Elsevier

Brain Research

Volume 956, Issue 2, 29 November 2002, Pages 183-193
Brain Research

Research report
Distribution of rSlo Ca2+-activated K+ channels in rat astrocyte perivascular endfeet

https://doi.org/10.1016/S0006-8993(02)03266-3Get rights and content

Abstract

Evidence that Ca2+-activated K+ (KCa) channels play a role in cell volume changes and K+ homeostasis led to a prediction that astrocytes would have KCa channels near blood vessels in order to maintain K+ homeostasis. Consistent with this thinking the present study demonstrates that rSlo KCa channels are in glial cells of the adult rat central nervous system (CNS) and highly localized to specializations of astrocytes associated with the brain vasculature. Using confocal and thin-section electron microscopic immunolabeling methods the distribution of rSlo was examined in adult rat brain. Strong rSlo immunolabeling was present around the vasculature of most brain regions. Examination of dye-filled hippocampal astrocytes revealed rSlo immunolabeling polarized in astrocytic endfeet. Ultrastructural analysis confirmed that the rSlo staining was concentrated in astrocytic endfeet ensheathing capillaries as well as abutting the pia mater. Immunostaining within the endfeet was predominantly distributed at the plasma membrane directly adjacent to either the vascular basal lamina or the pial surface. The distribution of the aquaporin-4 (AQP-4) water channel was also examined using dye-filled hippocampal astrocytes. In confirmation of earlier reports, intense AQP-4 immunolabeling was generally observed at the perimeter of blood vessels, and coincided with perivascular endfeet and rSlo labeling. We propose that rSlo KCa channels, with their sensitivity to membrane depolarization and intracellular calcium, play a role in the K+ modulation of cerebral blood flow. Additional knowledge of the molecular and cellular machinery present at perivascular endfeet may provide insight into the structural and functional molecular elements responsible for the neuronal activity-dependent regulation of cerebral blood flow.

Introduction

K+ redistribution is postulated to play a major role in coupling neuronal activation to changes in regional cerebral blood flow (CBF). The diameter of cerebral vasculature is sensitive to changes in extracellular K+ concentrations [19], [24], [43], however it is unclear whether the delivery of K+ occurs via a direct and/or indirect mechanism. A direct mechanism would involve the diffusion of K+ from the interstitial space surrounding neurons directly to the cerebral vasculature. Existence of a faster, astrocyte-dependent pathway between neurons and the vasculature that modulates CBF has been described [32], [36], which involves transportation of K+ from the area surrounding the neurons to the area surrounding the vasculature and the pial surfaces of the brain via an astrocytic K+ siphon. In support of this hypothesis, astrocytes have a greater K+ conductance than neurons, and the majority of K+ conductance is localized on astrocytic endfeet [32]. Using this information, Odette and Newman [46] proposed a one-dimensional spatial model of K+ dynamics incorporating several parameters (including extracellular and distribution space volume fractions, tortuosity, extracellular K+ concentrations and diffusion coefficients) to predict K+ dynamics in the brain and therefore the feasibility of astrocytic involvement in redistribution of K+ from the perineuronal to perivascular areas.

One type of K+ channel, the Ca2+-activated K+ channel (KCa), is present in several different cell types, including endocrine cells, muscle cells, neurons and glia [3], [10], [30], [39]. The high-conductance (Maxi-K) class of KCa channels is both Ca2+-dependent and voltage-dependent [41], and includes the slo family of alternatively spliced genes with differential sensitivities to Ca2+[40]. It has been suggested that KCa channels play a role in cell swelling and K+ homeostasis [23], [37].

In astrocytes, electrophysiological studies have demonstrated KCa currents in vitro [39]; however, there is comparatively little data concerning the localization of these channels in astrocytes in vivo. Extracellular K+ buildup near active neurons could be compensated for by K+ uptake by astrocytes, which would then be transported through KCa channels near blood vessels. These channels could shunt the K+ away from areas of neuronal activity, thereby maintaining K+ at acceptably low levels in neuropil.

The goals of present studies were to: (1) determine whether rSlo KCa channels are localized to perivascular astrocytic endfeet in the rat CNS; (2) determine if the patterns of rSlo distribution in the rat CNS produced with a different antibody produced by Knaus and coworkers [16] corresponded to our observations with the antibody produced to a different portion of rSlo[27]; and (3) confirm the distribution of AQP-4 water channels relative to astrocytic endfoot processes versus perineuronal processes of astrocytes, as these molecules might be expected to have a similar distribution since osmotic gradients induced by K+ buffering are likely accompanied by water flux [9].

