Increase of extracellular corticotropin-releasing factor-like immunoreactivity levels in the amygdala of awake rats during restraint stress and ethanol withdrawal as measured by microdialysis

Previous research has suggested a role for corticotropin-releasing factor (CRF) in the anxiogenic effects of stressful stimuli and ethanol withdrawal. This hypothesis was explored in a series of experiments using intracranial microdialysis to monitor CRF-like immunoreactivity (CRF-IR) in the extracellular compartment of the rat amygdala. The synaptic origin of CRF-IR release in the amygdala was determined in vitro by assessing the Ca²⁺ dependency of 4-aminopyridine stimulated CRF-IR release from tissue preparations of rat amygdala. In vivo experiments were performed in awake rats after the placement of microdialysis probes in the amygdala. In the first experiment, transient restraint stress (20 min) produced an increase of CRF-IR release (basal levels, 1.19 ± 0.15 fmol/50 μl; stress levels, 4.54 ± 1.33 fmol/50 μl; p < 0.05) that returned to basal values within 1 hr. When 4-aminopyridine (5 mM) was added to the perfusion medium, it consistently increased CRF-IR release (4.83 ± 0.92 fmol/50 μl, p < 0.05). In the second experiment, CRF-IR release was measured during ethanol withdrawal in rats previously maintained for 2-3 weeks on a liquid diet containing ethanol (8.5%). Basal CRF-IR levels were 2.10 ± 0.43 fmol/50 μl in ethanol exposed rats and 1.30 ± 0.19 fmol/50 μl in control rats. During withdrawal, a progressive increase of CRF-IR levels over time was observed, reaching peak values at 10-12 hr after the onset of withdrawal (10.65 ± 0.49 fmol/50 μl vs 1.15 ± 0.30 fmol/50 μl of control rats, p < 0.01). Since the peak of CRF-IR release corresponded to the time of appearance of anxiogenic behavioral effects in rats, the present data lend further support to the hypothesis that the CRF-IR system in the amygdala participates in the mediation of the emotional component of the response to exogenous or endogenous stressful stimuli.