Abstract
Purpose :
Dopaminergic amacrine cells (DACs) spontaneously generate action potentials that drive dopamine release, modulating visual function. Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels are known to regulate neuronal firing and excitability via the hyperpolarization-activated current (h-current). However, it is currently unclear the extent to which DACs express HCN channels to regulate their spontaneous activity generation. In this work, we sought to identify molecular and functional expression of HCN channels on DACs in the mouse retina.
Methods :
HCN channels consist of four subunits, HCN1–4, which can assemble in various combinations and conformations. We performed double immunohistochemical staining with antibodies against HCN1 channels and tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine biosynthesis, in vertical slices of the retinas obtained from developing and mature wild-type mice. To identify the functional expression of HCN channels, we carried out whole-cell current-clamp electrophysiology on DACs marked with a red fluorescent protein in flat-mounted retinas of transgenic mice.
Results :
Before eye-opening (postnatal days 4, 8, and 12), mouse DACs were labeled with a TH antibody but did not exhibit HCN1 immunostaining. However, by postnatal 22, TH and HCN1 were co-localized in the majority of DACs. The colocalization of TH and HCN1 was also revealed in adult retinas. In adult retinas, whole-cell current-clamp recordings demonstrated that 10 out of 12 DACs exhibited a depolarizing voltage sag, a signature of the HCN channel functional expression, in response to injections of hyperpolarizing currents (-80 pA to -100 pA). The depolarizing voltage sags were profoundly suppressed either by the application of HCN channel antagonist cesium (n=4) or ZD7288 (n=2).
Conclusions :
The after-eye-opening expression of HCN1 suggests that the development of HCN channels in DACs could be light-dependent. Although the expression of HCN2-4 channels on DACs remains to be determined, the molecular and functional expression of HCN1 on DACs indicates that activation of HCN channels may be involved in regulating their membrane excitability and spontaneous and light-induced firing, which could play an important role in dopamine-releasing in the retina.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.