As described above, only OX
1R has been detected by immunofluorescence in human and mammalian retinas.
5,6 Given that OX
1R has a greater affinity for orexin-A than orexin-B,
9,10 we used orexin-A to determine the effect of orexins on the retinal dopaminergic system. Light-induced excitatory postsynaptic currents (EPSCs) from RFP-labeled DACs were recorded in flat-mount retinas using a whole-cell voltage-clamp technique. Previous studies using C57BL/6J background wild-type mice have reported that in the majority of DACs (∼80%), light-induced EPSCs were completely blocked by L-AP4,
14,15 an agonist of mGluR6 receptors that selectively blocks the ON pathway of the retina.
36 This suggests that these cells receive input solely from rod and cone photoreceptors. In the present study, we used mixed C57BL/129 background wild-type
TH::RFP mice and found that L-AP4 completely blocked light-induced EPSCs in ∼50% of the recorded DACs.
Figure 1A shows a representative cell. This cell was clamped at −70 mV and exhibited an inward current at light onset (ON response) that decayed back to the baseline at light cessation (left trace). In the presence of 50 μM L-AP4, the initial EPSC was completely suppressed (middle trace). This suppression was fully reversed on washout (right trace).
Figure 1B illustrates one such L-AP4–sensitive cell. We found that 500 nM orexin-A reduced the peak current amplitude from 172 (left trace) to 124 pA (middle trace). This decrease was reversed on washout (right trace). However, the cell shown in
Figure 1C had no response to the same concentration of orexin-A. We tested 10 L-AP4–sensitive cells and found 7 of them were substantially suppressed by orexin-A, whereas the 3 other cells showed no response to orexin-A (
Fig. 1D; changes within ±10% were considered as having no effect). Although the results are inconsistent, average data show that the peak current amplitude was significantly reduced from 50.9 ± 15.1 to 35.0 ± 10.7 pA (or 71.6 ± 7.5% of control,
P < 0.01;
Fig. 1E). It is worth noting that in the presence of L-AP4, a delayed ON response (arrows) and an OFF response (arrowheads) became more evident (
Fig. 1A, middle trace), as we have previously reported.
20 Because these responses are inhibitory currents,
20 we did not test whether they are modulated by orexin-A.