Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 4668-4672, May 1992 Neurobiology Estrogen receptors colocalize with low-affinity receptors in of the (steroid autoradography/nonlsotopIc iu sift hybrdatlou/mo tohe sty/c t/aglng ahdAle r diease) C. DOMINIQUE TORAN-ALLERAND*t, RAJESH C. MIRANDA*, WAYNE D. L. BENTHAM*, FARIDA SOHRABJI*, THEODORE J. BROWNS, RICHARD B. HOCHBERG§, AND NEIL J. MACLUSKY* *Department of Anatomy and Cell Biology and Center for Reproductive Sciences, Columbia University College of Physicians and Surgeons, New York, NY 10032; tDivision of Reproductive Science, University of Toronto, Toronto, Canada M5G 1L4; and §Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT 06510 Communicated by Patricia S. Goldman-Rakic, February 28, 1992

ABSTRACT The rodent and primate basal forebrain is a effects of estrogen in the brain is whether these actions are target of a family of endogenous peptide signaling molecules, exerted directly. Alternatively, the steroid may potentiate the -nerve growth factor, brain-derived neu- synthesis ofendogenous growth and trophic factors and their rotrophic factor, and 3-and of the gonadal receptors (10), as in extraneural targets (11, 12), or it may steroid hormone estrogen, both of which have been implated interact with them in a stimulatory or inhibitory fashion to in cholinergic function. To investigate whether or not these promote the growth and differentiation ofspecific neuromod- ligands may act on the same neurons in the developing and ulatory or systems by autocrine or para- adult rodent basal forebrain, we combined autoradlography crine mechanisms, which may lead to shifts in the develop- with '2I-labeled estrogen and either nonisotopic in situ hy- mental patterns of resulting neural networks. bridization histochemistry or Im nohistochemistry. We now To test the hypothesis that estrogen and the neurotrophins report colocalization ofintranuclear estrogen binding sites with may act on the same basal forebrain neurons, we combined the mRNA and immunoreactive protein for the low-affinity autoradiography with 1251-labeled estrogen ("2'I-estrogen) nerve growth factor receptor, which binds all three neurotro- and nonisotopic in situ hybridization histochemistry or im- phins, and for the cholinergic marker enzyme choline acetyl- munohistochemistry to identify the mRNA and encoded transferase (acetyl-CoA:choline O-acetyltransferase, EC protein for the low-affinity (p75NOFR) NGF receptor and for 2.3.1.6). Colocalization of estrogen and low-affinity nerve choline acetyltransferase (ChAT; acetyl-CoA: choline O-ace- growth factor receptors implies that their ligands may act on tyltransferase, EC 2.3.1.6), the -synthesizing the same , perhaps synergistically, to regulate the enzyme, a marker ofcholinergic neurons. We report here the expression of specific genes or gene networks that may influ- colocalization of estrogen binding sites with the low-affinity ence neuronal survival, differentiation, regeneration, and plas- NGF receptor p75NGFR mRNA and immunoreactive protein ticity. That cholinergic neurons in brain regions subserving in neuronal subsets ofthe rodent medial septum, nuclei ofthe cognitive functions may be regulated not only by the neuro- diagonal band of Broca, and in the continuum of neurons of trophins but also by estrogen may have considerable relevance the ventral pallidum, substantia innominata, and nucleus for the development and maintenance of neural substrates of basalis of Meynert. Moreover, we also provide definitive cognition. Ifestrogen-neurotrophin interactions are important evidence that cholinergic basal forebrain neurons bind estro- for survival of target neurons, then clinical conditlons associ- gen as well. ated with estrogen deficiency could contribute to the atrophy or death ofthese neurons. These findgs have implications for the subsequent decline in those differentiated neural functions MATERIALS AND METHODS associated with aging and Alzheimer disease. Animals. Postnatal female Sprague-Dawley rats (Charles River Breeding Laboratories) 10 and 12 days old (n = 15) and The basal forebrain is