ELISA methods to measure cholinergic markers and nerve growth factor receptors in cortex, hippocampus, prefrontal cortex, and basal forebrain from rat brain.
The central cholinergic system has a fundamental role in normal cognitive function, and in diseases that exhibit cognitive dysfunction. The purpose of this study was to design ELISA methods to measure proteins that have essential functions in the central cholinergic system. We were particularly interested in quantifying proteins that respond directly or indirectly to nerve growth factor (NGF). ELISAs offer advantages over Western blot analyses and other methods, such as increased sensitivity, decreased assay variability, increased efficiency, and decreased cost. We developed indirect ELISA methods for: choline acetyltransferase (ChAT); the vesicular acetylcholine transporter (VAChT); the high affinity choline transporter (HACT/CHT); TrkA, the high affinity NGF receptor; the p75 neurotrophin receptor (p75(NTR)). A sandwich ELISA was developed to measure tyrosine-phosphorylated TrkA in brain lysates. We used these ELISAs to compare levels of the above proteins in important memory-related brain regions--basal forebrain, hippocampus, cortex, and prefrontal cortex--from old and young rats. We identified age-related differences in the levels of the aforementioned proteins (e.g., VAChT and HACT/CHT in hippocampus). Thus, these ELISA methods should be particularly useful for comparing the effects of age, disease, drugs, and toxicants on brain levels of key cholinergic and growth factor-related proteins.
Gearhart
Debra A
DA
Department of Clinical and Administrative Pharmacy, Program in Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, GA 30912-2450, USA. dgearhar@mcg.edu
Middlemore
Mary-Louise
ML
Terry
Alvin V
AV
D000109:Acetylcholine / Q000378:metabolism*; D000367:Age Factors; D000818:Animals; D015415:Biomarkers / Q000032:analysis*; D015153:Blotting, Western; D001923:Brain Chemistry / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D008297:Male; D020932:Nerve Growth Factor / Q000032:analysis*; D051381:Rats
D015415:Biomarkers; D020932:Nerve Growth Factor; D000109:Acetylcholine