Reducing l-lactate release from hippocampal astrocytes by intracellular oxidation increases novelty induced activity in mice.
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ABSTRACT:
SUBMITTER: Vaccari Cardoso B
PROVIDER: S-EPMC8576740 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Astrocytes support and modulate neuronal activity through the release of L-lactate. The suggested roles of astrocytic lactate in the brain encompass an expanding range of vital functions, including central control of respiration and cardiovascular performance, learning, memory, executive behaviour and regulation of mood. Studying the effects of astrocytic lactate requires tools that limit the release of lactate selectively from astrocytes. Here, we report the validation in vitro of novel molecular constructs derived from enzymes originally found in bacteria, that when expressed in astrocytes, interfere with lactate handling. When lactate 2-monooxygenase derived from M. smegmatis was specifically expressed in astrocytes, it reduced intracellular lactate pools as well as lactate release upon stimulation. D-lactate dehydrogenase derived from L. bulgaricus diverts pyruvate towards D-lactate production and release by astrocytes, which may affect signalling properties of lactate in the brain. Together with lactate oxidase, which we have previously described, this set of transgenic tools can be employed to better understand astrocytic lactate release and its role in the regulation of neuronal activity in different behavioural contexts.
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Project description:To date, neurons have been the primary focus of research on the role of glucocorticoids in the regulation of brain function and pathological behaviors, such as addiction. Astrocytes, which are also glucocorticoid-responsive, have been recently implicated in the development of drug abuse, albeit through as yet undefined mechanisms. Here, using a spectrum of tools (whole-transcriptome profiling, viral-mediated RNA interference in vitro and in vivo, behavioral pharmacology and electrophysiology), we demonstrate that astrocytes in the nucleus accumbens (NAc) are an important locus of glucocorticoid receptor (GR)-dependent transcriptional changes that regulate rewarding effects of morphine. Specifically, we show that targeted knockdown of the GR in the NAc astrocytes enhanced conditioned responses to morphine, with a concomitant inhibition of morphine-induced neuronal excitability and plasticity. Interestingly, GR knockdown did not influence sensitivity to cocaine. Further analyses revealed GR-dependent regulation of astroglial metabolism. Notably, GR knockdown inhibited induced by glucocorticoids lactate release in astrocytes. Finally, lactate administration outbalanced conditioned responses to morphine in astroglial GR knockdown mice. These findings demonstrate a role of GR-dependent regulation of astrocytic metabolism in the NAc and a key role of GR-expressing astrocytes in opioid reward processing.
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Project description:Astrocytes have a prominent role in metabolic homeostasis of the brain and can signal to adjacent neurons by releasing glutamate via a process of regulated exocytosis. Astrocytes synthesize glutamate de novo owing to the pyruvate entry to the citric/tricarboxylic acid cycle via pyruvate carboxylase, an astrocyte specific enzyme. Pyruvate can be sourced from two metabolic fuels, glucose and lactate. Thus, we investigated the role of these energy/carbon sources in exocytotic glutamate release from astrocytes. Purified astrocyte cultures were acutely incubated (1 h) in glucose and/or lactate-containing media. Astrocytes were mechanically stimulated, a procedure known to increase intracellular Ca2+ levels and cause exocytotic glutamate release, the dynamics of which were monitored using single cell fluorescence microscopy. Our data indicate that glucose, either taken-up from the extracellular space or mobilized from the intracellular glycogen storage, sustained glutamate release, while the availability of lactate significantly reduced the release of glutamate from astrocytes. Based on further pharmacological manipulation during imaging along with tandem mass spectrometry (proteomics) analysis, lactate alone, but not in the hybrid fuel, caused metabolic changes consistent with an increased synthesis of fatty acids. Proteomics analysis further unveiled complex changes in protein profiles, which were condition-dependent and generally included changes in levels of cytoskeletal proteins, proteins of secretory organelle/vesicle traffic and recycling at the plasma membrane in aglycemic, lactate or hybrid-fueled astrocytes. These findings support the notion that the availability of energy sources and metabolic milieu play a significant role in gliotransmission.
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Project description:Astrocytes, the most abundant glial cells in the central nervous system (CNS), sense synaptic activity and respond through the release of gliotransmitters, a process mediated by intracellular Ca2+ level changes and SNARE-dependent mechanisms. Ionotropic N-methyl-D-aspartate (NMDA) receptors, which are activated by glutamate along with D-serine or glycine, play a crucial role in learning, memory, and synaptic plasticity. However, the precise impact of astrocyte-released D-serine on neuronal modulation remains insufficiently characterized. To address this, we have used the dominant negative SNARE (dnSNARE) mouse model, which selectively inhibits SNARE-dependent exocytosis from astrocytes. We recorded field excitatory postsynaptic potentials (fEPSPs) in CA3-CA1 synapses within hippocampal slices obtained from dnSNARE mice and wild-type (Wt) littermates. Our results demonstrate that hippocampal θ-burst long-term potentiation (LTP), a critical form of synaptic plasticity, is impaired in hippocampal slices from dnSNARE mice. Notably, this LTP impairment was rescued upon incubation with D-serine. To further investigate the involvement of astrocytes in D-serine-mediated mechanisms of LTP maintenance, we perfused hippocampal slices with L-serine - a substrate used by both neurons and astrocytes for D-serine production. The enhancement in LTP observed in dnSNARE mice was exclusively associated with D-serine presence, with no effects evident in the presence of L-serine. Additionally, both D- and L-serine reduced basal synaptic strength in the hippocampal slices of both Wt and dnSNARE mice. These results provide compelling evidence that distinct processes underlie the modulation of basal synaptic transmission and LTP through D-serine. Our findings underscore the pivotal contribution of astrocytes in D-serine-mediated processes that govern LTP establishment and basal transmission. This study not only provides essential insights into the intricate interplay between neurons and astrocytes but also emphasizes their collective role in shaping hippocampal synaptic function.
