Research group produced transgenic mice tetO-I-1*. Mice with CaMKIIa promoter-rtTA and Cre-lacZ were used and double (I-I*/rtTA) and triple (I-I*/rtTA/lacZ)transgenic mice were obtained by heterzygous crossings. Before testing, mice were fed doxocyclin (Westward Pharmaceuticals Corp).
For RT-PCR used Superscript kit (Invitrogen)and oligonucleotides specific for the I-1* transgene. Phosphatase activity was measured on hippocami and cortex homogenates [lysis buffer: Tris-HCl,NaCl, magnesium chloride, DTT, NP-40, CaCl and protease inhibitors (Sigma)] using a Biomol green assay kit (BioMol). EGTA, okadaic acid, or inhibitor-2 (Bioconcept) were used to block calcineurin, PPI and PP2A or PPI activity, respectively.
Western blotting: For CREB, mice cortex extracts prepared as above. For CaMKII and GluR1, membrane-enriched preparations were obtained from hippocampi (sucrose, Tris-HCl, EGTA, EDTA, sodium pyrophosphate, b-glycerophosphate, NaF, PMSF and protease inhibitors). All samples were run on SDS–PAGE before electroblotting. Membranes were incubated either with anti-pSer 133 CREB (1:1,000), anti-CREB (1:1,000), anti-pThr 286 CaMKII (1:1,000), anti-CaMKII (1:2,000), anti-pSer 845 GluR1 (1:1,000), anti-GluR1 (1:1,000) or anti-actin (1:1,000) antisera (Upstate Biotechnology), and visualized by chemiluminescence before densitometric quantification (NIH Image).
b-galactosidase staining performed on 40-µm cryostat formaldehyde, glutaraldehyde fixed sagittal sections, incubated overnight in X-gal solution (5mM bromo-4-chloro-3-indolyl-b-D-galactoside)and then positive cells were counted.
mouse behavior measured using object recognition, water maze and probe trials.