AMPA receptors mediate acetylcholine release from starburst amacrine cells in the rabbit retina

Abstract

The light response of starburst amacrine cells is initiated by glutamate released from bipolar cells. To identify the receptors that mediate this response, we used a combination of anatomical and physiological techniques. An in vivo, rabbit eyecup was preloaded with [ 3 H]-choline, and the [ 3 H]-acetylcholine (ACh) released into the superfusate was monitored. A photopic, 3 Hz flashing light increased ACh release, and the selective AMPA receptor antagonist, GYKI 53655, blocked this light-evoked response. Nonselective AMPA/kainate agonists increased the release of ACh, but the specific kainate receptor agonist, SYM 2081, did not increase ACh release. Selective AMPA receptor antagonists, GYKI 53655 or GYKI 52466, also blocked the responses to agonists. We conclude that the predominant excitatory input to starburst amacrine cells is mediated by AMPA receptors. We also labeled lightly fixed rabbit retinas with antisera to choline acetyltransferase (ChAT), AMPA receptor subunits GluR1, GluR2/3, or GluR4, and kainate receptor subunits GluR6/7 and KA2. Labeled puncta were observed in the inner plexiform layer with each of these antisera to glutamate receptors, but only GluR2/3-IR puncta and GluR4-IR puncta were found on the ChAT-IR processes. The same was true of starburst cells injected intracellularly with Neurobiotin, and these AMPA receptor subunits were localized to two populations of puncta. The AMPA receptors are expected to desensitize rapidly, enhancing the sensitivity of starburst amacrine cells to moving or other rapidly changing stimuli. Indexing termsglutamate; ionotropic receptors; localization; cholinergic; interneuron; confocal microscopy Starburst amacrine cells are one of the most common types of amacrine cells in rabbit retina (MacNeil et al., 1999), and there are two subtypes. Type a cells have perikarya in the inner nuclear layer, processes ramifying in the outer half of the inner plexiform layer (IPL) in stratum 2 (S2) and OFF responses to light. Type b cells have perikarya displaced to the ganglion cell layer, processes ramifying in stratum 4 (S4) of the IPL and ON responses to light (Famiglietti, 1983;Tauchi and Masland, 1984;Vaney, 1984;Bloomfield and Miller, 1986). In rabbits, starburst amacrine cells are known to receive synaptic inputs from cone (Brandon, 1987;Millar and Morgan, 1987;Famiglietti, 1991).Starburst amacrine cells synthesize and release both γ-aminobutyric acid (GABA) and acetylcholine (ACh) when depolarized (Masland and Livingstone, 1976;Massey and Neal, 1979;O'Malley and Masland, 1989). ACh release from the rabbit retina increases at both light onset and light offset and is maximally stimulated by large stimuli at temporal frequencies of 1-4 Hz (Massey and Neal, 1979;Massey and Redburn, 1985;O'Malley and Masland, 1993). The majority of the synaptic output from the starburst cells is onto ganglion cell dendrites (Brandon, 1987;Millar and Morgan, 1987;Famiglietti, 1991 . The agonist α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) is more potent than kainate or...

Localization and Functional Mapping of AMPA Receptor Subunits in the Developing Rabbit Retina

“…Overall, the expression pattern of GluR4 in the developing rabbit retina is similar to that of GluR2/3 and shows equivalent immunoreactivity in both the IPL and OPL in the developing rat retina. 15,16 Analogous to GluR2/3, there were two bands of GluR4 immunoreactivity discernible in the IPL beginning on P2, which corresponds to GluR4 expression in cholinergic amacrine cells 44 and AII amacrine cells 35 in the adult rabbit retina, which indicates that this specific pattern of expression is already present at early developmental stages.…”

“…The expression pattern of GluR2/3 in the developing rabbit retina is similar to that in the developing rat retina, though the immunostaining in both the IPL and OPL was detected earlier in rabbits. [15][16][17] The fact that the immunoreactivity of GluR2/3 shows two bands in the IPL throughout the developmental stages implies that GluR2/3 is expressed early in both cholinergic amacrine cells 38,44 and AII amacrine cells, 35,37 which is similar to the adult rabbit retina.…”

Cellular Inhibitory Behavior Underlying the Formation of Retinal Direction Selectivity in the Starburst Network

“…This provides a plausible explanation why the cytosolic Ca 2+ signal is somatofugally motion biased [32]. The biophysical substrate of the nonlinearity favoring somatofugal motion of Ca 2+ remains the input-output segregation, with glutamatergic excitatory synaptic inputs from cone bipolar cells mediated by AMPA receptors [41], and cholinergic/GABAergic outputs localized at the distal end of the starburst dendrite [88,91]. The cytosolic Ca 2+ is locally distributed near the terminal end (as indicated by the arrows).…”

“…Zheng et al [120] used dual-patch clamp to show those GABA A receptors are on SACs. Firth et al [41] have used a combination of pharmacological methods and immunolabeling to show AMPA receptors are present on SACs. This is important because AMPA receptor would allow SACs to respond more effectively to rapidly changing stimuli than AMPA-KA receptors as was previously suggested by Poznanski [91].…”

“…Step 1: Somatic transient Na + spikes are initiated by light but rapidly shunted by the very thin proximal dendrite diameter under 1 µm; subsequent activation of glutamatergic excitatory synaptic inputs from cone bipolar cells mediated by AMPA receptors onto SACs [41] results in a sustained (prolonged) depolarization. Those SACs without direct connectivity with overlaying cone bipolars, the somatic Na + spikes, although shunted by the thin dendrites, may still contribute to the sustained prolonged depolarization within the dendrites.…”