethanol intoxication and acute withdrawal. These results provide insights into the enduring effects of ethanol on proteoglycan and calcium signaling genes in the brain.
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Project description:Previous work has characterized the properties of neurotransmitter release at excitatory and inhibitory synapses, but we know remarkably little about the properties of monoamine release, because these neuromodulators do not generally produce a fast ionotropic response. Since dopamine and serotonin neurons can also release glutamate in vitro and in vivo, we have used the vesicular monoamine transporter VMAT2 and the vesicular glutamate transporter VGLUT1 to compare the localization and recycling of synaptic vesicles that store, respectively, monoamines and glutamate. First, VMAT2 segregates partially from VGLUT1 in the boutons of midbrain dopamine neurons, indicating the potential for distinct release sites. Second, endocytosis after stimulation is slower for VMAT2 than VGLUT1. During the stimulus, however, the endocytosis of VMAT2 (but not VGLUT1) accelerates dramatically in midbrain dopamine but not hippocampal neurons, indicating a novel, cell-specific mechanism to sustain high rates of release. On the other hand, we find that in both midbrain dopamine and hippocampal neurons, a substantially smaller proportion of VMAT2 than VGLUT1 is available for evoked release, and VMAT2 shows considerably more dispersion along the axon after exocytosis than VGLUT1. Even when expressed in the same neuron, the two vesicular transporters thus target to distinct populations of synaptic vesicles, presumably due to their selection of distinct recycling pathways.
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Project description:Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate. VGLUTs were originally identified as sodium-dependent transporters of inorganic phosphate (Pi), but the physiological relevance of this activity remains unclear. Heterologous expression of all three VGLUTs greatly augments intracellular Pi levels. Using neuronal models, we show that translocation of VGLUTs to the plasma membrane during exocytosis results in highly increased Pi uptake. VGLUT-mediated Pi influx is counteracted by Pi efflux. Synaptosomes prepared from perinatal VGLUT2-/- mice that are virtually free of VGLUTs show drastically reduced cytosolic Pi levels and fail to import Pi. Glutamate partially competes with sodium (Na+)/Pi (NaPi)-uptake mediated by VGLUTs but does not appear to be transported. A nanobody that blocks glutamate transport by binding to the cytoplasmic domain of VGLUT1 abolishes Pi transport when co-expressed with VGLUT1. We conclude that VGLUTs have a dual function that is essential for both vesicular glutamate loading and Pi restoration in neurons.
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Project description:Adolescent alcohol use is the strongest predictor for alcohol use disorders. In rodents, adolescents have distinct responses to acute ethanol, and prolonged alcohol exposure during adolescence can maintain these phenotypes into adulthood. One brain region that is particularly sensitive to the effects of both acute and chronic ethanol exposure is the hippocampus. Adolescent intermittent ethanol exposure (AIE) produces long lasting changes in hippocampal synaptic plasticity and dendritic morphology, as well as in the susceptibility to acute ethanol-induced spatial memory impairment. Given the pattern of changes in hippocampal structure and function, one potential target for these effects is the ethanol sensitive GluN2B subunit of the NMDA receptor, which is known to be involved in synaptic plasticity and dendritic morphology. Thus we sought to determine if there were persistent changes in hippocampal GluN2B signaling cascades following AIE. We employed a previously validated GluN2B-targeted proteomic strategy that was used to identify novel signaling mechanisms altered by chronic ethanol exposure in the adult hippocampus. We collected adult hippocampal tissue (P70) from rats that had been given 2 weeks of AIE from P30-45. Tissue extracts were fractionated into synaptic and non-synaptic pools, immuno-precipitated for GluN2B, and then analyzed using proteomic methods. We detected a large number of proteins associated with GluN2B. AIE produced significant changes in the association of many proteins with GluN2B in both synaptic and non-synaptic fractions. Intriguingly the number of proteins changed in the non-synaptic fraction was double that found in the synaptic fraction. Some of these proteins include those involved in glutamate signaling cytoskeleton rearrangement, calcium signaling, and plasticity. Disruptions in these pathways may contribute to the persistent cellular and behavioral changes found in the adult hippocampus following AIE. Further, the robust change in non-synaptic proteins suggests that AIE may prime this signaling pathway for future ethanol exposures in adulthood.
