GABA: Circadian fluctuations in rat hypothalamus

THE CNS is involved in the regulation of the secretion of most endocrine glands and a major portion of this control is exerted via the hypothalamic regulation of the pituitary. This control is triggered by chemical agents secreted into the portal vessels (GANONG 1974). The cells that release hypothalamic hormones are probably innervated by fibers of the afferent ncuronal system converging on the hypothalamus. The neurotransmittcrs that are presumably secreted at the endings of the afferent fibers include catecholamines. serotonin. acetylcholine and GABA (ROSE & GANONG, 1976). Experimental evidence indicates the existence of a circadian rhythm of anterior pituitary hormones (SCAPAGNIXI ef a/., 1976). Total brain levels of catecholamines, serotonin (COCCHI et u/., 1976) and acetylcholine (HANIN rt a/., 1970) have also been shown to present circadian periodicity. Since brain monoamines play an important role in the neuroendocrinc control of hypothalamus and anterior pituitary functions, attempts have been made to correlate the circadian variations of brain monoamine lcvcls with the circadian rhythm of hypothalamic and pituitary hormones (ASANO 1971; SIMON & GEORGE, 1975). Recently, an inhibitory effect of GABA on ACTH secretion has been demonstrated (MAKAKA & STAKK, 1Y74). The presence of high levels of GABA in the hypothalamus has been well documented (KURIYAMA & KIMURA, 1976). However, an involvement of GABA in the hypothalamic regulatory mechanisms has not yet been established. As a first approach to this problem, we have investigated whether GABA levels in hypothalamus and other brain areas undergo circadian variations which might be correlated to hormone secretion. Male Sprague-Dawley rats (Charles River) weighing 120-15Og were used. The animals were kept at constant temperature (23-25'C) and housed 5 per cage in an air conditioned room for I S days prior to study, to allow adjustment to controlled conditions of light (06.0&21.00). Food and water were allowed ud lib. Animals were killed by exposing their heads for 3-4s to high energy microwave radiations (Oven: 2.0 kW. 2.45 GHz, 75 W/cm*, Med. Ing. Consult. U.S.A.) GUIDOTTI et ul. 1974). This procedure completcly inactivates thc post-mortem increase of GABA concentrations in the brain (CATTABEN1 rl d., 1975). Striatum, cortex. cerebellum and hippocampus were dissected according to GLOWINSKI & IVERSEN (1966). Substantia nigra was dissected after mounting the brain on a microtomc stage. Sections 400pm thick were then cut through whole substantia nigra. The brain slices were kept frozen and substantia nigra punched out with stainless steel tubing ( I mm, i.d.). Hypothalamus was dissected starting 400 pm after the disappearance of substantia nigra up to A 7,500 pm and 2 mm deep, according to the KONIG & KLIPPH (1967) stcrcotaxic atlas of the rat brain. GABA was determined with a mass fragmentographic method recently described (CATTABENI et a[., 1976). Tissues were homogenized in 1 ml of 0.1 M-Formic acid containing the internal standard 4-amino-valeric acid ( 2 pmol/ml). The homogenate was centrifuged at 15,OOOg for 15 min. An aliquot of lop1 was transferred and evaporated under reduced pressure. Thirty microlitres of a mixture of SilPrep and BSTFA (3: l ) (Applied Science, Lab. Inc. State College, Bellefonte. Penn.) was added. After 2 h at room temperature an aliquot of the reaction mixture was injected into a gas chromatograph mass spectrometer (Finnigan 3000 E), focused on m/e 304 and 174 for GABA-trimethyl-silyl derivative and 318 for the internal standard (CATTABENI el a/., 1976). Proteins were measured according to LOWRY et a/. (1951). Table 1 shows the distribution of GABA in different brain regions in animals killed at 9.00 a.m. A concentration gradient was dctected. with the highest levels in substantia nigra and the lowest in the cortex. The hypothalamus is the region with the second highest concentration. These findings arc in agreement with previous results reported by others (ROBERTS et a/., 1958; FAHN 1976). Figure 1 shows that during the 24 h cycle, GABA levels In cortcx, striatum, cerebellum, hippocampus and substantia nigra remained constant, without significant peaks and troughs. In contrast, in the hypothalamus the pattern of GABA concentration during the 24 h cycle shows a biphasic variation, with two peaks: one at 7.00a.m. and the other at 1l.00a.m. These results indicate that the hypothalamus is the only brain region, of those investigated, where GABA levels are not maintained at a steady-state level throughout the day. Since GABA is differently distributed in the various hypothalamic nuclei (KURIYAMA & KIMURA, 1976) we shall examine the possibility that individual nuclei are responsible for the observed changes. The physiological meaning of this finding in connection to the role of GABA in the hypothalamus still remains to be investigated. However, some working hypotheses can be put forward.