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Dynamic expression of homeostatic ion channels in differentiated cortical astrocytes in vitro.

ABSTRACT: The capacity of astrocytes to adapt their biochemical and functional features upon physiological and pathological stimuli is a fundamental property at the basis of their ability to regulate the homeostasis of the central nervous system (CNS). It is well known that in primary cultured astrocytes, the expression of plasma membrane ion channels and transporters involved in homeostatic tasks does not closely reflect the pattern observed in vivo. The individuation of culture conditions that promote the expression of the ion channel array found in vivo is crucial when aiming at investigating the mechanisms underlying their dynamics upon various physiological and pathological stimuli. A chemically defined medium containing growth factors and hormones (G5) was previously shown to induce the growth, differentiation, and maturation of primary cultured astrocytes. Here we report that under these culture conditions, rat cortical astrocytes undergo robust morphological changes acquiring a multi-branched phenotype, which develops gradually during the 2-week period of culturing. The shape changes were paralleled by variations in passive membrane properties and background conductance owing to the differential temporal development of inwardly rectifying chloride (Cl^-) and potassium (K⁺) currents. Confocal and immunoblot analyses showed that morphologically differentiated astrocytes displayed a large increase in the expression of the inward rectifier Cl^- and K⁺ channels ClC-2 and Kir4.1, respectively, which are relevant ion channels in vivo. Finally, they exhibited a large diminution of the intermediate filaments glial fibrillary acidic protein (GFAP) and vimentin which are upregulated in reactive astrocytes in vivo. Taken together the data indicate that long-term culturing of cortical astrocytes in this chemical-defined medium promotes a quiescent functional phenotype. This culture model could aid to address the regulation of ion channel expression involved in CNS homeostasis in response to physiological and pathological challenges.

SUBMITTER: Formaggio F

PROVIDER: S-EPMC8766406 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Recently, we have identified two astrocytic subpopulations in the cortex of GFAP-EGFP mice, in which the astrocytes are visualized by the enhanced green-fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promotor. These astrocytic subpopulations, termed high response- (HR-) and low response- (LR-) astrocytes, differed in the extent of their swelling during oxygen-glucose deprivation (OGD). In the present study we focused on identifying the ion channels or transporters that might underlie the different capabilities of these two astrocytic subpopulations to regulate their volume during OGD. Using three-dimensional confocal morphometry, which enables quantification of the total astrocytic volume, the effects of selected inhibitors of K? and Cl? channels/transporters or glutamate transporters on astrocyte volume changes were determined during 20 minute-OGD in situ. The inhibition of volume regulated anion channels (VRACs) and two-pore domain potassium channels (K(2P)) highlighted their distinct contributions to volume regulation in HR-/LR-astrocytes. While the inhibition of VRACs or K(2P) channels revealed their contribution to the swelling of HR-astrocytes, in LR-astrocytes they were both involved in anion/K? effluxes. Additionally, the inhibition of Na?-K?-Cl? co-transporters in HR-astrocytes led to a reduction of cell swelling, but it had no effect on LR-astrocyte volume. Moreover, employing real-time single-cell quantitative polymerase chain reaction (PCR), we characterized the expression profiles of EGFP-positive astrocytes with a focus on those ion channels and transporters participating in astrocyte swelling and volume regulation. The PCR data revealed the existence of two astrocytic subpopulations markedly differing in their gene expression levels for inwardly rectifying K? channels (Kir4.1), K(2P) channels (TREK-1 and TWIK-1) and Cl? channels (ClC2). Thus, we propose that the diverse volume changes displayed by cortical astrocytes during OGD mainly result from their distinct expression patterns of ClC2 and K(2P) channels.

Project description:Prolonged high-fat diet (HFD) exposure is associated with hyperphagia, excess caloric intake and weight gain. After initial exposure to a HFD, a brief (24-48 h) period of hyperphagia is followed by the regulation of caloric intake and restoration of energy balance within an acute (3-5 day) period. Previous studies have demonstrated this occurs via a vagally mediated signalling cascade that increases glutamatergic transmission via activation of NMDA receptors located on gastric-projecting neurons of the dorsal motor nucleus of the vagus (DMV). The present study used electrophysiological recordings from thin brainstem slice preparations, in vivo recordings of gastric motility and tone, measurement of gastric emptying rates, and food intake studies to investigate the hypothesis that activation of brainstem astrocytes in response to acute HFD exposure is responsible for the increased glutamatergic drive to DMV neurons and the restoration of caloric balance. Pharmacological and chemogenetic inhibition of brainstem astrocytes reduced glutamatergic signalling and DMV excitability, dysregulated gastric tone and motility, attenuated the homeostatic delay in gastric emptying, and prevented the decrease in food intake that is observed during the period of energy regulation following initial exposure to HFD. Understanding the mechanisms involved in caloric regulation may provide critical insights into energy balance as well as into the hyperphagia that develops as these mechanisms are overcome. KEY POINTS: Initial exposure to a high fat diet is associated with a brief period of hyperphagia before caloric intake and energy balance is restored. This period of homeostatic regulation is associated with a vagally mediated signalling cascade that increases glutamatergic transmission to dorsal motor nucleus of the vagus (DMV) neurons via activation of synaptic NMDA receptors. The present study demonstrates that pharmacological and chemogenetic inhibition of brainstem astrocytes reduced glutamatergic signalling and DMV neuronal excitability, dysregulated gastric motility and tone and emptying, and prevented the regulation of food intake following high-fat diet exposure. Astrocyte regulation of glutamatergic transmission to DMV neurons appears to involve release of the gliotransmitters glutamate and ATP. Understanding the mechanisms involved in caloric regulation may provide critical insights into energy balance as well as into the hyperphagia that develops as these mechanisms are overcome.

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Dynamic expression of homeostatic ion channels in differentiated cortical astrocytes in vitro.

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