Adrenal glucocorticoids as a required factor in barbitura-induced changes in functional tolerance and brainstem tryptophan hydroxylase

Male C57BL/6 mice were given phenobarbital via milled food for 9 days; control mice received unadulterated milled food. Blood phenobarbital levels rose to 116 μg/ml during the later days of the experiment. Twenty-fourth after withdrawal of the barbiturate, the animals were given a challenge dose of pentobarbital (PenB, 40 mg/kg, i.p.) and their sleep time was determined as a measure of tolerance. All control animals slept after the injection for an average of 57 min and woke up with brain PenB levels of 22 μg/g. However, none of the phenobarbital-treated animals slept after PenB injection despite brain PenB levels peaking 38% higher than control levels upon awakening (P < 0.001), thus indicating the development of functional tolerance to barbiturate. Adrenalectomized (Adx) animals receiving chronic phenobarbital slept for 13 min and woke up with low brain PenB levels. In the phenobarbital-treated Adx given glucocorticoid replacement (0.5 mg/mouse/day; s.c.), none of the animals slept after the PenB injection, similar to the intact animals. Since a large portion of the tolerance (from 57 min to 0 min) was metabolic in nature, the portion that remained in Adx animals represent lack of brainstem functional tolerance. Since previous studies suggested that functional tolerance to barbiturates may be due to adaptive changes in the serotonergic neurons, brainstem tryptophan hydroxylase activity was examined. The enzyme activity was increased by 64% (P < 0.001) following the chronic barbiturate administration. Interestingly, the barbiturate effect on tryptophan hydroxylase activity was totally abolished in Adx animals, and the effect was restored in Adx animals by hormone replacement with daily injections of corticosterone (0.5 mg/mouse). Thus, there was a correlation between the occurence of barbiturate functional tolerance and the changes in the activity of tryptophan hydroxylase, the rate-limiting enzyme in the synthesis of serotonin. Our findings support the notion that the development of functional tolerance to barbiturates may involved a serotonergic mechanism, and that adrenal glucocorticoids are a required factor for barbiturate-induced changes in both tolerance and tryptophan hydroxylase.