Histochemistry (1991) 95:303-314 030155649100013P Histochemist © Springer-Verlag 1991 Topographical localisation of endothelin mRNA and peptide immunoreactivity in neurones of the human brain A. Giaid 1, SJ Gibson 1, M.T. Herrero 3, S. Gentleman 1, S. Legon 2, M. Yanagisawa 4, T. Masaki 4, N.B.N. Ibrahim 5, G.W. Roberts 6, M.L. Rossi 7, and J.M. Polak 1 Departments of 1 Histochemistry and z ChemicaI Pathology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK 3 Department of Anatomy, University of Pamplona, Pamplona, Spain; 4 Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan 5 Department of Histopathology, Frenchay Hospital, Bristol BSI6 1LE, UK 6 Department of Anatomy, St. Mary's Hospital Medical School, London W12 1PG 7 Department of Neuropathology, Midland Centre for Neurosurgery and Neurology, Birmingham BG7 7JX, UK Accepted October 6, 1990 Summary. The distribution of endothelin mRNA and immunoreactivity in the human brain was investigated using the technique of in situ hybridization and immuno- cytochemistry. Cryostat sections from 22 cases of neu- rologically normal adult human brain, collected 3-7 h post-mortem were hybridized with 35S-labelled comple- mentary (c)RNA probes prepared from the 3' non-cod- ing region of endothelin-1 cDNA, and the chromosomal genes encoding endothelin-2 and -3. In situ hybridization with all three cRNA probes revealed labelled neuronal cell bodies in laminae IIIVI of the parietal, temporal and frontal cortices. Labelled cells were also seen, scat- tered throughout the para- and periventricular, su- praoptic and lateral hypothalamic nuclei, the caudate nucleus, amygdala, hippocampus, basal nucleus of Meynert, substantia nigra, raphe nuclei, Purkinje cell layer of the cerebellum and in the dorsal motor nuclei of the vagus of the medulla oblongata. The distribution of neurones immunoreactive to endothelin was similar to that of endothelin mRNA, although fewer immunore- active ceils throughout the brain, were noted. Immuno- reactive fibres were present mainly in the cortex and hypothalamus, and to a lesser extent in the brain stem. Combined in situ hybridization and immunocytochem- istry on the same section revealed the presence of en- dothelin-1 mRNA and immunoreactivity in the same cortical neuronal cell. Colocalisation studies in the cor- tex revealed endothelin-1 mRNA and immunoreactivity in a number of cells which also expressed neuropeptide Y mRNA and immunoreactivity. In the hypothalamus and basal nucleus of Meynert endothelin immunoreac- tivity was colocalised to a subset of neurophysin- and galanin-immunoreactive cell bodies respectively. En- dothelin mRNA and immunoreactivity was also seen in some blood vessel endothelial cells. The findings of endothelin mRNAs and immunoreactivity in heterogen- Offprint reqeusts to: S.J. Gibson ous neuronal populations further emphasises the poten- tial role of endothelin as a neuropeptide, probably hav- ing diverse actions in the nervous system of man. Introduction Within the last few years, a number of peptides isolated from tissues other than nervous system have subsequent- ly been found to occur in nerves and to possess neu- romodulatory and neurotransmitter actions (Snyder 1980; Gibson and Polak 1986). Endothelin represents one such example. Endothelin-1 is a 21 amino acid pep- tide which was originally isolated from the supernatant of porcine endothelial cell cultures (Yanagisawa et al. 1988a). This peptide is formed by cleavage of a 203 amino acid precursor to produce a 39 (porcine) or 38 (human) amino acid peptide, big endothelin. The latter, in the presence of a presumptive endothelin-converting enzyme produces mature endothelin (Yanagisawa and Masaki 1989). Recently, three endothelin-related genes have been identified in the human genome. These genes are known as endothelin-1 (identical to mature porcine endothelin), endothelin-2 and endothelin-3 (identical to rat endothelin) (Inoue et al. 1989). Endothelin-1 differs by two and six amino acids from endothelin-2 and -3 respectively although the C-terminal structure is pre- served among all three peptides (Inoue et al. 1989). The existence of three distinct endothelin-related loci have also been demonstrated in pig and rat genomic DNA (Inoue et al. 1989). Endothelin-I appears to be the most potent vasoconstrictor agent recognised to date (Yanag- isawa et al. 1988a, b) but in addition to these vascular mediated properties, the endothelin-related peptides are fast gaining recognition as neuropeptides. Endothelin has a direct action on neuronal excitabili- ty (Yanagisawa and Masaki 1989) and induces behavior-