Astrocytes are extensively coupled through gap junctions into a syncytium. However, the basic role of this major brain network remains largely unknown. Using electrophysiological and computational modeling methods, we demonstrate that the membrane potential (VM) of an individual astrocyte in a hippocampal syncytium, but not in a single, freshly isolated cell preparation, can be well-maintained at quasi-physiological levels when recorded with reduced or K+ free pipette solutions that alter the K+ equilibrium potential to non-physiological voltages. We show that an astrocyte’s associated syncytium provides powerful electrical coupling, together with ionic coupling at a lesser extent, that equalizes the astrocyte’s VM to levels comparable to its neighbors. Functionally, this minimizes VM depolarization attributable to elevated levels of local extracellular K+ and thereby maintains a sustained driving force for highly efficient K+ uptake. Thus, gap junction coupling functions to achieve isopotentiality in astrocytic networks, whereby a constant extracellular environment can be powerfully maintained for crucial functions of neural circuits.

Syncytial isopotentiality, resulting from a strong electrical coupling, emerges as a physiological mechanism that coordinates individual astrocytes to function as a highly efficient system in brain homeostasis. However, whether syncytial isopotentiality occurs selectively to certain brain regions or is universal to astrocytic networks remains unknown. Here, we have explored the correlation of syncytial isopotentiality with different astrocyte subtypes in various brain regions. Using a nonphysiological K+‐free/Na+ electrode solution to depolarize a recorded astrocyte in situ, the existence of syncytial isopotentiality can be revealed: the recorded astrocyte's membrane potential remains at a quasi‐physiological level due to strong electrical coupling with neighboring astrocytes. Syncytial isopotentiality appears in Layer I of the motor, sensory, and visual cortical regions, where astrocytes are organized with comparable cell densities, interastrocytic distances, and the quantity of directly coupled neighbors. Second, though astrocytes vary in their cytoarchitecture in association with neuronal circuits from Layers I–VI, the established syncytial isopotentiality remains comparable among different layers in the visual cortex. Third, neurons and astrocytes are uniquely organized as barrels in Layer IV somatosensory cortex; interestingly, astrocytes both inside and outside of the barrels do electrically communicate with each other and also share syncytial isopotentiality. Fourth, syncytial isopotentiality appears in radial‐shaped Bergmann glia and velate astrocytes in the cerebellar cortex. Fifth, although fibrous astrocytes in white matter exhibit a distinct morphology, their network syncytial isopotentiality is comparable with protoplasmic astrocytes. Altogether, syncytial isopotentiality appears as a system‐wide electrical feature of astrocytic networks in the brain.

BackgroundNeonatal astrocytes are diverse in origin, and undergo dramatic change in gene expression, morphological differentiation and  syncytial networking throughout development. Neonatal astrocytes also play multifaceted roles in neuronal circuitry establishment. However, the extent to which neonatal astrocytes differ from their counterparts in the adult brain remains unknown.ResultsBased on ALDH1L1-eGFP expression or sulforhodamine 101 staining, neonatal astrocytes at postnatal day 1–3 can be reliably identified in hippocampal stratum radiatum. They exhibit a more negative resting membrane potential (VM), −85 mV, than mature astrocytes, −80 mV and a variably rectifying whole-cell current profile due to complex expression of voltage-gated outward transient K+ (IKa), delayed rectifying K+ (IKd) and inward K+ (IKin) conductances. Differing from NG2 glia, depolarization-induced inward Na+ currents (INa) could not be detected in neonatal astrocytes. A quasi-physiological VM of −69 mV was retained when inwardly rectifying Kir4.1 was inhibited by 100 μM Ba2+ in both wild type and TWIK-1/TREK-1 double gene knockout astrocytes, indicating expression of additional leak K+ channels yet unknown. In dual patch recording, electrical coupling was detected in 74 % (14/19 pairs) of neonatal astrocytes with largely variable coupling coefficients. The increasing gap junction coupling progressively masked the rectifying K+ conductances to account for an increasing number of linear voltage-to-current relationship passive astrocytes (PAs). Gap junction inhibition, by 100 μM meclofenamic acid, substantially reduced membrane conductance and converted all the neonatal PAs to variably rectifying astrocytes. The low density expression of leak K+ conductance in neonatal astrocytes corresponded  to a ~50 % less K+ uptake capacity compared to adult astrocytes.ConclusionsNeonatal astrocytes predominantly express a variety of rectifying K+ conductances, form discrete cell-to-cell gap junction coupling and are deficient in K+ homeostatic capacity.

