Measuring of extracellular cortisol and corticotropin-releasing hormone in the amygdala using immunosensor coupled microdialysis.
A method is described for the measurement of cortisol or corticotropin-releasing hormone (CRH) from the brain, within, or in the dialysate of, a microdialysis probe using an antibody-linked assay. Polyclonal antibodies for either cortisol or CRH provide a specificity of measurement. These antibodies are affixed on a platinum electrode within the probe. Determination of bound cortisol or CRH is performed via an indirect assessment of competitive ligand also bound, and conjugated to HRP. This is activated and measured as current change. These probes were used for extracellular measurements in the amygdala, a limbic brain region, of sheep. Direct measurement in vivo compared favorably to the use of either the immunosensor or standard RIA techniques on dialysate emerging from a classical microdialysis probe, suggesting that the method could be used directly in-vivo. This is the first report in literature of rapid on-line measurement of CRH and cortisol from a discrete brain area. The probes have fast response times (>90% of maximum response within 30 s of start of analysis), high sensitivity (<0.1 pg/ml for CRH) and can acquire data every 2 min. They are stable in-vivo (>72 h) and regenerable, offering on-line measurement, with rapid time resolution, of neurohumoral substances.
Cook
C J
CJ
Technology Development Group, HortResearch, Private Bag 3123, Hamilton, New Zealand. ccook@hortresearch.co.nz
D000679:Amygdala / Q000378:metabolism*; D000818:Animals; D003346:Corticotropin-Releasing Hormone / Q000097:blood*; D004567:Electrodes, Implanted / Q000592:standards*; D005110:Extracellular Space / Q000378:metabolism; D005239:Fear / Q000502:physiology; D006854:Hydrocortisone / Q000097:blood*; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D017551:Microdialysis / Q000295:instrumentation / Q000379:methods*; D011188:Potassium / Q000494:pharmacology; D011863:Radioimmunoassay; D012756:Sheep; D013312:Stress, Physiological / Q000378:metabolism / Q000503:physiopathology
D003346:Corticotropin-Releasing Hormone; D011188:Potassium; D006854:Hydrocortisone