The Journal of Neurophysiology Vol. 85 No. 2 February 2001, pp. 986-994
Copyright ©2001 by the American Physiological Society

Müller Cell Ca²⁺ Waves Evoked by Purinergic Receptor Agonists in Slices of Rat Retina

Laboratory of Cellular and Molecular Neurophysiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Li, Yang, Lynne A. Holtzclaw, and James T. Russell. Müller Cell Ca²⁺ Waves Evoked by Purinergic Receptor Agonists in Slices of Rat Retina. J. Neurophysiol. 85: 986-994, 2001. We have measured agonist evoked Ca²⁺ waves in Müller cells in situ within freshly isolated retinal slices. Using an eye cup dye loading procedure we were able to preferentially fill Müller glial cells in retinal slices with calcium green. Fluorescence microscopy revealed that bath perfusion of slices with purinergic agonists elicits Ca²⁺ waves in Müller cells, which propagate along their processes. These Ca²⁺ signals were insensitive to tetrodotoxin (TTX, 1.0 µM) pretreatment. Cells were readily identified as Müller cells by their unique morphology and by subsequent immunocytochemical labeling with glial fibrillary acidic protein antibodies. While cells never exhibited spontaneous Ca²⁺ oscillations, purinoreceptor agonists, ATP, 2 MeSATP, ADP, 2 MeSADP, and adenosine readily elicited Ca²⁺ waves. These waves persisted in the absence of [Ca²⁺]_o but were abolished by thapsigargin pretreatment, suggesting that the purinergic agonists tested act by releasing Ca²⁺ from intracellular Ca²⁺ stores. The rank order of potency of different purines and pyrimidines for inducing Ca²⁺ signals was 2 MeSATP = 2MeSADP > ADP > ATP meATP = uridine triphosphate (UTP) > uridine diphosphate (UDP). The Ca²⁺ signals evoked by ATP, ADP, and 2 MeSATP were inhibited by reactive blue (100 µM) and suramin (200 µM), and the adenosine induced signals were abolished only by 3,7-dimethyl-1-propargylxanthine (200 µM) and not by 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine) or 8-cyclopentyl-1,3-dipropylxanthine at the same concentration. Based on these pharmacological characteristics and the dose-response relationships for ATP, 2 MeSATP, 2 MeSADP, ADP, and adenosine, we concluded that Müller cells express the P1A2 and P2Y₁ subtypes of purinoceptors. Analysis of Ca²⁺ responses showed that, similar to glial cells in culture, wave propagation occurred by regenerative amplification at specialized Ca²⁺ release sites (wave amplification sites), where the rate of Ca²⁺ release was significantly enhanced. These data suggest that Müller cells in the retina may participate in signaling, and this may serve as an extra-neuronal signaling pathway.