Section snippets

Antibodies

A previously characterized rabbit polyclonal antibody to the α-subunit of rSlo was used, as described by Mi and colleagues [25], [27]. Briefly, following the cloning of rSlo from a sciatic nerve mRNA library, a synthetic peptide corresponding to amino acids 1179–1196 of the mouse homolog of Slo (NQYKSTSSLIPPIREVEDEC) was used to raise polyclonal antiserum. Following antibody production and affinity purification, this antiserum successfully labeled sciatic nerve protein of appropriate weight

Light microscopic immunolocalization of rSlo KCa channels in the rat CNS

As previously reported [16], rSlo immunolabeling was observed throughout the rat CNS. The rSlo antibody used in the present studies produced a virtually identical pattern of labeling as that produced with the Knaus rSlo antibody (data not shown).

This labeling was particularly strong in regions of the cortex, cerebellum, hippocampus, thalamus, and basal ganglia. The characteristics of rSlo labeling greatly varied between brain regions and included labeling of neuronal somata and dendrites, as

Discussion

This study describes the first localization of rSlo KCa channels to astrocytic perivascular endfeet in the rat CNS. Using the previously characterized α-rSlo antibody [27], the distribution of rSlo immunolabeling within the adult rat brain was visualized using confocal microscopic and electron microscopy. Confocal imaging revealed distinct rSlo immunolabeling present around the blood vessels located within most brain regions. Immunofluorescent rSlo+GFAP double-labeling data further revealed

Acknowledgements

The authors wish to acknowledge Vickie Edelman, Thomas Deerinck, and Eric Bushong for their technical assistance. The rSlo antiserum against a different region of the KCa channel was generously provided by H.G. Knaus of the Institute of Biochemical Pharmacology, University of Innsbruck. We also thank Maryann Martone and Eric Bushong, for their comments on the manuscript. This work was supported by NIH grants RR04050, NS14718 and DC03192 to M.H.E., and NIH grant supplement RR04050-13S1 to D.L.P.

References (46)

  • J.E.J. Brian et al.

    Recent insights into the regulation of cerebral circulation

    Clin. Exp. Pharmacol. Physiol.

    (1996)
  • A. Bringmann et al.

    Mammalian retinal glial (Müller) cells express large-conductance Ca(2+)-activated K+ channels that are modulated by Mg2+ and pH and activated by protein kinase A

    Glia

    (1997)
  • E.H. Buhl, W.K. Schwertfeger, P. Germroth, Intracellular injection of neurons in fixed brain tissue combined with other...
  • E. Bushong et al.

    Protoplasmic astrocytes in CA1 stratum radiatum establish anatomical domains

    J. Neurosci.

    (2002)
  • N.O. Dalby et al.

    The process of epileptogenesis: a pathophysiological approach

    Curr. Opin. Neurol.

    (2001)
  • I. Dietzel et al.

    Transient changes in the size of the extracellular space in the sensorimotor cortex of cats in relation to stimulus-induced changes in potassium concentration

    Exp. Brain Res.

    (1980)
  • L. Fagni et al.

    Activation of a large-conductance Ca2+-dependent K+ channel by stimulation of glutamate phosphoinositide-coupled receptors in cultured cerebellar granule cells

    Eur. J. Neurosci.

    (1991)
  • T.S. Ha et al.

    Functional characteristics of two BKCa channel variants differentially expressed in rat brain tissues

    Eur. J. Biochem.

    (2000)
  • K. Higashi et al.

    An inwardly rectifying K+ channel, Kir4.1, expressed in astrocytes surrounds synapses and blood vessels in brain

    Am. J. Physiol. Cell Physiol.

    (2001)
  • M. Ito

    Cerebellar long-term depression: characterization, signal transduction, and functional roles

    Physiol. Rev.

    (2001)
  • J.H. Jaggar et al.

    Ca2+ channels, ryanodine receptors and Ca(2+)-activated K+ channels: a functional unit for regulating arterial tone

    Acta Physiol. Scand.

    (1998)
  • H.G. Knaus et al.

    Distribution of high-conductance Ca(2+)-activated K+ channels in rat brain: targeting to axons and nerve terminals

    J. Neurosci.

    (1996)
  • H.J. Knot et al.

    Regulation of membrane potential and diameter by voltage-dependent K+ channels in rabbit myogenic cerebral arteries

    Am. J. Physiol.

    (1995)
  • Cited by (0)

    1

    Current address: Celera Genomics, 850 Lincoln Center Drive, Mail Stop 431, Foster City, CA 94404, USA.

    View full text