Friday, May 3, 2024
5/3/2024
PROJECT SUMMARY This project proposes to study the roles of specific amacrine cells (ACs) in the visual signal processing performed by the mammalian retina. Electrophysiological recordings of light evoked activity will be made from ACs, ganglion cells (GCs), and bipolar cells in intact in vitro whole- mount and slice preparations of mouse retina maintained at photopic adaptation levels. The functional properties of the cells will be probed using images projected onto the photoreceptors through the microscope objective. Patch-clamp recordings from amacrine and ganglion cell somas will be performed to measure the stimulus-evoked postsynaptic currents, postsynaptic potentials, and spiking responses. The project focusses on the elucidating the synaptic mechanisms underlying the receptive field properties of 3 genetically labelled amacrine cell types. Aims 1 and 2 examine the functional properties of two types of amacrine cells, so called NOS-1 and NOS-2 amacrine cells, which are identified by their expression of nitric-oxide synthase (NOS). Aim 1 will test the hypothesis that the NOS-1 ACs are key interneurons for controlling the strength of surround antagonism in GCs at scotopic light levels and that they exert their effects via GABAergic synaptic connections to AII ACs. We will make dual recordings between the NOS- 1 ACs and specific types of GCs, to directly test for indirect synaptic connections consistent with the proposed circuit. Aim 2 will examine the role of NOS-2 ACs in conferring motion-sensitivity to specific type of small-field GCs in the mouse. We will use optogenetic stimulation of ChR2 expressing NOS ACs to identify the postsynaptic targets. The postsynaptic targets will be identified morphologically and physiologically and inputs arising from NOS-2 ACs will be confirmed by paired recordings. Aim 3 focuses on a novel amacrine cell type that is one of 2 AC types that can be identified by their expression of the gene Gbx2. We will focus on the Gbx2+ ACs that stratify in sublamina 3 (S3) of the inner plexiform layer. The S3-Gbx2+ ACs are highly unusual because they appear to express none of the conventional inhibitory or excitatory neurotransmitters, indicating that they represent novel populations of so-called non-GABAergic, non-glycinergic (nGnG) ACs. Preliminary data show that these nGnG ACs are tracer coupled to bipolar cells. We will quantify the spatio-temporal receptive field properties of these nGnG S3- Gbx2+ ACs and will test the hypothesis that they make output via electrical synapses with bipolar cells. To do so, we will make patch-clamp recordings from cone bipolar cells in slice and measure depolarizing responses elicited by optogenetic stimulation (ChR2 expression) of the S3-Gbx2+ ACs. Overall, the results will reveal the functional properties and connectivity of the three AC types and will determine their roles in visual processing in the retina.
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