Receptor autoradiography and quantification

Experimental procedures for 5-HT_2A binding autoradiography were based on those completed and reported previously.^45,65–67 5-HT_1A and 5-HT_2C binding autoradiography was also completed; however, binding results were too low and thus discounted from further analysis.

Brain sections for 5-HT_2A receptor binding were thawed at RT and then preincubated in 170 mM tris buffer (pH 7.4) for 15 minutes. Slides with sections were then incubated for 2 hours with 2 nM [³H]Ketanserin (specific activity: 47.3 Ci/mmol; PerkinElmer Inc., Waltham, MA, USA) in 170 mM tris buffer at RT to determine total binding. Nonspecific binding was determined with the addition of 2 µM Spiperone (Sigma-Aldrich Co.) to subsequent sections. Following incubation, sections were washed four times for 2 minutes in ice-cold buffer, dipped in ice-cold distilled water, and then air dried.^66,67

Following the completion of receptor binding experiments, all slides were exposed to Amersham Hyperfilm ECL for 2–3 months, along with autoradiographic standards ([³H] microscales from Amersham), in X-ray film cassettes. Quantitative analysis of binding images was conducted following the relevant exposure time, using the Multi-Analyst image analysis system (Bio-Rad Laboratories Inc.), connected to a GS-800 Imaging Densitometer (Bio-Rad Laboratories Inc.). Optical density measurement was then converted into femtomoles of [³H] ligand per milligram of tissue equivalent (TE) by comparing to the standard. Specific binding was calculated by subtracting nonspecific binding from total binding. A set of sections from each animal were stained with the 0.5% cresyl violet solution (Nissl staining), for the purpose of confirmation of anatomical structures. Specific brain regions in this project were identified by reference to the Nissl-stained sections, along with a standard rat brain atlas.⁶⁸