@ P N Vol.74,No.1,pp.19>218,1996 Copyright G1996IBRO.PublishedbyElsevier ScienceLtd PrintedinGreatBritain PII: S0306-4522(96)00116-9 0306-4522/96 $15.00+ 0.00 EXPRESSION OF (X2-ADRENERGIC RECEPTOR SUBTYPES IN THE MOUSE BRAIN: EVALUATION OF SPATIAL AND TEMPORAL INFORMATION IMPARTED BY 3 kb OF 5’ REGULATORY SEQUENCE FOR THE a2AAR-RECEPTOR GENE IN TRANSGENIC ANIMALS R. WANG,? L. B. MACMILLAN,? R. T. FREMEAU Jr,$ M. A. MAGNUSON,$ J. LINDNER$ and L. E. LIMBIRD*t tDepartments of Pharmacology and of ~MolecularPhysiologyand Biophysics,Vanderbilt University Medical Center, Nashville, TN 37232,U.S.A. $Departmentsof Pharmacologyand Neuroscience,Duke UniversityMedicalCenter, Durham, NC 27710, U.S.A. Abstra@-The present studies characterize the expression of the a c and U2Cadrenergicreceptor subtypesvia s hybridizationanalysis of messengerRNA expressionin the adult mouse brain, as well as the pattern of expressionof u2~adrenergicreceptormessengerRNA at embryonicday E9.5,the earliest time for detection of the messenger RNA encoding this receptor subtype. am adrenergic receptor messengerRNA is highlyexpressedin the sixth layer of the cortex and the locuscoeruleus,az~adrenergic receptor messengerRNA predominantlyin the thalamus and in the Purkinje layer of the cerebellum,and a - m W region,ofthe mou~ brain. Both C a r m d s e w c hypothalamus, olfactory system and the hippocampalformation. To developa molecular understanding of the unique cellularexpressionof messengerRNA encodingthe azAadrenergicreeeptor subtype,2.83kb of the upstream regulatory sequencefor the a2~adrenergic receptor gene was fused to the LacZ gene as a reporter gene and expressionof /?-galactosidaseactivity was assessedin transgenicoffspring.Although the spatial expression of the transgene in the adult brain often overlaps that for the endogenous adrenergic receptor, both eetopic expression and the absence of appropriate expressionwere noted; in contrast five of the six lines show temporal expressioncharacteristic of the endogenous adrenergic receptor gene. The present studies provide the first characterization of messengerRNA localization for the three adrenergic receptor subtypesin the mouse CNS. Becausethe functionalroles of the prazosin-sensitive adrenergic receptor and azc adrenergicreceptor subtypeshave been masked in most earlier physiological and pharmacologicalanalysesof adrenergicreceptor function,identifyingthe multipleloci adrenergic reeeptor subtype expressionis an important prelude to understandingthe functional roles of these three independent receptor populations in the mouse CNS. The findingsin the transgenic animals indicating that -3 kb of regulatory sequencehas imparted faithful temporal but not spatial expressionfor the adrenergic receptor gene suggestthat additional regulatory information is neeessaryfor appropriate cell specificexpressionof messengerRNA for the adrenergicreceptor subtype. Copyright ~ 1996IBRO. Published by Elsevier Science Ltd. w a2-adrenergicreceptor subtypes, s hybridization,transgenicmice, regulation of temporal expression,regulation of cell-specificmRNA expression,catecholamines. *To whom correspondenceshould be addressed. S c a u The studies reported in this manuscript were undertaken to reveal temporal and spatial expressionof the genesencodingthe and azc subtypes of the az-adrenergicreceptor during devel- opment and in the adult brain. It was not possible, given the goals of these studies, to utilize non-animal materials. It shouldbe emphasized,however,the animals were anesthetized prior to being killed and all efforts were made to prevent, and thereby eliminate, animal suffering. A b a2AR,alpha2-adrenergicreceptorand a c and receptor subtypes; E, embryonic day of gestation, such as E9.5; EDTA, ethylenediaminetetra- acetate; kb, kilobase; LacZ, lactose operon gene encoding beta-galactosidase; RNase, ribonuclease A; SSC, standard saline citrate; X-Gal, 5-bromo-4-chloro- 3-indolyl-/?-mgalactoside. The endogenous catecholamines epinephrine and norepinephrine mediate their effects via interaction with three distinct receptor families, dubbed #- adrenergic, al-adrenergic and a2-adrenergic receptors. The ctz-adrenergic receptors (ct2AR) modulate meta- bolic effects in adipose, renal and intestinal epithelial cells and suppress insulin release from ~ cells of the pancreas. The prominent effects of a2AR, however, are in the central and peripheral nervous system, where they mediate suppression of neurotransmitter release .19 The rt2AR have been shown to exist as three independent subtypes. Although U2ARsubtypes had been postulated to exist based on earlier physiological and pharmacological studies,3 definitive identification 199