Abstract
Purpose :
Retinal motion detection is dependent on the excitatory and inhibitory outputs of the starburst amacrine cell (SAC), Acetylcholine (Ach) and GABA respectively. We recently found that blockade of alpha-7 nicotinic acetylcholine receptors (α7-nAChRs) decreased the direction selectivity of on-off direction-selective ganglion cells, which was attributable to cholinergic feedback to bipolar cells. To examine whether the direction selectivity of SACs is controlled by cholinergic feedback to bipolar cells, we carried out two-photon calcium imaging on light-evoked responses from SACs in the presence and absence of Methyllycaconitine (MLA).
Methods :
Oregon Green BAPTA (OGB-1) or GCaMP6m was used for calcium imaging. Electrophoretic injection of OGB-1 was conducted with a sharp electrode placed onto tdTomato labeled SACs. Recording of calcium responses in labeled SACs took place 1-2 hours after injection of OGB1. For GCaMP6 introduction, AAV9-GCaMP6M was injected into the intravitreal space of ChAT-Cre mouse eyes. Distal dendrites of SACs were imaged, and a 180° bidirectional moving bar stimulus was projected onto the photoreceptors on the ventral half of the retina by a UV LED. After calcium imaging was captured in a control solution, MLA (100 nM) was bath applied. Two-photon imaging occurred at 15 frames per second in a recording window of 256x124 pixels. Calcium fluorescence was analyzed using Fluoanalyzer in MATLAB.
Results :
In response to two-directional stimulation, we observed brighter responses to preferred motion, centripetal direction, and dimmer responses during null, centrifugal direction. The application of MLA decreased the calcium responses in both directions for the SAC. After washout of MLA, preferred directional response increased to near control conditions, but null directional responses remained dimmer. These results indicate decreased synaptic output from the SACs, which is consistent with our previous observation of reduced direction selectivity in on-off direction-selective ganglion cells.
Conclusions :
Our results show that α7-nAChR blockade by MLA decreases the overall calcium response in distal SAC dendrites. These new results confirm our previous work that cholinergic feedback to bipolar cells from SACs plays a role in SACs’ direction selectivity, resulting in a reduced direction selectivity in on-off direction-selective ganglion cells.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.