Developmental Regulation of Glutamate Receptor Function

The goal is to understand the cell and molecular mechanisms of activity-dependent synapse development and plasticity in the visual pathway and facilitate the development of pharmaceuticals and therapies to ameliorate life-long visual dysfunctions resulting from early abnormal visual experience, trauma or disease. This research uses rodent visual pathways and focuses on the membrane associated guanylate kinases (MAGUKS), SAP102 and PSD-95. These synaptic scaffolds hold N-methyl-D-aspartate subtypes of glutamate neurotransmitter receptors (NRs) and the many molecules they signal through during visually driven synaptogenesis and during NR-dependent long-term synaptic potentiation and depression (LTP & LTD). We postulate that PSD-95 and SAP102 organize separate NR signaling modules at the post-synaptic density (PSD), that the major switch from SAP102 to PSD-95 at visual system PSD's occurs at eye opening, and that PSD-95 bound NRs drive synaptic change that also requires extrasynaptic SAP102-bound NRs.

There are 3 specific aims:

(1) To test the hypothesis that SAP102 and PSD-95 have distinct roles in establishing and sorting synapses during the neonate and early post-eye-opening period respectively using lentiviral mediated over- expression of PSD-95 or SAP102 or siRNAs to knock down the expression of these scaffolds. Whole-cell patch-clamping in slices from the visual layers of the superior colliculus will determine the effect of these manipulations on synaptic current changes and on electrically evoked LTP and LTD.

(2) To test the hypothesis that SAP102 and PSD-95 hold different signaling modules adjacent to NRs during visual development we will examine a staged series of post-synaptic density fractions and immunoprecipitates of proteins associated with each of the MAGUKS using liquid chromatography and tandem mass spectrometry in collaboration with Dr. Steven Carr, Director the Proteomics Platform at the Broad Institute.

(3) We have evidence that the scaffold and signaling modules held near the NR receptor is determined by a specific association between NR subunits and either SAP102 and PSD-95. Therefore, will use molecularly engineered NR receptor subunits that should hold NRs with different ion pore characteristics adjacent to either SAP102 or PSD-95. In visual cortex cultures we will characterize the engineered receptors' binding characteristics and targeting. Subsequently, we will use lentiviral vectors to introduce these constructs into the superficial visual layers of the superior colliculus (sSC) in a mouse strain lacking one of the normal NR subunits (the NR2A-/- mouse).

We will determine if the chimeric subunits alter or eliminate a deficit in LTP that we have found in the sSC of these mice after eye opening. We will also determine whether the normal, highly stereotyped glutamate current changes, we have characterized in normal animals after eye-opening are maintained or modified according to our predictions in NR2A-/- neurons carrying the engineered subunits.

PUBLIC HEALTH RELEVANCE Two to three in 100 children are impaired by a condition known as amblyopia in which inputs between the two eyes and the brain are imbalanced and one eye loses visual discrimination. Studies of the cell and molecular mechanisms through which early vision strengthens appropriate connections between the eyes and the brain will facilitate the development of treatments for this wide-spread impairment. Work identifying mechanisms of functional connectivity in visual development will also help in integrating visual prosthetics for retinal dysfunction into the brain circuits that produce vision.