Figure 4. CB1R-mediated increase in RGC responsiveness requires GlyRs and NKCC1 inhibition by AMPK.

(a–h) Extracellular multi-unit spike rates in RGCs were evoked with a 500 ms light-OFF stimulus. After establishing baseline response rates (Before), various pharmacological agents were washed on sequentially. (a–c) The CB1R inverse agonist AM-251 prevented (a) WIN 55,212-2-, (b) ACEA- and (c) URB597-mediated increases in spiking rates (n = 10 animals for each group), confirming the requirement for CB1R activation. (d) The GABA-A receptor blocker gabazine did not prevent the enhancement of RGC excitability, but the selective GlyR antagonist strychnine completely abolished the enhancement when applied either (e) before or (f) after WIN 55,212-2. (g) The NKCC1 blocker bumetanide mimicked and occluded the effects of WIN 55,212-2, and (h) no additional effect was observed if bumetanide was applied after WIN 55,212-2 (n = 10 animals for d–g, n = 9 animals for h). (i) RT-PCR confirmed expression of the cation-chloride cotransporters NKCC1 and KCC2 in the Xenopus retina at stage 45. (j) Western blots show a 60 kD band staining for phospho-SPAK/phospho-OSR1, and β-tubulin, in controls or following treatment with WIN-55,212-2 (1 µM) in the Xenopus retina at stage 45. (k) Bar graph showing fold-change in phosphoprotein levels following treatment with WIN-55,212-2 (1 µM) of NKCC1_{phospho Thr212+Thr217}, and phospho-SPAK/phospho-OSR1 normalized to control levels (n = 4). (l) In the presence of dorsomorphin (10 µM) to block AMPK, WIN-55,212-2 (1 µM) treatment no longer enhances, but instead reduces RGC firing rates (n = 10 animals). (a–h) *p<0.05 one-tailed RM ANOVA with Holm-Sidak posttest, (l) **p<0.01, paired t-test. RGC, Retinal ganglion cell.

DOI: http://dx.doi.org/10.7554/eLife.15932.007

Figure 4—figure supplement 1. Similar trend of pharmacological agents on light-ON responses to that observed for light-OFF stimuli.

Normalized spike rates of RGCs in response to light-ON stimuli before and after application of the CB1R inverse agonist AM-251, followed by the application of (a) WIN 55,212-2, or (b) URB597. Normalized spike rates of RGCs in response to light-ON stimuli, before and after the application of (c) the GABA-A receptor antagonist gabazine. (d) the GlyR antagonist strychnine, followed by the application of WIN 55,212, (e) WIN 55,212-2, followed by the application of strychnine, (f) the NKCC1 blocker bumetanide, followed by the application of WIN 55,212-2 or (g) WIN 55,212-2, followed by the application of bumetanide. (h) Summary of fold differences in extracellular RGC spike rates in response to light-ON and light-OFF stimuli before and after WIN 55,212-2 application for the various inhibitors used in a–g. (n = 10 animals for each condition). (i) Western blots of stage 45 retina showing a 130 kD band stained for phospho-NKCC1 (left) and total NKCC1 (right), in control condition (top) or following treatment with WIN-55,212-2 (1 µM) (bottom). *p<0.05, one-tailed, two-way RM ANOVA. Holm-Sidak tests were used to correct for multiple comparisions. RGC, Retinal ganglion cell.

DOI: http://dx.doi.org/10.7554/eLife.15932.008