Brain Research, 506 (1990) 9-13 9 Elsevier BRES 15077 Acetylcholine stimulates the secretion of corticotropin-releasing factor from primary dissociated cell cultures of the rat telencephalon and diencephalon Edward Hillhouse 2 and Seymour Reichlin 1 1Division of Endocrinology, Tufts University, School of Medicine, New England Medical Center, Boston, MA 02111 (U.S.A.) and 2Department of Medicine, King's College School of Medicine, London (U. K.) (Accepted 30 May 1989) Key words: Corticotropin-releasing factor; Cell culture; Rat telencephalon; Rat diencephalon, Acetylcholine; Arg vasopressin Corticotropin-releasing factor (CRF-41) immunoreactive neurones in the CNS are widely distributed; although the major concentration is in the parvocellular division of the paraventricular nucleus, there are also large clusters of CRF-containing neurones in the brainstem, limbic system and cerebral cortex. In this study, we sought to determine whether dispersed rat brain cells cultured in vitro are capable of secreting CRF immunoreactivity. In both telencephalic and diencephalic cultures CRF content of the cells increased from day 7 to 28. This effect was greatest in cells raised in media containing 10% fetal calf serum and 10% heat inactivated horse serum as compared with media containing 10% fetal calf serum and 10% NuSerum. Exposure of the cells to 56 mM K + or to 10 9 M acetylcholine caused a release of CRF which was calcium dependent. The effect of acetylcholine was antagonised by atropine (10 6 M). The CRF immunoreactivity in both types of cell produced two peaks on gel chromatography; one of which eluted with the void volume and the other of which co-eluted with standard CRF-41. However, only the mature peptide was detected in media from cells stimulated with potassium. We conclude that cell cultures of the rat diencephalon and telencephalon produce immunoreactive CRF-41 and that immunoreactive CRF-41 secretion in both diencephalic and telencephalic cells is regulated by acetylcholine. INTRODUCTION Neurotransmitter and hormonal control of CRF secre- tion has been studied extensively using both whole animal 4 and isolated hypothalamic fragments 7'j7. In early work by Hillhouse and Jones t6 corticotropin-releasing factor (CRF) secretion was shown by bioassay to be regulated by a number of neurotransmitters, of which acetylcholine (ACh) was the most potent. However, at the time the work was done, which was prior to the elucidation of the chemical structure of CRF by Vale and colleagues 23, it had not been established that CRF and vasopressin were both potent releasers of adrenocortico- tropic hormone (ACTH) ~5, and that the two peptides were synergistic in their activity 5'2x. In light of the more recent findings, it became necessary to re-study this question using more specific measures of CRF secretion. In this paper we report the effects of ACh on immuno- reactive (Jr) CRF and immunoreactive vasopressin secre- tion. We have utilized a dispersed diencephalic cell culture system previously shown to be useful for study of factors regulating the secretion of somatostatin x2~22 and CCK ~. The use of a similar approach has recently been reported by Clark, Lawry and Gillies 1°. Since CRF-ir neurones are also localized in the telencephalon 2°, we have also determined the effect of ACh on CRF secretion by cortical neurones. METHODS Radioimmunoassay of CRF-41 The radioimmunoassay was developed using antiserum RC70 (kindly donated by Dr. W. Vale) which is highly specific for rat/human CRF-41. In the assay the antiserum (final dilution 1:420,000) and synthetic rat CRF-41 standards or test samples were added to tubes containing a buffer consisting of 100 mM NaCI, 50 mM NazHPO 4, 25 mM EDTA, 0.1% BSA and 0.1% Triton X-100. After 48 h incubation at 4 °C, the tracer was added and the incubation allowed to continue for a further 24 h before separation of the bound tracer using the second antibody technique. The tracer was prepared by gentle chloramine T iodination of synthetic rat Tyrosine CRF and purified initially on a Sep-Pak C~ column followed by HPLC according to the method of Vale et alfl4. Using this technique, the sensitivity of the assay was 20 pg/ml with intra-assay coefficient of variation of 6.4% and an inter-assay coefficient of variation of 10.6%. Radioimmunoassay of arginine vasopressin (AVP) The radioimmunoassay was developed using a specific antiserum for AVP donated by Dr. F. Sanchez-Franco. The antiserum (final dilution 1:360,000) was added to tubes containing either standard or Correspondence: E. Hillhouse, Department of Medicine, King's College School of Medicine, Denmark Hill, London SE5 8RX, U.K.