Retrograde Endocannabinoid Effects Exerted on IK(V) of Cones in Goldfish Retina

Abstract

Abstract: : Purpose: Endocannabinoids act as retrograde signal for numerous synapses in the CNS (see review, Physiol. Rev. ’03, 83:1017). We showed that an endocannabinoid system is present in goldfish retina (Vis. Neurosci. ’00, 17:391; IOVS Abs. ’03, 4168). Voltage–dependent outward K–current elicited by depolarizing pulses (I_K(V)) of goldfish cones is biphasically modulated by cannabinoid CB1 receptor agonist WIN 55212–2; enhanced at < 1 µM and suppressed at > 1µM (Vis. Neurosci. ’03, 20:177). Here, we determined if retrograde effects of endocannabinoids could be measured on I_K(V) of cones by K⁺ depolarization of cells in the inner nuclear layer (INL). Methods: Experiments were performed on goldfish retinal slices, from which whole–cell recordings were obtained from the inner segment of long single cones under voltage clamp. A single, short puff (50 msec, 15 psi) of physiological saline with 70 mM KCl was directed through a pipette (tip diameter ∼2 µm) to depolarize bipolar cell bodies in the middle of the INL. Results: Following a single K⁺ puff in the INL, effects on I_K(V) of cones were observed after ∼200 msec; the change in I_K(V) reached the maximum at ∼400 msec and gradually returned to control in minutes. I_K(V), recorded ∼400 msec after the K⁺ puff, either decreased to 77 ± 5% (mean ± S.E., n = 10) or increased to 139 ± 15% (n = 5) relative to control. The direction of the effect reflected the biphasic effect of WIN 55212–2 and presumably was determined by the local concentration of endocannbinoid around the cone terminal. The effect of the K⁺ puff on I_K(V) was blocked by the CB1 antagonist SR 141716A (3 µM, n = 5) but not by combined 300 µM picrotoxin and 3 µM CNQX (n = 7). Endocannabinoids, anandamide and 2–arachidonylglycerol, are degraded in vitro by cyclooxygenase–2 (COX–2). Application of COX–2 inhibitor nimesulide had no effect on the amplitude of I_K(V) but it prolonged the effects of the K⁺ puff on I_K(V). In control conditions, I_K(V) returned to baseline in 6.7 ± 2.7 min (n =10) following a K⁺ puff. However, in 30 µM nimesulide, full recovery to baseline was not observed, rather the half–recovery of I_K(V) increased to 36.0 ± 14.6 min (n = 5). Conclusions: Depolarization of cells in the INL releases an endocannabinoid that exerts a retrograde effect on the amplitude I_K(V) of long single cones. COX–2 appears to one limiting factor of endocannabinoid action in the retina. Based on the position of the puff pipette in the middle of the INL, we suggest that bipolar cells are a source of endocannabinoids.

Keywords: photoreceptors • electrophysiology: non-clinical • neurotransmitters/neurotransmitter systems