Identification of a novel metabotropic glycine receptor and an excitatory pathway in retina
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Glycine is a major inhibitory neurotransmitter in central nervous system that acts by opening a chloride channel. Ionotropic glycine receptors are blocked by strychnine, a potent competitive antagonist. Glycine receptors belong to a family of ionotropic receptors that include acetylcholine, GABA, and serotonin receptors. Curiously, only glycine does not seem to activate a metabotropic receptor. The first part of my study focused on looking for evidence for a metabotropic glycine receptor in retina. To explore the possible existence of a metabotropic glycine receptor in retina, we examined the effects of glycine when ionotropic receptors were blocked by strychnine. Experiments were performed using whole-cell patch clamp techniques in the retinal slice preparation. Glycine in the presence of 10 μM strychnine suppressed voltage-gated calcium channel current. Lack of a chloride current indicated ionotropic receptors were blocked. This effect was observed in both bipolar and ganglion cells in salamander retina. 10 μM strychnine appeared to completely block glycine-induced chloride current, so this phenomenon suggested that the inhibition was not related to the traditional glycine-gated chloride current. The dose response of glycine revealed an EC 50 of 3 μM, suggesting the metabotropic glycine receptor is ten-fold more sensitive than the ionotropic receptor to glycine. Studies of the second messenger cascade indicated that activation of metabotropic glycine receptors inhibited PKA activity and subsequently suppressed calcium channel current. To determine if synaptic transmission was affected by this novel inhibitory effect of glycine on calcium channel current, we recorded from ganglion cells in the dark-adapted retinal slice preparation. The input from bipolar cell to ganglion cell was assessed by recording the light evoked ganglion cell EPSCs in response to full-field green (550 nm) or red (660 nm) illumination. Both 50 μM picrotoxin and 10 μM strychnine were applied to continuously block inhibitory input to ganglion cells from GABA A , GABA C , and glycine receptors. Glycine suppressed light-evoked EPSCs of ganglion cells. In addition, a paired stimulus protocol was used consisting of two red stimuli of 500 ms duration separated by a 3 s interval. The P 2 /P 1 ratio was compared before and after adding glycine in the presence of picrotoxin and strychnine. Glycine reduced the first response but increased the P 2 /P 1 ratio. The results indicate that glycine inhibits neurotransmitter release from bipolar to ganglion cells by activating presynaptic strychnine-insensitive glycine receptors, suggesting the presence of metabotropic glycine receptor in vertebrate retina. In a second study, I found that the AMPA/Kainate receptor antagonists and NMDA receptor antagonists each suppressed more than 50% of light-evoked post-synaptic currents (EPSCs) in ganglion cells. This suggested a novel excitatory pathway in retina that included a glutamatergic amacrine cell and an excitatory disynaptic serial pathway in inner retina. Block of N-type calcium channels or voltage-activated sodium channels, both parameters associated with amacrine cells, also suppressed glutamatergic EPSCs in ganglion cells. The excitation from amacrine cells activates NMDA receptors in ganglion cells. Without NMDA receptors, ganglion cells are unable to produce graded increases in response to bright light stimuli. Comparing ganglion cell light responses with light intensity suggests that one function of glutamatergic amacrine cells is to extend their dynamic range to high light levels.