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Mature astrocytes exhibit a linear current-to-voltage K(+) membrane conductance (passive conductance) and an extremely low membrane resistance (Rm) in situ. The combination of these electrophysiological characteristics establishes a highly negative and stable membrane potential that is essential for basic functions, such as K(+) spatial buffering and neurotransmitter uptake. However, astrocytes are coupled extensively in situ. It remains to be determined whether the observed passive behavior and low Rm are attributable to the intrinsic properties of membrane ion channels or to gap junction coupling in functionally mature astrocytes. In the present study, freshly dissociated hippocampal tissues were used as a new model to examine this basic question in young adult animals. The morphologically intact single astrocytes could be reliably dissociated from animals postnatal day 21 and older. At this animal age, dissociated single astrocytes exhibit passive conductance and resting membrane potential similar to those exhibited by astrocytes in situ. To precisely measure the Rm from single astrocytes, dual-patch single-astrocyte recording was performed. We show that dissociated single astrocytes exhibit a low Rm similarly to syncytial coupled astrocytes. Functionally, the symmetric expression of high-K(+) conductance enabled rapid change in the intracellular K(+) concentrations in response to changing K(+) drive force. Altogether, we demonstrate that freshly dissociated tissue preparation is a highly useful model for study of the functional expression and regulation of ion channels, receptors, and transporters in astrocytes and that passive behavior and low Rm are the intrinsic properties of mature astrocytes.

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We have recently shown that a linear current-to-voltage (I-V) relationship of membrane conductance (passive conductance) reflects the intrinsic property of K+ channels in mature astrocytes. While passive conductance is known to underpin a highly negative and stable membrane potential (VM) essential for the basic homeostatic function of astrocytes, a complete repertoire of the involved K+ channels remains elusive. TREK-1 two-pore domain K+ channel (K2P) is highly expressed in astrocytes, and covalent association of TREK-1 with TWIK-1, another highly expressed astrocytic K2P, has been reported as a mechanism underlying the trafficking of heterodimer TWIK-1/TREK-1 channel to the membrane and contributing to astrocyte passive conductance. To decipher the individual contribution of TREK-1 and address whether the appearance of passive conductance is conditional to the co-expression of TWIK-1/TREK-1 in astrocytes, TREK-1 single and TWIK-1/TREK-1 double gene knockout mice were used in the present study. The relative quantity of mRNA encoding other astrocyte K+ channels, such as Kir4.1, Kir5.1, and TREK-2, was not altered in these gene knockout mice. Whole-cell recording from hippocampal astrocytes in situ revealed no detectable changes in astrocyte passive conductance, VM, or membrane input resistance (Rin) in either kind of gene knockout mouse. Additionally, TREK-1 proteins were mainly located in the intracellular compartments of the hippocampus. Altogether, genetic deletion of TREK-1 alone or together with TWIK-1 produced no obvious alteration in the basic electrophysiological properties of hippocampal astrocytes. Thus, future research focusing on other K+ channels may shed light on this long-standing and important question in astrocyte physiology.

Membrane potential (V) depolarization occurs immediately following cerebral ischemia and is devastating for the astrocyte homeostasis and neuronal signaling. Previously, an excessive release of extracellular K and glutamate has been shown to underlie an ischemia-induced V depolarization. Ischemic insults should impair membrane ion channels and disrupt the physiological ion gradients. However, their respective contribution to ischemia-induced neuronal and glial depolarization and loss of neuronal excitability are unanswered questions. A short-term oxygen-glucose deprivation (OGD) was used for the purpose of examining the acute effect of ischemic conditions on ion channel activity and physiological K gradient in neurons and glial cells. We show that a 30 min OGD treatment exerted no measurable damage to the function of membrane ion channels in neurons, astrocytes, and NG2 glia. As a result of the resilience of membrane ion channels, neuronal spikes last twice as long as our previously reported 15 min time window. In the electrophysiological analysis, a 30 min OGD-induced dissipation of transmembrane K gradient contributed differently in brain cell depolarization: severe in astrocytes and neurons, and undetectable in NG2 glia. The discrete cellular responses to OGD corresponded to a total loss of 69% of the intracellular K contents in hippocampal slices as measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). A major brain cell depolarization mechanism identified here is important for our understanding of cerebral ischemia pathology. Additionally, further understanding of the resilient response of NG2 glia to ischemia-induced intracellular K loss and depolarization should facilitate the development of future stroke therapy.