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Project description:The hippocampus is the brain's center for episodic memories. Its subregions, the dentate gyrus and CA1-3, are differentially involved in memory encoding and recall. Hippocampal principal cells represent episodic features like movement, space, and context, but less is known about GABAergic interneurons. Here, we performed two-photon calcium imaging of parvalbumin- and somatostatin-expressing interneurons in the dentate gyrus and CA1-3 of male mice exploring virtual environments. Parvalbumin-interneurons increased activity with running-speed and reduced it in novel environments. Somatostatin-interneurons in CA1-3 behaved similar to parvalbumin-expressing cells, but their dentate gyrus counterparts increased activity during rest and in novel environments. Congruently, chemogenetic silencing of dentate parvalbumin-interneurons had prominent effects in familiar contexts, while silencing somatostatin-expressing cells increased similarity of granule cell representations between novel and familiar environments. Our data indicate unique roles for parvalbumin- and somatostatin-positive interneurons in the dentate gyrus that are distinct from those in CA1-3 and may support routing of novel information.
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Project description:In hypothalamic astrocytes obtained from adult female rats, estradiol rapidly increased free cytoplasmic calcium concentrations ([Ca(2+)](i)) that facilitate progesterone synthesis. The present study demonstrated that estradiol (1 nm) significantly and maximally stimulated progesterone synthesis within 5 min, supporting a rapid, nongenomic mechanism. The group I metabotropic glutamate receptor (mGluR1a) antagonist LY 367385 [(S)-(+)-a-amino-4-carboxy-2-methylbenzeneacetic acid] attenuated both the estradiol-induced [Ca(2+)](i) release and progesterone synthesis. To investigate membrane-associated estrogen receptors (mERs), agonists for ER?, ER?, STX-activated protein, and GPR30 were compared. The selective ER? agonist propylpyrazole triole (PPT) and STX most closely mimicked the estradiol-induced [Ca(2+)](i) responses, where PPT was more potent but less efficacious than STX. Only high doses (100 nm) of selective ER? agonist diarylpropionitrile (DPN) and GPR30 agonist G-1 induced estradiol-like [Ca(2+)](i) responses. With the exception of DPN (even at 100 nm), all agonists stimulated progesterone synthesis. The PPT- and STX-induced [Ca(2+)](i) release and progesterone synthesis were blocked by LY 367385. While the G-1-stimulated [Ca(2+)](i) release was blocked by LY 367385, progesterone synthesis was not. Since GPR30 was detected intracellularly but not in the membrane, we interpreted these results to suggest that G-1 could activate mGluR1a on the membrane and GPR30 on the smooth endoplasmic reticulum to release intracellular calcium. Although STX and G-1 maximally stimulated [Ca(2+)](i) release in astrocytes from estrogen receptor-? knock-out (ERKO) mice, estradiol in vivo did not stimulate progesterone synthesis in the ERKO mice. Together, these results indicate that mER? is mainly responsible for the rapid, membrane-initiated estradiol-signaling that leads to progesterone synthesis in hypothalamic astrocytes.
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Project description:Aims/hypothesisHypoglycaemia is a major barrier to good glucose control in type 1 diabetes. Frequent hypoglycaemic episodes impair awareness of subsequent hypoglycaemic bouts. Neural changes underpinning awareness of hypoglycaemia are poorly defined and molecular mechanisms by which glial cells contribute to hypoglycaemia sensing and glucose counterregulation require further investigation. The aim of the current study was to examine whether, and by what mechanism, human primary astrocyte (HPA) function was altered by acute and recurrent low glucose (RLG).MethodsTo test whether glia, specifically astrocytes, could detect changes in glucose, we utilised HPA and U373 astrocytoma cells and exposed them to RLG in vitro. This allowed measurement, with high specificity and sensitivity, of RLG-associated changes in cellular metabolism. We examined changes in protein phosphorylation/expression using western blotting. Metabolic function was assessed using a Seahorse extracellular flux analyser. Immunofluorescent imaging was used to examine cell morphology and enzymatic assays were used to measure lactate release, glycogen content, intracellular ATP and nucleotide ratios.ResultsAMP-activated protein kinase (AMPK) was activated over a pathophysiologically relevant glucose concentration range. RLG produced an increased dependency on fatty acid oxidation for basal mitochondrial metabolism and exhibited hallmarks of mitochondrial stress, including increased proton leak and reduced coupling efficiency. Relative to glucose availability, lactate release increased during low glucose but this was not modified by RLG. Basal glucose uptake was not modified by RLG and glycogen levels were similar in control and RLG-treated cells. Mitochondrial adaptations to RLG were partially recovered by maintaining euglycaemic levels of glucose following RLG exposure.Conclusions/interpretationTaken together, these data indicate that HPA mitochondria are altered following RLG, with a metabolic switch towards increased fatty acid oxidation, suggesting glial adaptations to RLG involve altered mitochondrial metabolism that could contribute to defective glucose counterregulation to hypoglycaemia in diabetes.
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