Characterization of inhibitory metabotropic receptors in the rodent retina
Garaycochea, Jay Albert
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Unlike many regions of the central nervous system, the retina contains both of the major inhibitory fast neurotransmitters: GABA and glycine. In the inner plexiform layer of the retina, the excitatory bipolar cells connect to the ganglion cells, which are the retinal output neurons. Interposed are inhibitory amacrine cells, about half glycinergic and the other half GABAergic. These amacrine cells activate chloride channels on bipolar and ganglion cells as well as other amacrine cells. In addition to these well studied channels, GABA also activates metabotropic receptors (GABA B Rs) linked to G-protein pathways. Less established are the putative glycine metabotropic receptors. Much of the work on both GABA and putative glycine metabotropic receptors in retina have been performed in non-mammalian systems. The goals of the studies described here were to characterize the mechanisms of action and pathways of these two metabotropic receptor systems in the rat retina. The GABA B Rs were found in about two-thirds of amacrine and ganglion cells, collectively referred to as third-order neurons. Activation of these receptors led to a reduction of voltage-gated calcium current which was not dependent on activation of the cAMP pathway. This suppression was voltage-dependent, characteristic of direct G-protein interaction with the calcium channel. Direct inhibition of N-type channels with omega-conotoxin occluded the effect of GABA B R stimulation and indicated that a saturating activation of the GABA B R led to inhibition of approximately half of the channels. However, under more physiological conditions the activation of GABA B Rs led to a reduction in the outward potassium current. This was found to be an indirect effect on calcium-sensitive voltage activated BK channels due to the effect being occluded by the calcium channel pore blocker cadmium, and by two drugs that block BK channels: low doses of tetraethylammonium (TEA) or iberiotoxin. Furthermore, direct block of N-type channels with omega-conotoxin replicated the effects of GABA B R stimulation. I suggest a model mechanism in which activation of GABA B Rs generates a direct interaction of the G-protein beta-gamma subunit with the N-type calcium channel that leads to their suppression. The N-type calcium channel is closely juxtaposed to the BK channel so that the reduced calcium influx leads to decreased BK channel activation. The physiological ramification was tested by injected currents into single cells to produce a graded depolarization. Compared to control conditions, the activation of GABA B Rs produced little effect with small depolarizations, but enhanced large depolarizations. The increase of depolarization was due to inhibition of BK channels, which reduced outward currents that normally limit depolarization. Thus, GABA B Rs reduced the influence of the BK channel current on large depolarizing events. The second project was to study the putative metabotropic glyince receptor (mGlyR) in rat retina. The mGlyR receptor has been described in salamander retina (Hou et al, 2008) based on the action of glycine in the presence of the ionotropic glycine receptor blocker strychnine. My goals were to extend this finding to the mammalian retina, identify selective agonists and antagonists, and begin identifying genes associated with the receptor. The results in this section are interesting but limited due to the response in rat retina being observed for only a few months, thus the results are incomplete. During the several months, the response to glycine in the presence of strychnine was robust and repeatable. Unlike the response reported in salamander retina, the mGlyR acted to enhance, not suppress, a calcium current. Furthermore, the glycine enhancement and the GABA B R suppression were often observed in the same cell. The effect of glycine was not blocked by a GABA B R antagonist, CGP55845. Forskolin enhanced the calcium current and occluded the effect of glycine, suggesting that glycine leads to a phosphorylation of the calcium channel. A number of glycine analogs were tested as potential agonists or antagonists, of which only the compound 4-chlorophenylglycine (CPG) duplicated and occluded the effect of glycine. CPG is particularly intriguing because it has a structure analogous to a potent GABA B R agonist, baclofen.