All CPGs have been partially sequenced and subjected to a search for homologies in DNA and protein sequence data bases. Various sequence analysis and search programs that we and others have designed have allowed us not only to separate CPGs into known genes (with sequences already in the databases) and novel CPGs, but also to identify new members of known functional categories, such as transcription factors, kinases, phophatases, 7 transmembrane receptors, etc. Through chromosomal mapping of the CPGs using ESTs and sequence information from the Human Genome Project, we hope to match CPG loci with those of known neurological disease markers. In addition we are using cross species screens of invertebrate genomes that are fully or partially sequenced to identify CPGs that can be tested for function using genetic approaches.

     Additional screens of the CPG pool have used in situ hybridization to temporally and spatially localize expression of approximately 80 individual CPGs. Thus far, 30 CPGs chosen because of their expression patterns, have been full-length cloned and sequenced. The sequence analysis of these CPGs predicts the function of some and hints at possible functions for several others.

     In principle, to select CPGs of interest the CPG pool can be cross-screened by any criteria relevant to plasticity, for example up or down regulation with aging/disease/trauma. Two criteria that we used for selecting CPGs were: (1) cortical expression during activity dependent phases of development and (2) induction by a physiological sensory stimulus (light) specifically in the visual cortex. Six CPGs were found to be both developmentally regulated and modulated by light; with temporal and spatial patterns that are consistent with the ability of the cortex to undergo long-term activity-dependent changes (Nedivi et al., 1996). The implication is that gene expression regulated by activity may be a mechanism by which the brain can adapt not only to peripheral manipulations, but to everyday demands for efficient function.

     Novel CPGs highly prioritized for further analysis are those that are strongly induced from relatively low levels, are specific to the brain or to parts of the brain, are temporally and spatially localized correctly for participating in activity-dependent phenomena, and have a sequence prediction for a testable function.

     Our goal is to elucidate the cellular mechanisms of activity-dependent synaptic plasticity through identification and characterization of participating genes and their protein functions. We previously cloned a large number of candidate plasticity genes (CPGs). Two of these, cpg15 and cpg2, were selected for in-depth analysis because their expression and regulation patterns, and the cellular function of their protein products are consistent with a role in formation and restructuring of synapses. Our studies show that the cpg15 gene product, CPG15, functions as a membrane-bound growth factor that promotes dendritic growth in neighboring neurons (Nedivi et al. 1998). CPG2 is a structural protein that is localized to dendritic spines and interacts with the actin cytoskeleton (Cottrell et al., 2004). We propose to continue analysis of cpg15 and cpg2 with the aim of revealing previously unknown molecular details of the mechanisms that underlie activity-dependent plasticity.

     Manipulating expression levels of specific proteins is a standard strategy for studying their function in the context of a normal cellular environment. Virally-mediated gene transfer has been successfully used in neural tissues to obtain high levels of ectopically expressed recombinant protein. Our main approach to testing CPG participation in cellular events related to plasticity involves manipulating their expression in mammalian cell or slice preparations or in vivo using virally mediated gene transfer. The effect of cellular manipulation is then monitored by light or electron microscopy for changes in neuronal morphology, or electrophysiologically for changes is synaptic properties. This approach has been successful in elucidating a function of CPG15 as a membrane bound ligand that can promote dendritic arbor growth in the Xenopus retinotectal system (Nedivi et al. 1998) and for CPG2 which can regulate the endocitic machinery of dendritic spines (Cottrell et al., 2004).

     Ultimately, the most interesting CPGs will be used for gene 'knock out' experiments where their in vivo effect on development of brain anatomy and physiology, as well as adult cortical plasticity, will be determined. The first CPG that we are attempting to knock out in a tissue specific manner is cpg15.