Glutamate-induced Ca2+ influx in third-order neurons of salamander retina is regulated by the actin cytoskeleton

Abstract

Ligand-gated ion channels (ionotropic receptors) link to the cortical cytoskeleton via specialized scaffold proteins and thereby to appropriate signal transduction pathways in the cell. We studied the role of filamentous actin in the regulation of Ca influx through glutamate receptor-activated channels in third-order neurons of salamander retina. Staining by Alexa-Fluor 488-phalloidin, to visualize polymerized actin, we show localization of filamentous actin in neurites, and the membrane surrounding the cell soma. With Ca(2+) imaging we found that in dissociated neurons, depolymerization of filamentous actin by latrunculin A, or cytochalasin D significantly reduced glutamate-induced intracellular Ca(2+) accumulation to 53+/-7% of control value. Jasplakinolide, a stabilizer of filamentous actin, by itself slightly increased the glutamate-induced Ca(2+) signal and completely attenuated the inhibitory effect when applied in combination with actin depolymerizing agents. These results indicate that in salamander retinal neurons the actin cytoskeleton regulates Ca(2+) influx through ionotropic glutamate receptor-activated channels, suggesting regulatory roles for filamentous actin in a number of Ca(2+)-dependent physiological and pathological processes.

Fig. 1

Distribution of F-actin in retinal ganglion cells. Ganglion cells and axon fibers were retrogradely labeled with rhodamine-dextran (A), and then flat-mounts stained with Alexa Fluor 488-phalloidin (B). F-actin was present in axonal fibers and numerous processes of ganglion cells; often appearing as bright spots (inset, arrowheads). Merger of two confocal images is illustrated in C. The high-power view of ganglion cells (inset) was obtained by focusing on cell soma labeled with dextran (red), and phalloidin (green).

Fig. 2

Disruption of the actin cytoskeleton in retinal neurons by latrunculin A. (A) Confocal images of dissociated third-order retinal neurons exposed for 30 min to either vehicle (Control), or 10 μM cytochalasin D, or 5 μM latrunculin A before fixation and staining with Alexa-Fluor488-phalloidin. Note intense fluorescence ring surrounding the cell perimeter (arrowheads) in control cells, and predominantly punctate character of staining, or loss of fluorescence in cytochalasin-, and latrunculin-treated cells, respectively. (B) F-actin staining in dissociated rhodamine-dextran-labeled ganglion cells in control saline (a), and after incubation with cytochalasin D (b), or latrunculin A (c). (a′–c′) Shows the phalloidin fluorescence intensity profile across the white line marked in a–c.

Fig. 3

Effect of latrunculin A on the glutamate-induced Ca²⁺ influx. In control solution glutamate (100 μM) induced Ca²⁺ influx (A), which was significantly (P<0.05) reduced following 30 min incubation of cells with 5 μM latrunculin A (B).

Fig. 4

Jasplakinolide attenuates the inhibitory effect of latrunculin A on glutamate-induced Ca²⁺ influx. Cells were incubated for 30 min in Ringer solution containing 5 μM latrunculin A+5 μM jasplakinolide (B), or jasplakinolide alone (D), and glutamate-induced Ca²⁺ signals were compared with those measured in control solution (A and C). Neither jasplakinolide alone nor in combination with latrunculin A had statistically significant effect on the glutamate-induced Ca²⁺ signal (P>0.3 and P>0.1 respectively).

Fig. 5

Effect of cytochalasin D on Ca²⁺ influx via glutamate receptor-activated channels, and voltage-activated Ca²⁺ channels. (A) High-temporal resolution Ca²⁺ signals induced by glutamate (300 μM), and KCl (20 mM) were recorded with a confocal microscope before and after incubation of cells with 10 μM cytochalasin D. Cytochalasin D itself caused a transient increase in [Ca²⁺]_i (arrowhead), and reduced both glutamate-, and KCl-induced Ca²⁺ signals. The amplitude of spontaneous Ca²⁺ transients (asterisks) was also reduced following exposure to cytochalasin D. (B) Bar graph summarizing the effect of F-actin depolymerization on glutamate-induced Ca²⁺ accumulation.