In Vivo imaging of CaMKII activity in the primary visual cortex of ferrets using FRET.

A.F. Mower*, S.M. Kwok, A. Majewska, H. Yu, K. Okamoto, Y. Hayashi and M. Sur.

MIT, Cambridge , MA 02139

 Most excitatory synapses in the brain are made on small dendritic protrusions called spines. Spines are motile structures that undergo activity-dependent morphological plasticity involving Ca +2 influx through glutamate receptors and voltage-gated Ca +2 channels. Rapid structural changes at the level of spines have been shown to occur following brief periods of monocular deprivation (MD). At the molecular level, this activity-dependent plasticity requires the transformation of synaptic depolarization into changes in synaptic weight. CaMKIIa and ß are known to play a role in this transformation through their interaction with the actin cytoskeleton. Autophosphorylation of CaMKIIa is also known to be required for ocular dominance (OD) plasticity. To examine activity dependent changes in CaMKIIa and ß activation in the primary visual cortex of ferrets in vivo, we performed intracortical injections of engineered FRET probes, Camuia and ß, packaged into herpes simplex virus. An optical window was then implanted to allow chronic 2-photon microscopic imaging and optical imaging of intrinsic signals for the derivation of OD maps. HSV-Camuia and ß specifically allow for the detection of calmodulin binding and autophosphorylation at threonine 286(a)/287(ß), which renders the enzyme constitutively active. Chronic imaging in normal animals, and following MD, showed robust expression of HSV-Camuia and ß in both dendrites and spines. This expression lasted for several days and allowed for high resolution imaging as indicated by the clear visibility of individual spine structure. Chronic in vivo imaging of HSV-Camuia and ß showed little fluctuation in FRET signal over time in normal animals. These experiments demonstrate the feasibility of utilizing FRET for studying CaMKII activition during OD plasticity in vivo .

Supported by NIH EY15068, EY07023, RIKEN and the Ellison Medical Foundation