European Journal of Neuroscience, Vol. 10, pp. 3257–3268, 1998 © European Neuroscience Association The murine Y 1 receptor 5' upstream sequence directs cell-specific and developmentally regulated LacZ expression in transgenic mice CNS Alessandra Oberto, Emanuela Tolosano, 1 Rossella Brusa, Fiorella Altruda, 1 Giancarlo Panzica 2 and Carola Eva Section of Pharmacology, Department of Anatomy, Pharmacology and Forensic Medicine, 1 Department of Genetics, Biology and Biochemistry, and 2 Section of Anatomy, Laboratory of Neuroendocrinology, Department of Anatomy, Pharmacology and Forensic Medicine, University of Torino, Italy Keywords: β-galactosidase, brain, neuropeptide Y, transgenic mouse, Y 1 receptor Abstract The Y 1 receptor for neuropeptide Y (NPY) is highly expressed in mammalian CNS where it mediates the activation of several neurobiological functions. We have previously demonstrated that a 1.3-kb fragment upstream of the transcription initiation sites of the murine Y 1 receptor gene is able to direct specific expression of reporter genes in neuronal cell cultures. In the present study transgenic mice harbouring this putative promoter region linked to the LacZ reporter gene were generated and analysed for temporal and spatial distribution. Ten transgenic lines expressed β-galactosidase in the CNS but not in other organs such as heart, liver and kidney. Histochemical analysis of brain from adult transgenic mice showed specific expression of the transgene in specific brain regions with little variation. Four transgenic lines showed characteristic patterns of β- galactosidase activity in the brain that are consistent with the expression of the endogenous gene. Prominent LacZ activity was present in several telencephalic and diencephalic structures, including deeper layers of cerebral cortex, amygdaloid complex, hippocampus, preoptic area, several thalamic and hypothalamic nuclei and habenula. The ontogeny analysis indicates that the LacZ expression agrees with the temporal expression pattern of rat Y 1 receptor mRNA. These data demonstrate that the 1.3-kb upstream region of the murine Y 1 receptor gene contains the cis acting elements required for establishing a CNS-restricted and developmental stage- specific pattern of expression in vivo. Moreover they provide further information on the distribution of this NPY subtype receptor in mammalian brain. Introduction Neuropeptide Y (NPY) is a 36-amino-acid polypeptide with potent central as well as peripheral biological effects, including stimulation of food intake, anxiolysis, memory effects and cardiovascular effects (Wahlestedt & Reis, 1993). NPY receptors are G-protein-coupled receptors, all of which are associated with inhibition of adenylate cyclase. At least six NPY receptor subtypes have been described on the basis of their ability to bind NPY analogues, termed Y 1 ,Y 2 ,Y 3 , Y 4 /PP, Y 5 and y 6 (Blomqvist & Herzog, 1997). Except for the Y 3 receptors, molecular cloning of cDNAs encoding corresponding receptor proteins has been carried out (Eva et al., 1990; Larhammar et al., 1992; Bard et al., 1995; Gerald et al., 1995, 1996; Rose et al., 1995; Weinberg et al., 1996). They all belong to the G-protein- coupled receptor superfamily and are coupled to more than one second-messenger system. The functional y 6 receptor protein is only expressed in mouse and rabbit and its role still remains to be determined (Blomqvist & Herzog, 1997). In the peripheral nervous system the Y 1 receptor is found predomi- nantly at the sympathetic postjunctional sites in blood vessels, where Correspondence: Dr Carola Eva, Sezione di Farmacologia, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Universita ` di Torino, Via P. Giuria, 13-10125 Torino, Italy. E mail: eva@medfarm.unito.it Received 12 February 1998, revised 28 May 1998, accepted 2 June 1998 it mediates the contractile response to NPY of vascular smooth muscle, both directly and indirectly by potentiating the action of other pressure agents, such as noradrenaline (Wahlestedt et al. 1986; Grundemar et al., 1992; Malmstro ¨m, 1997). In the rat central nervous system the distribution of Y 1 receptors has been investigated using autoradiographical, immunohistochemical and in situ hybridization techniques. Specific Y 1 binding sites are mostly located in various cortical areas, olfactory anterior nucleus, molecular layer of the dentate gyrus, claustrum and most of thalamic nuclei (Dumont et al., 1990; Larsen et al., 1993; Dumont et al., 1996). High levels of Y 1 receptors have been also identified in the arcuate nucleus, and in the basket cells and Purkinje cells of cerebellum by immunohistochemical studies (Zhang et al., 1994a; Fuxe et al., 1997; Kopp et al., 1997). This distribution is not fully confirmed by in situ hybridization, suggesting also a possible presynaptic role of the Y 1 receptor (Eva et al., 1990; Mikkelsen & Larsen, 1992; Larsen et al., 1993). In addition Y 1 receptor and its mRNA were found in dorsal horn of lumbar spinal cord and in dorsal root ganglia (Jazin et al., 1993; Ji et al., 1994; Zhang et al., 1994a, b).