Expression And Modulation Of Ion Channels On Amacrine Cells In Rat Retina

Amacrine cells are important intemeurons in the vertebrate retina, which mediate lateral interaction in the inner plexiform layer. There are a variety of amacrine cells according to their morphological and physiological properties. In this dissertation, we first studied the expression of voltage-gated and inwardly rectifying K~+ channels on rat cholinergic and dopaminergic amacrine cells using immunohistochemistry. Furthermore, we studied modulation by C-type natriuretic peptide of glutamate receptors on cultured rat GABAergic amacrine cells using patch clamp recording techniques in combination with double immnofluorescence labeling.Part One: Voltage-gated and inwardly rectifying potassium channels on cholinergic and dopaminergic amacrine cells in rat retinaPotassium channels are one of the most important ion channels in the central nervous system, and are mainly divided into two families: voltage-gated potassium (Kv) channels and inwardly rectifying potassium (Kir) channels. Kv and Kir channels play pivotal roles in regulation of the excitability of neurons and signaling in the nervous system. K~+ channels, composed of different subunits, exhibit distinct pharmacology and kinetics and play different physiological functions. Using double immunofluorescence labeling and confocal laser scanning microscopy, we first studied the expression of Kv subunits (Kv 1.1-1.6, 2.1, 3.1b, 3.2, 3.4, 4.2 and 4.3) and Kir subunits (Kir 1.1, 2.1, 2.3, 3.1, 3.2, 3.3, 4.1 and 4.2) on rat cholinergic and dopaminergic amacrine cells. Staining for Kv3.1b was found in the cholinergic cells,being present on the membrane of somata, and on the processes, but not in the dopaminergic cells. Kv4.3-immunoreactivity was localized on the somatodendritic compartment of the dopaminergic cells, but was not found in the cholinergic cells. Differential expression between the two cell types was not found for 10 other Kv subunits and all of the Kir subunits tested. These results suggest that the Kv3.1b and Kv4.3 subunits may differentially contribute to the electrophysiological properties underlying distinct functions of these two retinal interneurons. Moreover, we also investigated expression patterns of Kir subunits (Kir 1.1, 2.1, 2.3, 3.1, 3.2 3.3, 4.1, and 4.2) in rat retina. Except for Kir4.1, the subunits tested were all expressed on neurons in the inner nuclear and ganglion cell layers. While Kir2.1 was densely present in the cytoplasm, the staining for all other subunits was predominantly expressed on the cell membrane. Most subunits, but not Kir 2.1 and 2.3, were present in the outer and inner plexiform layers to different extents. Kir4.1-immunoreactivity was found in astrocytes wrapping retinal blood vessels, in addition to the Miiller cell endfoot. The differential expression of the Kir subunits suggests the diversity of physiological roles these channels may play.Part Two: Modulation by C-type natriuretic peptide of glutamate receptors on cultured rat retinal amacrine cellsNatriuretic peptides (NPs) constitute a family of three structurally related hormones: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). They are widely distrbuted in the central nervous system and may act as neuromodulators. There are three classes of receptors that have been characterized: natriuretic peptide receptor (NPR)-A, which is sensitive to ANP and BNP, NPR-B, which is highly specific for CNP, and NPR-C, which binds the three natriuretic peptides with similar affinities. NPR-A and NPR-B have an intrinsic guanylyl cyclase (GC) domain and transduce their biological effects via cGMP. In thiswork, we used patch clamp recording techniques in combination with double immnofluorescence labeling to study modulation by CNP of glutamate receptors on cultured retinal amacrine cells. We first developed the culture method for rat retinal amacrine cells. After 10 days in culture, virtually all of the large multipolar cells >10 um were immunoreactive for HPC-1, an amacrine cell marker, and GABA, a common neurotransmitter for amacrine cells, and these GABAergic amacrine cells were used throughout the work. Immunocytochemical assay revealed that NPR-B was expressed on the membrane of cultured GABAergic amacrine cells, whereas NPR-A was mainly located in the cytoplasm. NPR-C was neither present on the membrane nor in the cytoplasm of these cells. Using whole cell recording, we demonstrated glutamate-induced currents from cultured amacrine cells with fast desensitization were abolished by GYKI 52466, a specific AMPA receptor antagonist, suggesting that these cultured amacrine cells mainly expressed the AMPA-prefering glutamate receptor subtypes. We further showed that CNP suppressed glutamate-induced currents from cultured amacrine cells in a dose-dependent manner, by decreasing the efficacy and apparent affinity of the glutamate receptor. The effects of CNP were reversed by HS-142-1, a guanylyl cyclase-coupled NPR-A/B antagonist, indicating the involvement of the NPR-B receptor. Meanwhile, the selective NPR-C agonist cANF could not change the glutamate currents, suggesting no involvement of NPR-C. The inhibitory effects of CNP on the glutamate current were mimicked by application of 8-Br-cGMP and were not observed in the 8-Br-cGMP incubated amacrine cells. Moreover, incubation in the medium containing CNP increased cGMP immunoreactivity in these amacrine cells. These results suggest that the CNP effects may be mediated by increasing the intracellular concentration of cGMP through activation of NPR-B. CNP/cGMP-mediated modulation may modify synaptic functions of GABAergic amacrine cell.