Research reportDistribution of rSlo Ca2+-activated K+ channels in rat astrocyte perivascular endfeet
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.
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