Adjacent, coronal sections through the hippocampus were processed for the in situ hybridization detection of BDNF, NT-3, trkB, and trkC mRNAs by using 35S-labeled cRNA probes as described previously (Seroogy et al. 1994; Seroogy and Herman 1997; Numan and Seroogy 1999). Briefly, the slide-mounted sections were brought to room temperature, placed in 4% paraformaldehyde for 10 min, and washed sequentially in 0.1 m phosphate buffer (PB), 0.1␣m PB/0.2% glycine, and 0.25% acetic anhydride in 0.1 m triethanolamine. The sections were then dehydrated with increasing concentrations of ethanol, delipidated in chloroform, and air-dried. Sections were hybridized at 60°C overnight in a hybridization solution consisting of 50% formamide, 10% dextran sulfate, 1 × Denhardt's solution, 0.15 mg/mL yeast tRNA, 0.33 mg/mL denatured salmon sperm DNA, 40 mm dithiothreitol, 1 mm EDTA, 20 mm Tris-HCl and the 35S-labeled cRNA probe at a concentration of 1.0 × 106 cpm/50 µL/slide. Both sense and antisense cRNA probes for each neurotrophin and trk receptor were prepared by in vitro transcription using linearized DNA constructs in the presence of RNA polymerase (T3, T7 or SP6) and [35S]UTP (New England Nuclear; Boston, MA, USA). BDNF and NT-3 cDNA constructs (generous gifts from C. Gall and J. Lauterborn, University of California at Irvine) resulted in antisense transcripts that were 540 and 550 bases long, respectively. The cDNA constructs for trkB and trkC (kindly supplied by D. McKinnon, State University of New York at Stony Brook) resulted in antisense RNA transcripts that were 196 and 300 bases long, respectively. The trkB cRNA probe detects only the kinase-specific, full-length catalytic form of the receptor mRNA (Klein et al. 1990; Middlemas et al. 1991; Sternini et al. 1996), whereas the trkC cRNA probe recognizes mRNA transcripts for both the catalytic and non-catalytic isoforms of the receptor (Valenzuela et al. 1993; Dixon and McKinnon 1994; Albers et al. 1996). For posthybridization treatment, sections were washed several times in 4 x saline sodium citrate buffer (SSC; 1 × SSC = 0.15 m sodium chloride, 0.015 m sodium citrate, pH 7.0) containing 10 mm sodium thiosulfate, at␣37°C. The sections were then incubated in ribonuclease A (0.05 mg/mL) for 30 min at 45°C, followed by several washes in decreasing concentrations of SSC (2 ×, 0.5 × and 0.1 ×) at 37°C. The sections were then briefly rinsed in dH20, dipped in 95% ethanol, and air-dried. To generate film autoradiograms the sections were exposed to β-Max Hyperfilm (Amersham; Arlington Heights, IL, USA) for 11 days (BDNF and NT-3) or 7 days (trkB and trkC). In control procedures, prehybridization treatment of tissue with ribonuclease A (0.05 mg/mL; 45°C for 30 min), processing tissue with 35S-labeled sense strand transcripts for each probe, and processing tissue with no probe at all (positive chemography control), resulted in no specific hybridization signal. Film autoradiograms were analyzed with NIH Image public domain software (Image 1.62) to compare the densities of hybridization (mean corrected gray level) of each probe in various hippocampal subfields (dentate gyrus, CA1 and CA3) and in parietal cortex in each treatment paradigm. We did not attempt to quantify hybridization levels in specific subpopulations of cortical neurons; the analysis was made on the entire thickness of the cortex to provide a measure of overall levels of mRNA in the cortex. At least six measurements were taken for each probe from each animal. Statistical analysis included Student's unpaired t-test, and analysis of variance (anova) followed by Fisher's protected least significant differences procedure where appropriate. The NIH image software was also used to acquire images of representative sections from film autoradiograms.