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In the brain, the complement system plays a crucial role in the immune response and in synaptic elimination during normal development and disease. Here, we sought to identify pathways that modulate the production of complement component 4 (C4), recently associated with an increased risk of schizophrenia. To design a disease-relevant assay, we first developed a rapid and robust 3D protocol capable of producing large numbers of astrocytes from pluripotent cells. Transcriptional profiling of these astrocytes confirmed the homogeneity of this population of dorsal fetal-like astrocytes. Using a novel ELISA-based small-molecule screen, we identified epigenetic regulators, as well as inhibitors of intracellular signaling pathways, able to modulate C4 secretion from astrocytes. We then built a connectivity map to predict and validate additional key regulatory pathways, including one involving c-Jun-kinase. This work provides a foundation for developing therapies for CNS diseases involving the complement cascade., Competing Interests: Conflict of interests B.S. serves on the scientific advisory board of Annexon LLC and is a minor shareholder of Annexon LLC; however, this is unrelated to the submitted work. L.L.R. is a founder of Elevian, Rejuveron, and Vesalius Therapeutics, a member of their scientific advisory boards, and a private equity shareholder. All are interested in formulating approaches intended to treat diseases of the nervous system and other tissues. He is also on the advisory board of Alkahest, a Grifols company, focused on the plasma proteome. None of these companies provided any financial support for the work in this paper. The current work is unrelated to work carried out at any of these companies. All other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: We studied Na+ channel expression and the ability to generate action potential (AP)-like responses in primary cultures of human astrocytes by whole cell patch-clamp recording techniques., Methods: Tissue samples from 22 patients with various classifications of temporal lobe epilepsy (TLE) were plated to form separate astrocyte cultures from three regions; the hippocampus, parahippocampus, and anterolateral temporal neocortex., Results: The resting membrane potential of seizure focus astrocytes (MTLE, mesial TLE) was significantly depolarized (approximately -55 mV) as compared with cortical astrocytes (-80 mV). Hippocampal astrocytes from other substrates for TLE (MaTLE, mass-associated TLE; PTLE, paradoxical TLE) in which the hippocampus is not the seizure focus displayed resting membrane potentials similar to those of neocortical astrocytes (approximately -75 mV). Astrocytes from the seizure focus (MTLE) displayed much larger tetrodotoxin (TTX)-sensitive Na+ currents with -66-fold higher Na+ channel density (113.5 +/- 17.41 pA/pf) than that of comparison neocortical astrocytes (1.7 +/- 3.7 pA/pf) or than that of the hippocampal and parahippocampal astrocytes of the MaTLE and PTLE groups. As a consequence of this higher channel density, seizure focus astrocytes were capable of generating AP-like responses. However, at the resting potential, most Na+ channels are inactive and no spontaneous firing was observed. In contrast, astrocytes in the comparison neocortex from all groups and the hippocampus and parahippocampus from the MaTLE and PTLE groups could not fire AP-like responses even after large current injections., Conclusions: The function of Na+ channels in these astrocytes is unclear. However, the marked differences in seizure focus astrocytes as compared with cortical and nonseizure focus hippocampal astrocytes suggest a more active role for astrocytes associated with hyperexcitable neurons at a seizures focus.

We have studied the optimal conditions to maintain the astrocyte GSH levels under normal and oxidative stress conditions. The rate of GSH synthesis from L-methionine was statistically lower than from L-cystine or N-acetyl-cysteine in astrocytes treated with diethyl-maleate, which is a substrate of GSH S-transferases. This is in accordance with the fact that cystathionase activity was not detectable. The transport of L-cystine mediated by the Na(+)-independent system Xc- is the limiting step in GSH synthesis in astrocytes. Incubation with tert-butyl hydroperoxide (t-booH) reduced GSH concentration in astrocytes. This reduction was ameliorated in part by the addition of ascorbate or dehydroascorbate. When L-cystine and ascorbate were added together to the t-booH-treated astrocytes, the GSH concentration was indistinguishable from controls. Electron micrographs of astrocytes treated with t-booH showed an increased number of vacuoles and mitochondrial swelling. This was prevented by ascorbate and dehydroascorbate. The physiological implications of the availability of GSH precursors and ascorbate in the maintenance of GSH in astrocytes are discussed.

