CONFORMATIONAL CHANGES IN THE EXTRACELLULAR DOMAIN OF GLUTAMATE RECEPTORS

The family of membrane protein called glutamate receptors play an important role in the central nervous system in mediating signaling between neurons. Glutamate receptors are involved in the elaborate game that nerve cells play with each other in order to control movement, memory, and learning. Neurons achieve this communication by rapidly converting electrical signals into chemical signals and then converting them back into electrical signals. To propagate an electrical impulse, neurons in the brain launch bursts of neurotransmitter molecules like glutamate at the junction between neurons, called the synapse. Glutamate receptors are found lodged in the membranes of the post-synaptic neuron. They receive the burst of neurotransmitters and respond by fielding the neurotransmitters and opening ion channels. Glutamate receptors have been implicated in a number of neuropathologies like ischemia, stroke and amyotrophic lateral sclerosis. Specifically, the NMDA subtype of glutamate receptors has been linked to the onset of Alzheimer’s disease and the subsequent degeneration of neuronal cells. While crystal structures of AMPA and kainate subtypes of glutamate receptors have provided valuable information regarding the assembly and mechanism of activation; little is known about the NMDA receptors. Even the basic question of receptor assembly still remains unanswered. Therefore, to gain a clear understanding of how the receptors are assembled and how agonist binding gets translated to channel opening, I have used a technique called Luminescence Resonance Energy Transfer (LRET). LRET offers the unique advantage of tracking large scale conformational changes associated with receptor activation and desensitization. In this dissertation, LRET, in combination with biochemical and electrophysiological studies, were performed on the NMDA receptors to draw a correlation between structure and function. NMDA receptor subtypes GluN1 and GluN2A were modified such that fluorophores could be introduced at specific sites to determine their pattern of assembly. The results indicated that the GluN1 subunits assembled across each other in a diagonal manner to form a functional receptor. Once the subunit arrangement was established, this was used as a model to further examine the mechanism of activation in this subtype of glutamate receptor. Using LRET, the correlation between cleft closure and activation was tested for both the GluN1 and GluN2A subunit of the NMDA receptor in response to agonists of varying efficacies. These investigations revealed that cleft closure plays a major role in the mechanism of activation in the NMDA receptor, similar to the AMPA and kainate subtypes. Therefore, suggesting that the mechanism of activation is conserved across the different subtypes of glutamate receptors.

Evidence for glutamatergic ganglion cells in the avian retina

This dissertation presents structural, immunochemical and neurochemical evidence for glutamatergic retinotectal synaptic transmission, augmenting and extending previous physiological and anatomical studies. The evidence is especially striking when the laminar patterns of ($\sp3$H) L-glutamate receptor binding, ($\sp3$H) L-glutamate high affinity uptake (HAU) and glutamate immunoreactivity (GLIR) of the dorsal tectum are compared. All show high activity in the tectal SGFS, with a peak in the most superficial laminae of SGFS followed by dip in the b-c region, and a second broad peak in deeper SGFS. Uptake and immunoreactivity bear a stronger resemblance to one another than either does to receptor binding, consistent with the fact that HAU and GLIR are localized in the same structures: glutamatergic terminals, intrinsic cell bodies and their processes. Receptor binding, as attested by the lack of enucleation effects, is a marker of postsynaptic receptors. In summary, these results are consistent with the hypothesis that most of the retinal projection to the optic tectum is glutamatergic: (1) A glutamate/aspartate HAU system exists in the superficial laminae, and it is dependent upon an intact retinal input, as shown developmentally and by retinal ablation; (2) Glutamate-like immunoreactivity appears in retinorecipient tectal regions (partially responsive to enucleation), in cell bodies of retinal ganglion cells and displaced ganglion cells, and in a non-tectal ganglion cell projection, the ectomammilary nucleus; (3) Sodium-independent glutamate receptor binding (which remains unchanged by enucleation) is most intense in the retinorecipient regions of the tectum and the ectomammilary nucleus. This binding is pharmacologically typical of a CNS sensory structure, being dominated by the quisqualate/kainate receptor subclass. Thus, as with other sensory systems, a portion of the retinotectal projection has been shown to include glutamatergic afferents with the distribution and properties expected of the primary projection ^

Characterization of neurotransmitter inputs to cholinergic amacrine cells of the rabbit retina: Analysis of ACh release

The cholinergic amacrine cells of the rabbit retinal are the only neurons which accumulate choline and also synthesize acetylcholine (ACh). It is widely accepted that the physiologically evoked release of acetylcholine can be taken as a measure of the activity of the entire cholinergic population. Initially, we examined the possibility that these cells receive excitatory input via glutamate receptors from glutamatergic neurons. Glutamate analogs were found to cause massive ACh release from the rabbit retina. Glutamate was found to activate several different receptor subtypes. Selective glutamate antagonists were used to separate the responses evoked by the different glutamate receptor subtypes. The kainate receptor was determined pharmacologically to be the subtype activated physiologically. Since bipolar cells make direct contact with cholinergic amacrine cells, our results support the hypothesis the bipolar cell neurotransmitter is glutamate. Although NMDA receptors can be activated by NMDA analogs, they are not activated during the physiologically evoked release of ACh. A separate study examined the possibility that L-homocysteate could be the bipolar cell neurotransmitter and the results placed serious constraints on this possibility.^ GABA$\sb{\rm A}$ agonists and antagonists are known to have powerful effects on ACh release from the rabbit retina. By pharmacologically blocking the excitatory input from bipolar cells, we attempted to determine the site of GABA$\sb{\rm A}$ input. Our results suggest that the predominant site of GABA$\sb{\rm A}$ input is onto the bipolar cells presynaptic to cholinergic amacrine cells. In a separate study, we found SR-95531 to be a potent and selective GABA$\sb{\rm A}$ receptor antagonist. In addition, GABA$\sb{\rm B}$ agonists and antagonists were found to have minor or no effects on ACh release. Glycine was also examined, its inhibitory effects were found to be very similar to GABA$\sb{\rm A}$ agonists. In contrast, strychnine was found to increase basal but inhibit light evoked ACh release. Additional results indicated that the predominant site of glycinergic input is onto the presynaptic bipolar cells. Our results suggest a different role for glycine compared to GABA in shaping the light evoked release of ACh from the rabbit retina. ^