The presence of nerve growth factor (NGF) mRNA and protein in the rat central nervous system is documented. Blot-hybridization analysis showed an abundance of NGF mRNA in the hippocampus, cerebral cortex, and olfactory bulb. Enzyme immunoassay confirmed significant levels of a NGF-like protein in the hippocampus and cerebral cortex. Bioassay of a NGF-like immunoaffinity-purified protein from these regions was physiologically indistinguishable from NGF. Immunohistochemistry revealed a widespread distribution of NGF-like reactivity in the adult brain, preferentially in fiber tracts. NGF mRNA accumulation began at birth, with adult levels reached 3 weeks postnatally. Enzyme immunoassay detected the presence ofa NGF-like protein in the embryonic rat brain. Postnatally, the level of NGF-like protein reached a maximum at 3 weeks. Additionally, a distinct fetal form of NGF may exist. ,B nerve growth factor (NGF) is essential for the development and maintenance of sensory and sympathetic neurons in the peripheral nervous system (PNS) (1, 2). However, both the presence and potential physiological function of NGF in the central nervous system (CNS) has remained elusive (3). The characteristic responses of peripheral adrenergic neurons following NGF or anti-NGF antibody injection have not been observed after similar application into the CNS (4-11). In the PNS, NGF is taken up by adrenergic terminals and also retrogradely transported to the cell body (2). In the CNS, injected NGF was specifically retrogradely transported from the hippocampus to the medial septum and the diagonal band of Broca (9) and also from the neocortex to the nucleus basalis (12), presumably in cholinergic neurons. NGF induced choline acetyltransferase in neonatal rat cortex, hippocampus, septum (10), and striatum (11), but not in adult hippocampus (13). Thus, NGF may have a physiological function in the CNS, and affect cholinergic neurons. Initial indication of endogenous NGF in the CNS was obtained by immunohistochemistry in the fetal rat (14, 15). NGF cDNA clones have been isolated from male mouse submaxillary glands (16, 17), and Shelton and Reichardt (18) recently detected NGF mRNA in the rat brain. Subsequently, NGF mRNA and an NGF-like protein have been described in some regions of the adult rat brain (19). In the present study, we demonstrate the developmental time course as well as the adult regional distribution of both NGF mRNA and protein in the adult rat brain. Finally, using a bioassay, we show that the adult brain contains nerve growth-stimulating activity typical of purified NGF. Our results demonstrate that NGF is expressed in the adult rat CNS with regional specificity and suggests the existence of a fetal form of NGF. MATERIALS AND METHODS Preparation of RNA and Blot-Hybridization Analysis. Total RNA from the brains of Sprague-Dawley rats (Alab, Stockholm, Sweden) was prepared (20), and poly(A)+ RNA, isolated by oligo(dT) chromatography (21), was separated on 1% agarose gels containing 0.7% formaldehyde and transferred to nitrocellulose filters. The double-stranded DNA probe used to detect /3-NGF (hereafter referred to as NGF) was a 900-base-pair (bp) Pst I fragment derived from a NGF cDNA clone (16); the probe to detect c-myc was 1.3-kilobase (kb) Cla I-EcoRI fragment from the 3' exon of the human MYC gene (22); and the probe to detect a-actin was a 1.5-kb Pst I DNA fragment (23). Purified DNA fragments were nick-translated to a specific activity of 109 cpm/,ug, hybridized to the blotted nitrocellulose filters overnight, and washed at high stringency. Filters were exposed on Kodak XAR-5 x-ray film at -80°C. Known dilutions of poly(A)+ RNA from male mouse submaxillary glands were always included as standards. Autoradiograms were quantified by densitometry [Shimadzu (Kyoto, Japan) CS-390]. Immunoafflnity Chromatography ofRat Brain NGF. Cortex and hippocampus (100 g wet weight) from 100 adult female Sprague-Dawley rats were homogenized in 0.1 M Tris HCl, pH 8.0/0.5 M NaCl and centrifuged. The supernatant was applied to a 3-ml column of afflinity-purified anti-mouse NGF antibodies coupled to CNBr-activated Sepharose (Pharmacia). The column was washed extensively, and bound NGF was eluted with 0.1 M glycine HCl (pH 2.5) containing 0.02% bovine serum albumin, concentrated to 4 ml in the culture medium used for bioassay by pressure dialysis, sterilized by filtration, and tested for NGF activity. Quantitation of NGF in Rat Brain. Explanted ganglia bioassay. Chicken sympathetic ganglia (day 9 embryos) were explanted into a collagen matrix, and the rat brain immunoelutant was tested for NGF activity (24, 25). Cultures were examined with dark-field microscopy and scored for fiber outgrowth. Enzyme-linked immunoassayfor NGF. Immunoplates (96well black Microfluor plates, Dynatech, Alexandria, VA) were coated with affinity-purified antibodies against mouse NGF (25-27), and nonspecific binding was blocked with 1% bovine serum albumin. Brain samples in TBS buffer (0.02 M Tris HCl, pH 7.5/0.5 M NaCl) containing aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, 0.1% bovine serum albumin, 10 mM EDTA, and 0.5% Tween 20 were added to the wells Abbreviations: NGF, nerve growth factor; PNS, peripheral nervous system; CNS, central nervous system; bp, base pair(s); kb, kilobase(s). §To whom reprint requests should be addressed. 817 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 818 Neurobiology: Whittemore et al.