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. 2005 Jun 8;25(23):5502-10.
doi: 10.1523/JNEUROSCI.1354-05.2005.

Calcium increases in retinal glial cells evoked by light-induced neuronal activity

Eric A Newman  1
Affiliations

Calcium increases in retinal glial cells evoked by light-induced neuronal activity

Eric A Newman. J Neurosci. .

Abstract

Electrical stimulation of neurons in brain slices evokes increases in cytoplasmic Ca(2+) in neighboring astrocytes. The present study tests whether similar neuron-to-glial signaling occurs in the isolated rat retina in response to light stimulation. Results demonstrate that Müller cells, the principal retinal glial cells, generate transient increases in Ca(2+) under constant illumination. A flickering light stimulus increases the occurrence of these Ca(2+) transients. Antidromic activation of ganglion cell axons also increases the generation of Müller cell Ca(2+) transients. The increases in Ca(2+) transients evoked by light and antidromic stimulation are blocked by the purinergic antagonist suramin and by TTX. The addition of adenosine greatly potentiates the response to light, with light ON evoking large Ca(2+) increases in Müller cells. Suramin, apyrase (an ATP-hydrolyzing enzyme), and TTX substantially reduce the adenosine-potentiated response. NMDA, metabotropic glutamate, GABA(B), and muscarinic receptor antagonists, in contrast, are mainly ineffective in blocking the response. Light-evoked Ca(2+) responses begin in Müller cell processes within the inner plexiform (synaptic) layer of the retina and then spread into cell endfeet at the inner retinal surface. These results represent the first demonstration that Ca(2+) increases in CNS glia can be evoked by a natural stimulus (light flashes). The results suggest that neuron-to-glia signaling in the retina is mediated by neuronal release of ATP, most likely from amacrine and/or ganglion cells, and that the response is augmented under pathological conditions when adenosine levels increase.