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Project description:AimGlutamate has been considered as neurotransmitter that is critical in triggering relapse to drugs of abuse, including ethanol and cocaine. Extracellular glutamate concentrations are tightly regulated by several mechanisms, including reuptake through glutamate transporters. Glutamate transporter type 1 (GLT-1) is responsible for clearing the majority of extracellular glutamate. The astrocytic cystine/glutamate antiporter (xCT) regulates also glutamate homeostasis. In this study, we investigated the effects of cocaine exposure and ampicillin/sulbactam (AMP/SUL), a β-lactam antibiotic known to upregulate GLT-1 and xCT, on relapse-like ethanol intake and the expression of astrocytic glutamate transporters in mesocorticolimbic brain regions.MethodsMale alcohol-preferring (P) rats had free access to ethanol for 5 weeks. On Week 6, rats were exposed to either cocaine (20 mg/kg, i.p.) or saline for 12 consecutive days. Ethanol bottles were then removed for 7 days; during the last 5 days, either AMP/SUL (100 or 200 mg/kg, i.p.) or saline was administered to the P rats. Ethanol bottles were reintroduced, and ethanol intake was measured for 4 days.ResultsCocaine exposure induced an alcohol deprivation effect (ADE), which was associated in part by a decrease in the expression of GLT-1 and xCT in the nucleus accumbens (NAc) core. AMP/SUL (100 mg/kg, i.p.) attenuated the ADE, while AMP/SUL (200 mg/kg, i.p.) reduced ethanol intake during 4 days of ethanol re-exposure and upregulated GLT-1 and xCT expression in the NAc core, NAc shell and dorsomedial prefrontal cortex (dmPFC).ConclusionThis study suggests that these astrocytic glutamate transporters might be considered as potential targets for the treatment of polysubstance abuse.
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Project description:Inositol pyrophosphates have been implicated in cellular signaling and membrane trafficking, including synaptic vesicle (SV) recycling. Inositol hexakisphosphate kinases (IP6Ks) and their product, diphosphoinositol pentakisphosphate (PP-IP5 or IP7), directly and indirectly regulate proteins important in vesicle recycling by the activity-dependent bulk endocytosis pathway (ADBE). In the present study, we show that two isoforms, IP6K1 and IP6K3, are expressed in axons. The role of the kinases in SV recycling are investigated using pharmacologic inhibition, shRNA knockdown, and IP6K1 and IP6K3 knockout mice. Live-cell imaging experiments use optical reporters of SV recycling based on vesicular glutamate transporter isoforms, VGLUT1- and VGLUT2-pHluorins (pH), which recycle differently. VGLUT1-pH recycles by classical AP-2 dependent endocytosis under moderate stimulation conditions, while VGLUT2-pH recycles using AP-1 and AP-3 adaptor proteins as well. Using a short stimulus to release the readily releasable pool (RRP), we show that IP6K1 KO increases exocytosis of both VGLUT1-and VGLUT2-pH, while IP6K3 KO decreases the amount of both transporters in the RRP. In electrophysiological experiments we measure glutamate signaling with short stimuli and under the intense stimulation conditions that trigger bulk endocytosis. IP6K1 KO increases synaptic facilitation and IP6K3 KO decreases facilitation compared to wild type in CA1 hippocampal Schaffer collateral synapses. After intense stimulation, the rate of endocytosis of VGLUT2-pH, but not VGLUT1-pH, is increased by knockout, knockdown, and pharmacologic inhibition of IP6Ks. Thus IP6Ks differentially affect the endocytosis of two SV protein cargos that use different endocytic pathways. However, while IP6K1 KO and IP6K3 KO exert similar effects on endocytosis after stimulation, the isoforms exert different effects on exocytosis earlier in the stimulus and on the early phase of glutamate release. Taken together, the data indicate a role for IP6Ks both in exocytosis early in the stimulation period and in endocytosis, particularly under conditions that may utilize AP-1/3 adaptors.
