Microaspiration and TC RNA amplification procedures have been described in detail elsewhere (Ginsberg and Che 2002, 2004; Che and Ginsberg 2004) and are diagrammed in Fig. 1. Linearity and fidelity of the TC RNA amplification procedure has been published, including the use of CBF neurons as input sources of RNA (Che and Ginsberg 2004; Ginsberg 2005). Moreover, variability between single cell expression profiles and reproducibility of expression levels has been evaluated extensively by our laboratory group and published previously (Che and Ginsberg 2004; Ginsberg and Che 2004; Ginsberg 2005). Briefly, individual p75NTR-immunoreactive NB neurons were microaspirated from paraformaldehyde-fixed 40 µm thick frozen cut sections of the basal forebrain using a micromanipulator and microcontrolled vacuum source (Eppendorf, Westbury, NY, USA) attached to an inverted microscope (E800, Nikon, Japan). The amplification of RNA from individual NB neurons was performed using a new terminal continuation (TC) RNA amplification methodology (Che and Ginsberg 2004; Ginsberg and Che 2004; Ginsberg 2005). The TC RNA amplification protocol is available at http://cdr.rfmh.org/pages/ginsberglabpage.html. Individual, not pooled, CBF neurons were extracted in 250 µL of Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNAs were reverse transcribed in the presence of the poly d(T) primer (10 ng/µL) and TC primer (10 ng/µL) in 1X first strand buffer (Invitrogen), 1 mm dNTPs, 5 mm DTT, 20 U of RNase inhibitor and 5 U reverse transcriptase (Superscript III, Invitrogen). The synthesized single stranded cDNAs were converted into double stranded cDNAs by adding into the reverse transcription reaction the following: 10 mm Tris (pH 8.3), 50 mm KCl, 1.5 mm MgCl2, and 0.5 U RNase H (Invitrogen) in a total volume of 99 µL. Samples were placed in a thermal cycler and second strand synthesis proceeded as follows: RNase H digestion step 37°C, 10 min; denaturation step 95°C, 3 min; annealing step 50°C, 3 min; elongation step 75°C, 30 min 5 U (1 µL) Taq polymerase (PE Biosystems, Foster City, CA, USA) was added to the reaction at the initiation of the denaturation step (i.e. hot start) (Che and Ginsberg 2004). The reaction was terminated with 5 m ammonium acetate. The samples were extracted in phenol:chloroform:isoamyl alcohol (25 : 24 : 1) and ethanol precipitated with 5 µg of linear acrylamide (Ambion, Austin, TX, USA) as a carrier. The solution was centrifuged at 14 000 r.p.m and the pellet washed once with 95% ethanol and air-dried. The cDNAs were resuspended in 20 µL of RNase free H2O and drop dialyzed on 0.025 µm filter membranes (Millipore, Billerica, MA, USA) against 50 mL of 18.2 MegaOhm RNase-free H2O for 2 h. The sample was collected off the dialysis membrane and hybridization probes were synthesized by in vitro transcription using 33P incorporation in 40 mm Tris (pH 7.5), 7 mm MgCl2, 10 mm NaCl, 2 mm spermidine, 5 mm of DTT, 0.5 mm of ATP, GTP, and CTP, 10 µm of cold UTP, 20 U of RNase inhibitor, T7 RNA polymerase (1000 U, Epicentre, Madison, WI, USA), and 40 µCi of 33P-UTP (GE Healthcare, Piscataway, NJ, USA). The reaction was performed at 37°C for 4 h. Radiolabeled TC RNA probes were hybridized to custom-designed cDNA arrays without further purification.