CORTICOTROPIN-RELEASING FACTOR RELEASE FROM THE MEDIOBASAL HYPOTHALAMUS OF THE RAT AS MEASURED BY MICRODIALYSIS

Procedures were developed to permit the measurement of corticotropin-releasing factor in perfusate collected from microdialysis probes implanted in various brain areas of anesthetized and awake rats. Initially in vitro experiments were carried out to optimize the recovery of corticotropin-releasing factor and the radioimmunoassay conditions. Addition of a specific antiserum against corticotropin-releasing factor to the perfusion medium (artificial cerebrospinal fluid) increased the relative in vitro recovery over a range of different flow rates (1-10 mul/min) using commercially available microdialysis probes with a membrane cutoff of 20,000 mol. wt. This procedure increased recovery from 3% to 6% at flow rate of 2.5 mul/min, and from 4% to 8% at a flow rate of 5 mul/min. In vivo experiments were performed with a flow rate of 3.3 mul/min and 50-mul fractions were used for radioimmunoassay. In each experiment, the standard curve of the radioimmunoassay was constructed from aliquots of the same medium used to perfuse the probe. Basal levels of corticotropin-releasing factor in dialysate collected from the mediobasal hypothalamus of anesthetized rats were estimated to be 0.75 +/- 0.07 fmol/50 mul. Raising the concentration of potassium (60 mM) in the perfusate increased corticotropin-releasing factor levels to 2.04 +/- 0.37 fmol/50 mul. Hypertonic stress induced by intraperitoneal injection of 1.5 M NaCl (20 ml/kg) elevated the levels to 1.32 +/- 0.07 fmol/50 mul. A marked increase of corticotropin-releasing factor levels was also produced by a 10-min pulse of the potassium-channel blocker 4-aminopyridine (10 mM) included in the perfusate. A second stimulation pulse with 4-aminopyridine, administered 2 h after the first pulse again increased the levels, with a mean ratio between the first and second pulse of 0.97. Corticotropin-releasing factor efflux produced by the second stimulation pulse was completely inhibited by perfusion with calcium-free medium containing calcium-chelating agent ethyleneglycol tetraacetic acid (10 mM). In separate experiments, microdialysis probes were implanted in several brain areas of anesthetized rats. Basal and potassium-evoked levels of corticotropin-releasing factor were measured in dialysate collected from the amygdala (1.20 +/- 0.22 and 2.05 +/- 0.48 fmol/50 mul, respectively) and frontal cortex (0.51 +/- 0.10 and 1.64 +/- 0.15 fmol/50 mul, respectively). Corticotropin-releasing factor levels in the dorsal part of the third ventricle and in the striatum were below the detection limits. In awake rats, corticotropin-releasing factor levels in the mediobasal hypothalamus were 0.98 +/- 0.03 fmol/50 mul. Restraint (20 min) and hypertonic stress increased the levels to 1.14 +/- 0.07 fmol/50 mul and 1.51 +/- 0. 10 fmol/50 mul, respectively. Significant changes in plasma adrenocorticotropin and corticosterone levels paralleled those of corticotropin-releasing factor levels measured in dialysate. Additional estimates of corticotropin-releasing factor content in dialysate from mediobasal hypothalamus were obtained by controlling for the possible reduction of antiserum levels associated with the in vivo microdialysis procedure. The absence of corticotropin-releasing factor in the striatum permitted the use of perfusion medium collected from this site to estimate the reduction in the maximal binding capacity of antiserum contained in the medium after passage through the microdialysis probe and tubing connections in vivo. Estimates showed that there was a 36% reduction of maximal binding capacity of the antiserum. When standards curves were constructed using antiserum containing perfusion medium to interpolate values obtained in freely moving animals, the reduction of basal and stimulated levels was uniform. In summary, this paper describes the development of an original microdialysis procedure to measure the release of corticotropin-releasing factor from the rat brain. The recovery of this high molecular weight peptide was improved by adding a specific antiserum to the perfusion medium to increase the concentration gradient across the membrane. Basal levels of corticotropin-releasing factor in fractions collected from the mediobasal hypothalamus and amygdala, but not striatum or third ventricle, were detectable by radioimmunoassay. Estimates of corticotropin-releasing factor concentrations were obtained by correcting for the reduction of maximal binding capacity of the antiserum after passage through microdialysis probes in vivo. The potassium-induced increase of extracellular corticotropin-releasing factor in the mediobasal hypothalamus was calcium-dependent, confirming its synaptic origin. in freely moving rats corticotropin-releasing factor efflux increased after exposure to restraint and hypertonic stress, in parallel with plasma adrenocorticotropin levels. These results demonstrate the potential of intracranial microdialysis for studying the regulation of corticotropin-releasing factor release under physiological conditions, and its relevance in behavioral models of anxiety, depression and endocrine disorders.