A Model for Combined Morphological and Functional Investigations on the Isolated Mediobasal Rat Hypothalamus

Abstract

We have developed a model for combined morphological and functional in vitro studies of the isolated mediobasal hypothalamus (MBH) by considering two prerequisites: (1) the tissue must be well preserved, free of morphological artefacts and functionally unimpaired until the end of the in vitro incubation, and (2) the tissue must be processed for morphology in optimal conditions. To test our model we have studied some aspects of the luteinizing hormone-releasing hormone (LHRH) system in 4-month-old male Sprague-Dawley rats. After decapitation the MBH was isolated and put in a flask containing 0.5 ml Hepes-buffered Locke’s medium gassed by 5 ml/min of O₂/CO₂ (95%/5%) and shaken in a water bath at 37 °C. After a 10-min washing, the medium was changed twice at an interval of 20 min. After the in vitro incubation the tissue was satisfactorily preserved as judged by light- and electron-microscopic analysis. LHRH, somatostatin and thyrotropin-releasing hormone could be demonstrated by alkaline phosphatase or peroxidase-antiperoxidase im-munohistochemistry on semithin sections and by immunogold technique on thin sections. The LHRH secretion was close to basal values after 30 min of incubation (22.1 ± 4.8 pg/MBH) and then remained constant for another period of 20 min (17.6 ± 2.6 pg/MBH). During the second 20 min of incubation LHRH secretion increased in presence of 61.6 mM K⁺ (110.7 ± 8.7 pg/MBH). Thus the isolated hypothalamus was excitable until the end of the in vitro incubation. We conclude that this model can be successfully used for combined morphological and functional studies.

Functional and Morphological Changes in Mediobasal Hypothalamus of Streptozocin-induced Diabetic Rats: In Vitro Study of LHRH Release

“…The in vitro system has been previously described (18). Briefly, each MBH was washed for 10 min, preincubated for 20 min in basal medium (0.4 ml), then incubated for two 20-min periods in basal (5.6 mM KCI, 154 mM NaCI) or K + -enriched medium (61.6 mM KCI, 98 mM NaCI).…”

“…The LHRH released into the media during the two periods of incubation was quantified by radioimmunoassay (RIA) as previously described (18).…”

“…The postincubation processing of the hypothalamic fragments has been previously described (18). Briefly, the tissue fragments were fixed in 2% glutaraldehyde/1.5% paraformaldehyde in phosphate buffer (pH 7.4).…”

“…Immunohistochemistry. Standard semithin sections from non-postfixed specimens were stained for LHRH as previously reported (18) with the same anti-LHRH serum as used in the RIA. One standard immunohistochemically stained semithin section containing half of the MBH was considered in eight randomly selected animals of each group.…”

“…This evidence would be crucial to understanding the pathogenesis of the diabetic gonadal axis impairment. Therefore, we undertook a combined functional and morphological study of the mediobasal hypothalamus (MBH) in 1-mostreptozocin-induced diabetic (STZ-D) male rats, using a recently developed in vitro model (18). We could thus study successively 1) basal and depolarization-induced hypothalamic LHRH release and 2) morphology, morphometry, and immunohistochemistry in the same isolated MBH.…”