Adenosine A1 Receptor-Mediated Neuromodulation of Rat ipRGC Light Responses

Abstract

Purpose: Receptors for the neuromodulator adenosine are found throughout the mammalian retina, with the A1 receptor especially prevalent in the ganglion cell layer. Our aim was to determine whether adenosine alters the light responses of intrinsically photosensitive retinal ganglion cells (ipRGCs) and to characterize the underlying intracellular signaling mechanisms.

Methods: Retinas dissected from adult and neonatal (P8-P13) Long Evans rats were placed RGC-side down on a multi-electrode array (MEA) while being continuously superfused with heated, oxygenated Ames medium. At the neonatal ages tested, ipRGCs are functional, but rod/cone-driven signalling to RGCs is absent. In the adult retinas, a synaptic blocker cocktail was used to isolate ipRGC responses. Each retina was stimulated with 20s pulses of blue light (470nm; 470nm; 2x10¹⁴ photons/s/cm²) in the presence and absence of compounds known to increase extracellular adenosine levels (500μM adenosine plus 20μM adenosine transport inhibitor NBMPR) and affect A1-mediated signaling (10μM agonist CPA and 20μM antagonist DCPCX). Fura-2 calcium imaging was used to examine light-evoked calcium influx in isolated ipRGC cultures prepared from neonatal rats using an immunopanning technique.

Results: In neonatal ipRGCs (N=3 retinas, n=130 ipRGCs), treatment with adenosine plus NBMPR significantly (p=0.03) decreased both the duration of the light response and the total number of spikes fired as compared to control and washout. A similar inhibitory effect (p=0.04) for adenosine was observed in adult ipRGCs (N=3, n=12). CPA application also significantly reduced (p=0.02; p=0.01) the duration of light-evoked spike firing in neonate (N=3, n=51) and adult ipRGCs (N=3, n=13). Pre-treatment of neonatal ipRGCs (N=3, n=32) with DPCPX blocked the effect of adenosine. Moreover, adenosine inhibited light-evoked calcium influx in isolated ipRGCs, and this effect was reversed with DPCPX pre-treatment (n=3).

Conclusions: These results indicate that adenosine can modify ipRGC function through A1 receptor activation. Taken together with our previous findings, we propose that the effect of adenosine involves cAMP/PKA-mediated inhibition of voltage-gated calcium channels. As retinal adenosine levels rise at night, adenosinergic modulation of ipRGCs may serve as an endogenous mechanism to limit transmission of nocturnal photic signals by ipRGCs to the brain.

Keywords: 410 adenosine • 531 ganglion cells • 648 photoreceptors