1. We probed for the expression of electrogenic Na+/HCO3- co-transport in cultured mammalian astrocytes by recording voltage and current changes induced by bath application of HCO3-, with the use of patch-clamp electrophysiology. Application of 25 mM HCO3-, at a constant pHo, to astrocytes bathed in a nominally HCO3(-)-free solution, produced an abrupt and reversible change in membrane potential ranging from +3 to -30 mV [-11.8 +/- 9.34 (SD) mV]; 55% of cells showed relatively large hyperpolarizing responses (-18.8 +/- 6.23 mV), whereas 45% showed only small shifts in membrane potential (range of -5 to +3 mV; -1.9 +/- 1.96 mV). 2. The size of the HCO3(-)-induced hyperpolarization was strongly related to the cell's initial resting membrane potential in HCO3(-)-free solution; the larger responses were seen in cells with relatively low resting membrane potentials (-48.5 +/- 9.4 mV), and the smaller responses were seen in cells with more negative potentials (-68.1 +/- 6.5 mV). The membrane potentials of hippocampal astrocytes were highly variable in HCO3(-)-free solution (range -38 to -80 mV; -60.9 +/- 12.53); this variability was greatly reduced in HCO3(-)-containing solution (range -59 to -82 mV; -68.5 +/- 4.8). 3. The magnitude of the HCO3(-)-induced response was less strongly correlated with cell input resistance, which was higher in the larger responder cells than in the small responders. However, the differences in input resistance were insufficient to account for the different HCO3(-)-induced responses observed. 4. In the presence of extracellular Ba2+, which by blocking K+ conductance depolarized cells by 30-50 mV, cells that initially showed a small response, showed a large and completely reversible hyperpolarization (-18.4 +/- 6.13 mV) to application of 25 mM HCO3-. In Na(+)-free solution, the HCO3(-)-induced hyperpolarization was reduced by 66%, and the response was not sustained, as in Na(+)-containing solution. Removal of extracellular Cl- had no effect on the HCO3- response The stilbene derivative 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS), a blocker of anion transport, eliminated the HCO3(-)-induced hyperpolarization. Blockers of Na+/K+ ATPase and Na(+)-H+ exchange were without effect. These observations indicated the presence of an electrogenic Na+/HCO3- co-transporter in hippocampal astrocytes. 6. Voltage-clamp recording demonstrated that the HCO3(-)-induced hyperpolarization was caused by outward currents averaging 335 +/- 104 pA. The reversal potential of the HCO3(-)-induced current ranged between -80 and -99 mV with an average = -86.1 +/- 6.2 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Primary astrocyte cultures exposed to hypotonic media undergo a rapid initial swelling followed by a regulatory volume decrease (RVD), which is associated with the release of ions and amino acids. The Ca2+ dependence of RVD was investigated. Using a method that measures extracellular electrical resistance to measure cell volume changes in substratum-attached cells, we found that when astrocytes were exposed to hypotonic media without calcium, RVD was abolished. The addition of CaCl2 to astrocytes swollen in hypotonic media without calcium caused an almost immediate initiation of volume regulation, with an EC50 of approximately 0.1 mM CaCl2. Swelling of astrocytes in hypotonic medium caused an increased influx of 45Ca2+, which was partially blocked (60%) by 1 microM nimodipine, suggesting that voltage-gated L-type calcium channels were being opened. Previous work had shown that hypotonic media-induced swelling of astrocytes caused membrane potential depolarizations sufficient to open such channels (Kimelberg and O'Connor, 1988). By measuring intracellular free calcium with fura-2, we found that astrocytes swollen in hypotonic medium showed a rapid increase in [Ca2+]i, reaching a peak of approximately 600 nM, followed by a decrease to a sustained plateau (approximately 250 nM) mirroring the time course of volume regulation. The removal of extracellular calcium totally abolished, and the addition of 1 microM nimodipine partially abolished the elevated plateau, while neither affected the initial [Ca2+]i peak. These data suggest that the initial peak of the hypotonic-induced rise in [Ca2+]i is caused by release from intracellular stores and that the sustained elevated plateau is due to extracellular calcium influx. The removal of extracellular calcium also abolished swelling-induced K+(86Rb) and 36Cl- efflux, but did not affect the swelling-induced release of 3H-D-aspartate, or 3H-taurine (data not shown). These data indicate that hypotonic-induced aspartate