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Project description:Neurogenesis in hippocampal dentate gyrus (DG) and subventricular zone (SVZ) matures during adolescence to adult levels. Binge drinking is prevalent in adolescent humans, and could alter brain neurogenesis and maturation in a manner that persists into adulthood. To determine the impact of adolescent binge drinking on adult neurogenesis, Wistar rats received adolescent intermittent ethanol (AIE) exposure (5.0 g/kg/day, i.g., 2 days on/2 days off from postnatal day, P25-P54) and sacrificed on P57 or P95. Neural progenitor cell proliferation, differentiation, survival and maturation using immunohistochemistry was determined in the DG and SVZ. We found that AIE exposure decreased neurogenesis in both brain regions in adulthood (P95). In the DG at P57, AIE exposure resulted in a significant reduction of SOX2+, Tbr2+, Prox1+ and parvalbumin (PV)+IR expression, and at P95 decreased DCX+ and PV+IR expression. AIE exposure also reduced the expression of two cell proliferation markers (Ki67+ and BrdU+IR with 300 mg/kg, 2 h) at P95. The immune signaling molecule β-2 microglobulin+ and the cell death marker activated caspase-3+IR were significantly increased in the DG by AIE exposure. In the SVZ, AIE exposure decreased SOX2+, Mash1+, DCX+ and Dlx2+IR expression at P95, but not at P57. Thus, in adulthood both brain regions have reduced neurogenesis following AIE exposure. To assess progenitor cell survival and maturation, rats were treated with BrdU (150 mg/kg/day, 14 days) to label proliferating cells and were sacrificed weeks later on P95. In the hippocampus DG, AIE exposure increased survival BrdU+ cells which differentiated into Iba1+ microglia. In contrast, SVZ had decreased BrdU+ cells similar to decreased DCX+ neurogenesis. These data indicate that AIE exposure causes a lasting decrease in both adult hippocampal DG and forebrain SVZ neurogenesis with brain regional differences in the AIE response that persist into adulthood.
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Project description:Glutamate is the main excitatory neurotransmitter in the auditory system, but associations between glutamatergic neuronal populations and the distribution of their synaptic terminations have been difficult. Different subsets of glutamatergic terminals employ one of three vesicular glutamate transporters (VGLUT) to load synaptic vesicles. Recently, VGLUT1 and VGLUT2 terminals were found to have different patterns of organization in the inferior colliculus, suggesting that there are different types of glutamatergic neurons in the brainstem auditory system with projections to the colliculus. To positively identify VGLUT-expressing neurons as well as inhibitory neurons in the auditory brainstem, we used in situ hybridization to identify the mRNA for VGLUT1, VGLUT2, and VIAAT (the vesicular inhibitory amino acid transporter used by GABAergic and glycinergic terminals). Similar expression patterns were found in subsets of glutamatergic and inhibitory neurons in the auditory brainstem and thalamus of adult rats and mice. Four patterns of gene expression were seen in individual neurons. 1) VGLUT2 expressed alone was the prevalent pattern. 2) VGLUT1 coexpressed with VGLUT2 was seen in scattered neurons in most nuclei but was common in the medial geniculate body and ventral cochlear nucleus. 3) VGLUT1 expressed alone was found only in granule cells. 4) VIAAT expression was common in most nuclei but dominated in some. These data show that the expression of the VGLUT1/2 and VIAAT genes can identify different subsets of auditory neurons. This may facilitate the identification of different components in auditory circuits.
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