Effects of Protein Restriction, Melatonin Administration, and Short Daylength on Brain Benzodiazepine Receptors in Prepubertal Male Rats

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Abstract
The possibility that there are changes in brain benzodiazepine binding sites controlled by photoperiod was investigated in two strains of male rats. The hypothesis was tested by 3H-diazepam binding studies in various brain regions of prepubertal rats maintained in 14 or 10 h of light or treated with late-afternoon injections of melatonin (50 .mu.g/da). Protein restriction was applied during the experiment to sensitise the animals to the treatments. Under the conditions employed, rats kept in short daylength throughout or kept on long photoperiod and given late-afternoon melatonin injections showed evidence of delayed puberty (seminal vesicle, ventral prostate, and testis weight decreased by 45%, 55%, and 60% respectively, compared to control rats). Binding measurements were made 1 h before and 2 and 5 h after the onset of darkness in the pubertal (42-day-old) or experimentally prepubertal rats. In the rats of the Porton strain (for which protein restriction was obligatory for the gonadal response) there was no consistent treatment or time effects on specific binding of 3H-diazepam to washed membranes of the hypothalamus, midbrain, or striatum. Similarly, there were no differences in the stimulation of 3H-diazepam binding by 100 .mu.M GABA or the inhibition of binding by 50 .mu.M N-acetyl 5 methoxy kynurenamine. By contrast, in Wistar rats, specific binding to midbrain membranes was reduced 5 h after dark compared to 2 h (37% saline; 20% melatonin) and the extent of stimulation by GABA in the hypothalamus was increased 5 h after darkness (35.6% to 46.7% saline; 37.4% to 50% melatonin). Melatonin treatment resulted in significantly higher specific binding in the hypothalamus 2 h after dark (10%, controd fed; 20%, protein restricted) but reduced the GABA induced stimulation of binding in the midbrain (35.5% to 25%, control fed; 33.7% to 23.5%, protein restricted). The Bmax of benzodiazepine binding to unwashed cortical P2 synaptosomal membranes has been reported to increase twofold in adult Wistar rats at mid-dark. By contrast the Bmax of juvenile Wistar rats in this study increased only 17% (116 .+-. 2.4 fmol/mg protein to 140 .+-. 3 fmol/mg protein) between 2 and 5 h after darkness. In melatonin-treated animals the increase in Bmax of 3H-diazepam binding was blocked (124 .+-. 5 fmol/mg protein at 2 h; 127 .+-. 3 fmol/mg protein at 5 h) and the Kd reduced (4.5 .+-. 0.5 to 4.0 .+-. 0.2 nM). When the cortical membranes from these animals were extensively washed and analysed for binding using a single concentration of ligand (3 nM) the rise in binding during the night was still observed (430 .+-. 27 fmol/mg protein to 505 .+-. 60 fmol/mg protein), but there was no blocking effect of melatonin treatment. These results indicate that some aspects of benzodiazepine binding may be influenced by melatonin treatment during the prepubertal period in the rat.