ONTOGENY OF DIAZEPAM-BINDING INHIBITOR-RELATED PEPTIDES (ENDOZEPINES) IN THE RAT-BRAIN

Benzodiazepine receptors are expressed very early in the brain during embryonic life, suggesting that endogenous ligands for these receptors may play an important role during ontogenesis in the central nervous system. In the present study, the distribution and characterization of diazepam-binding inhibitor-related peptides (endozepines) in the rat brain was investigated during embryonic and postnatal development using an antibody raised against the biologically active region of the precursor molecule. Immunohistochemical labelling showed that, in newborn rats, endozepine-like immunoreactivity was present in ependymal cells of the hypothalamus. Although the number of positive cells increased by day 5, the intensity of the immunoreaction in each cell diminished. In 15-day-old rats, both the number of endozepine positive cells and the intensity of the immunoreaction increased in the ependymal layer. At day 40, a dense accumulation of immunoreactive tanycytes and glial cells was observed in the median eminence and the arcuate nucleus. Endozepines were detected by radioimmunoassay in all regions of the brain as early as embryonic day 18. The concentration of endozepine-related peptides increased in the hypothalamus and olfactory bulb during late gestation. Between birth and postnatal day 5, the levels of endozepines decreased two- to four-fold in all brain regions studied. Thereafter, endozepine concentration increased gradually until day 25. Reversed-phase high-performance liquid chromatography analysis of tissue extracts revealed that the olfactory bulb, pituitary, hypothalamus and cerebellum contained only one immunoreactive peak eluting at 39 min (peak C). In the telencephalon two peaks were observed: peak C and a second one eluting at 34 min (peak B). Peak B was present as early as embryonic day 20 and the ratio peak B/peak C gradually increased until day 25. At day 25 peak B was also detected in hippocampus, medulla oblongata, cortex and striatum extracts. In any brain region, no immunoreactivity co-eluting with the octadecaneuropeptide was observed. Sephadex G-50 gel filtration of hypothalamus extracts of 25-day-old animals, confirmed the existence of only one immunoreactive compound with an apparent molecular weight of 10,000. In the telencephalon two major species were resolved, with apparent molecular weights of 10,000 and 8800, and a minor one of 6500 mol. wt. In conclusion, the present study shows that endozepines are expressed in the rat brain as early as embryonic day 18 and the amount of endozepine-like material increases rapidly during the two days preceding birth. The results also indicate that diazepam-binding inhibitor is processed to different molecular forms depending on the brain region. Taken together these data support the concept that endozepines may play important functions during brain development.