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Figures

Figure 1.
Figure 1.
Calcium transients in Müller cells. A-C show Ca2+ fluorescence ratio images of the retina that have been thresholded. The red and yellow areas indicate retinal regions in which Ca2+ has increased transiently during the acquisition period. The red areas are regions <12 μm2. The yellow areas exceed this size and are scored as Ca2+ transients within Müller cells. Images in A-C were acquired before, during, and after a flickering light stimulus, respectively. The light stimulus protocol, a 16 s episode of flickering light superimposed on a dim background light, is shown at the bottom. The dashed line represents 0 intensity. The gray bars indicate the timing of the three acquisition periods, and the arrows point to the corresponding images (see Materials and Methods for details). Müller cell Ca2+ transients are shown in supplemental movie 1 (available at www.jneurosci.org as supplemental material).
Figure 2.
Figure 2.
Calcium transients in individual Müller cells in retinas under constant illumination. In some Müller cells, Ca2+ rose rapidly from a flat baseline. In others, the rapid rise in Ca2+ was preceded by a smaller, slow Ca2+ increase (arrows). The four traces were obtained in different trials. Calcium fluorescence images were acquired at 30 Hz.
Figure 3.
Figure 3.
Light-evoked Ca2+ increases in Müller cells. A, Calcium fluorescence measured simultaneously in eight Müller cells. Calcium transients are more likely to be generated during the flickering light stimulus. B, Mean Ca2+ fluorescence increase evoked by a flickering light. The response represents transient Ca2+ increases averaged over 84 trials. The Ca2+ fluorescence from ∼260 Müller cells was monitored in each trial. A linear downward slope of the trace, caused by dye bleaching, has been subtracted. The light stimulus is shown at the bottom in both A and B.
Figure 4.
Figure 4.
Effect of agonists and antagonists on Ca2+ transients in Müller cells. A, Both a flickering light stimulus and addition of ATP to the superfusate evoke increases in the generation of Ca2+ transients. B, A flickering light evokes an increase in the generation of Ca2+ transients (light stimulation; control). The response is blocked by 100 μm suramin and 200 nm TTX. Antidromic activation of ganglion cell axons evokes an increase in the occurrence of Ca2+ transients (antidromic stimulation; control). This response is also blocked by 30 μm suraminand 200 nm TTX. Asterisks indicate a significant difference from controls (*p < 0.05). The flickering light protocol used in A and B is illustrated in Figure 1.
Figure 5.
Figure 5.
Adenosine potentiates the light-evoked Müller cell Ca2+ increase (100 μm adenosine in superfusate). A, B, Ca2+ fluorescence images within the ganglion cell layer. During the first 0.8 s after light ON (A), Ca2+ remains low within Müller cells. At 3.1 s after light ON (B), Müller cell Ca2+ has risen substantially. Müller cell processes surrounding ganglion cell somata (dark circles) are visible. Scale bar, 20 μm. C, Time course of Ca2+ rise in Müller cells for the experiment illustrated in A and B. The light stimulus is shown at the bottom. The dashed line represents 0 intensity. This trial is shown in supplemental movie 2 (available at www.jneurosci.org as supplemental material).
Figure 6.
Figure 6.
Light-evoked, propagated Ca2+ wave in Müller cells (100 μm adenosine in superfusate). Calcium florescence images in the ganglion cell layer are shown. Within the first seconds after light ON, there is a large Ca2+ increase in all Müller cells (data not shown). By 12.5 s after light ON, this Ca2+ increase has decayed (12.5 s). At 14.2 s, a secondary rise in Ca2+ occurs in two Müller cells (short arrows). This Ca2+ increase propagates into adjacent Müller cells at 15.9 and 18.4 s (long arrows). Numbers indicate elapsed time after light ON. Scale bar, 10 μm. This trial is shown in supplemental movie 3 (available at www.jneurosci.org as supplemental material).
Figure 7.
Figure 7.
Light-evoked Müller cell Ca2+ increases within different retinal layers (5 μm NECA in superfusate). The images are from an oblique optical section through an everted eyecup and show retinal layers within the inner half of the retina. The vertical lines indicate boundaries between retinal layers. INL, Inner nuclear layer. The drawing above A shows the location of a Müller cell (M), a ganglion cell soma (G), and neuronal somata within the INL (unlabeled). A, Ca2+ fluorescence image in the unstimulated retina. Müller cells are selectively labeled. Müller cell processes surrounding ganglion cell somata in the GCL, Müller cell stalk processes in the IPL, and Müller cell processes surrounding neuronal somata in the INL are shown. B-D, Ca2+ fluorescence ratio images showing the change in Ca2+ after light ON. At 1.6 s after light ON (B), no increase in Ca2+ is seen. At 2.4 s (C), Ca2+ rises in Müller cell processes within the inner and middle IPL and, to a lesser extent, in the GCL. At 3.2 s (D), Müller cell Ca2+ increases have spread throughout the GCL and into Müller cell endfeet at the inner retinal surface (far left). Müller cell Ca2+ increases also spread throughout the IPL and into the inner portion of the INL.
Figure 8.
Figure 8.
Intensity-response relationship of the light-evoked Ca2+ increase in Müller cells. The peak Ca2+ fluorescence amplitude is plotted as a function of the intensity of the light stimulus (2 μm NECA in superfusate). Mean amplitude ± SEM is shown; n = 7.
Figure 9.
Figure 9.
Effect of agonists and antagonists on Müller cell Ca2+ increases potentiated by NECA (adenosine agonist) (2 μm NECA in all solutions). A, Effect of agonists. The addition of ATP but not trans-ACPD (mGluR agonist) evokes Müller cell Ca2+ increases. Control response to light is shown at the left. B, Effect of antagonists. The addition of suramin (30 μm; purinergic antagonist) and apyrase (100 U/ml; ATP-hydrolyzing enzyme) blocks the light-evoked Ca2+ response. TTX (1 μm) reduces the light response. CPP (10 μm; NMDA antagonist) and scopolamine (10 μm; muscarinic antagonist) have no effect on the light response. E4CPG (200 μm; group I/II mGluR antagonist), E4CPG plus CPPG (2 μm; group III mGluR antagonist), and saclofen (200 μm; GABAB antagonist) have little effect on the light response. Cyclopiazonic acid (CPA; 30 μm), which depletes internal Ca2+ stores, abolishes the light response. Control response to light ON is shown at the left. Asterisks indicate significant difference from controls (p < 0.05).
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