Cats were used for differential display PCR, cloning, sequencing, northern and western blotting. Cats were reared in a normal 12 hour light/dark (N) cycle or in complete darkness from birth to 5 or 20 weeks of age (D). For each of these four rearing/age conditions (N5, D5, N20, D20), three animals were used (12 cats in total). Fresh brain regions (visual cortex [all of Area 17 and possibly a small part of 18], frontal cortex [the anterior quarter of the cerebral hemisphere], and cerebellum [entire structure]) were dissected, immediately frozen in liquid nitrogen, and stored at -80°C until used for extraction of total RNA and protein.
Mice were used for western blot analysis of α-chimaerin isoform expression. They were reared in a normal 12 hour light/dark cycle or in complete darkness from birth to 3.5 or 9.5 weeks of age. The ages were based on published studies of the time course of the critical period in mouse visual cortex [[37–39]]. Three independent samples of mice including all four ages and rearing conditions (N3.5, D3.5, N9.5, D9.5) were collected. Within each sample three animals were pooled for each rearing/age condition (36 mice in total) to provide sufficient tissue and reduce individual variability. To provide a complete description of normal postnatal development of chimaerin expression in visual cortex, additional mice were reared in a normally lit environment until 1, 2, 6.5 weeks, and adult (18 weeks). Three animals were pooled for each age sample as above (total 12 additional mice). Visual cortex (monocular and binocular regions), frontal cortex and in several normal 9.5 week mice cerebellum were dissected, immediately frozen in liquid nitrogen, and stored at -80°C until used for western blots. Cats and mice were killed by an overdose of sodium pentobarbital prior to tissue dissection (75 mg/kg, intraperitoneal injection). All procedures in cats and mice conformed to the guidelines of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee.
Differential Display PCR (ddPCR)
RNA was extracted from cat visual cortical samples and its amount and integrity determined as described previously . DNA contamination was removed from the RNA (MessageClean, GenHunter Corp., Nashville, TN) and cDNA was synthesized (SMART, Clontech, Palo Alto, CA or RNAimage, GenHunter Corp.). PCR on cDNA from each RNA sample was done using the appropriate oligo-dT-X (oligo-dT-C; dT-G; dT-A) primer and 80 arbitrary primers as provided in a commercially available kit (RNAimage, GenHunter Corp.). The ddPCR products showing bidirectional expression due to age and dark rearing were amplified in a PCR reaction using the same primers as in the initial reaction, then cloned (PCR-TRAP, GenHunter) for use in sequencing and as probes for northern blots. The resultant sequences were run against gene and expressed sequence tag databases to determine similarity to known genes. 5'RACE PCR and cloning (SMART, Clontech) were used to generate additional sequence for the ddPCR fragment.
Northern Blot Analysis
The cat ddPCR fragment (153 bp) obtained from the screen was used as the probe for northern blotting. Total RNA was extracted from the tissue samples as described above. Filters with lanes from all experimental conditions were hybridized (stringency: 0.1x sodium chloride-sodium citrate buffer, 0.1x sodium dodecyl sulphate (SDS), 42°C) using these probes according to our standard procedures . The filters were stripped and re-hybridized with a probe to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to correct for loading errors (American Type Culture Collection, ATCC 57090). The relative intensity of signals in northern blots was determined by densitometric scanning, corrected against GAPDH.
Western Blot Analysis
Antibodies against α1- and α2-chimaerin were developed in collaboration with Quality Controlled Biochemicals (Hopkinton, MA.) The antibody to the α1 isoform was developed against amino acids 9-29 and the antibody to α2 against amino acids 151-173. Both antibodies were affinity purified. Homogenates of visual and frontal cortex were prepared in the presence of a standard protease inhibitor cocktail (Sigma-Aldrich Co., St. Louis, MO). Centrifugation was done to yield a crude synaptosomal fraction and equal amounts of protein were added to each well of a 4-15% polyacrylamide SDS gel (Bio-Rad Laboratories, Inc., Hercules, CA), separated by electrophoresis, and then electrophoretically transferred to a polyvinylidene fluoride membrane using a Transblot cell (Bio-Rad Laboratories, Inc., Hercules, CA). Nonspecific binding sites were blocked with 5% non-fat milk and blots were then incubated with the primary antibody (rabbit polyclonal, diluted 1:1000). Blots were extensively washed and incubated with appropriate horseradish peroxidase conjugated secondary antibodies at room temperature for 2 hr. Specific protein bands were visualized by enhanced chemiluminescence (ECL) detection reagents (Amersham Biosciences, Piscataway, NJ). The blots processed for ECL were then stripped and reprobed with the other chimaerin antibody, then stripped again and reprobed with a mouse monoclonal antibody to β-actin (Sigma-Aldrich Co., St. Louis, MO) to control for loading errors. The second and third immunostaining were carried out as described above.
Exposed films from western blots were quantified by computerized densitometric analysis. High resolution TIF images of each film were obtained with an AlphaImager EP, MultiImage I. Protein expression levels for each individual antibody were calculated as an average pixel density by AlphaView software (version 184.108.40.206) from AlphaInnotech. All densitometric measures for α-chimaerin protein were corrected against β-actin. Statistical analysis included two way ANOVA and Tukey post hoc comparisons.