and taurine release is not necessary for RVD in astrocytes swollen by exposure to hypotonic media, since RVD is completely inhibited by the omission of external Ca2+. The addition of 1 mM quinine HCl, which is known to block Ca(2+)-activated K+ channels, also abolished both volume regulation and 86Rb+ efflux in hypotonic media-swollen astrocytes in the presence of medium calcium, but did not affect 3H-D-aspartate efflux. We suggest that the swelling of astrocytes in hypotonic media which leads to a rapid membrane depolarization first opens voltage-gated calcium channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Exposure of primary astrocyte cultures to isosmotic ethanol from 10-100 mM led to both swelling of the cells and release of [3H]taurine and D-[3H]aspartate. Exposure to hyperosmotic ethanol, in the same concentration range, caused neither swelling nor release. Release was inhibited by the anion transport blocker L-644,711, already shown to inhibit amino acid release evoked by hypoosmotic or high-potassium medium, conditions that also cause astrocytic swelling. Ethanol-induced release generally showed a decline in response to successive exposures to ethanol, and release was not dependent on extracellular calcium. Thus, the characteristics of swelling-induced release of amino acids by isosmotic ethanol seem to correspond to those of swelling-induced release from astrocytes due to exposure to hypotonic or high-K+ media. We discuss whether such effects may contribute to CNS damage after head injury and stroke.

An electrical resistance method was developed to measure volume changes in substratum-attached monolayer cultures. Astrocytes in primary monolayer cultures prepared from neonatal rat cerebral cortex were placed in a confined channel containing a balanced salt solution, and the electrical resistance of the channel was measured using an applied alternating current. If the volume of the cells increases, then the volume of the solution within the channel available for current flow decreases by the same amount, resulting in an increase in the measured resistance through the channel. If the volume of the cells decreases, a decrease in resistance would be recorded. This method allows continuous measurements of volume changes in real time. When primary astrocyte monolayers were exposed to hyposmotic solutions (93-193 mosmol/kgH2O), they showed a rapid initial swelling and, in the continued presence of hyposmotic media, a characteristic regulatory volume decrease (RVD) in which there was a return to normal cell volume within approximately 20 min. Astrocytes exposed to hyperosmotic media (343-493 mosmol/kgH2O) gave a decrease in electrical resistance, indicating shrinkage. Putative endogenous effectors of astrocytic swelling, such as high extracellular K+ and glutamate, resulted in a much slower onset of swelling and no sign of RVD. This system can reliably measure the average change in cell monolayer volume to 1-2% and thus provides a sensitive means of continuous measurements of changes in cell volume in monolayer cultures.

Rat brain astrocytes growing in primary monolayer cultures were swollen by exposing them to media of decreasing osmolality caused by removal of NaCl, and the effects of this treatment on their membrane potentials were measured by intracellular recording. Depolarizations were seen that were proportional to the degree of swelling, reaching a maximum of around 60 mV when 80-100 mM NaCl was removed from the reaction media, which had an original total osmolality of 290 mosmolal. These effects were completely reversible, since restoring the cells to iso-osmotic medium after a 2-min exposure caused an immediate repolarization back to the original membrane potentials, and depolarizations were not seen when isotonicity was maintained by replacing NaCl with sucrose. Partial repolarization was seen during an extended period (30 min) of exposure to hypo-osmotic medium, mirroring a regulatory volume decrease we have previously described in these cells under identical conditions. In ion-replacement studies depolarizations were seen when the solution was made hypo-osmotic with the large cation N-methyl-D-glucamine totally replacing Na+ in the medium. Removal of Cl- from the medium also had no effect on the initial swelling-induced depolarization. These results show that even moderate swelling of astrocytes in primary culture results in marked depolarization of their membrane potentials; possible mechanisms for this effect and the potentially profound implications for the swelling of astrocytes seen in situ under pathological conditions are discussed.