a region that, in the human, is impor- adult female mice (RIII) from our breeding colony, ovariec- tant for learning, memory, and other cognitive functions and tomized under methoxyflurane (Metofane) for 1 week before in which the cholinergic neurons are affected early in Alz- use (n = 5), were injected s.c. or i.v. in thejugular [1 1ACi per heimer disease and age-related cognitive impairment (1, 2). g of body weight (1 Ci = 37 GBq); =0.2 ng per g of body Developing and adult basal forebrain neurons are targets not weight] with ll(-methoxy[16a-125I]iodoestradiol ([125I]MIE2) only of the family of neurotrophins-nerve growth factor synthesized by R.B.H. as described (13) (100 1.d; 506% pro- (NGF), brain-derived neurotrophic factor (BDNF), and neu- pylene glycol/50% saline) and killed 1 h later by transcardial rotrophin 3 (NT-3)-but of the gonadal steroid estrogen as perfusion under deep ether anesthesia with 4% paraformal- well. Basal forebrain cholinergic neurons of the developing dehyde in 0.1 M phosphate buffer (pH 7.6) containing 2.5% rodent and primate have been shown to express the mRNA (vol/vol) dimethyl sulfoxide (DMSO) and 0.1% ofthe RNase (3, 4) or encoded protein (5, 6) for the low-affinity form ofthe inhibitor diethylpyrocarbonate. Specific binding of this iodi- NGF receptor, which binds the neurotrophins (7, 8). Over- nated estrogen is completely abolished by concurrent expo- lapping basal forebrain regions have also been shown to sure to 100-fold molar excess of the unlabeled nonsteroidal exhibit estrogen-receptor mRNA, estrogen binding sites, and estrogen diethylstilbestrol (14). Brains were postfixed for 2 h high levels of the estrogen synthesizing enzyme cytochrome at 40C in the same fixative, without DMSO, and then equil- P450-dependent aromatase (for refs. see ref. 9), although the ibrated overnight at 40C in 15% buffered sucrose before neurotransmitter phenotype(s) ofthese neurons has not been reported. A critical question concerning the developmental Abbreviations: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin 3; ChAT, choline acetyl- The publication costs of this article were defrayed in part by page charge transferase; [125I]MIE2, 11.8-methoxy[16a-125I]iodoestradiol; ERE, payment. This article must therefore be hereby marked "advertisement" estrogen response element. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

4668 Downloaded by guest on September 23, 2021 Neurobiology: Toran-Aflerand et aL Proc. Natl. Acad. Sci. USA 89 (1992) 4669 embedding in M-1 embedding matrix (Lipshaw Manufac- Controls for the specificity of p75NGFR and ChAT mRNA turing, Detroit) and freezing on dry ice/acetone as described detection included, as described (9, 17): (i) the demonstration (14). on Northern blot analysis that signal detection by each probe Steroid Autoradiography. Steroid autoradiography was hybridized only to a species of RNA of a size corresponding carried out first. Coronal sections (10-12 ,um) through the to that of the mRNA encoding the appropriate protein; (ii) basal forebrain region were cut on a cryostat and thaw- incubation with noncomplementary (sense) digoxigenin- mounted onto NTB-3 (Kodak) emulsion-coated slides and labeled probes; (iii) pretreatment with excess unlabeled 90- processed for steroid autoradiography combined with either base oligonucleotides synthesized against sequences that immunohistochemistry (14) or nonisotopic (digoxigenin) in overlapped the experimental probes; and (iv) analyses of situ hybridization histochemistry as described (9). After thermal stability, which showed a close match (-14C) be- photographic development, the autoradiograms were pro- tween theoretical and experimentally calculated melting tem- cessed for immunohistochemistry with antibodies raised peratures, indicating hybridization to a single species of against either p75NGFR [monoclonal antibody IgG 192 (15); mRNA containing the expected sequence. Neurons express- 1:25; gift ofEugene M. Johnson, Jr., Washington University, ing p75NGFR or ChAT immunoreactivity or the blue cytoplas- St. Louis] or ChAT (rabbit polyclonal antibody; 1:500; Chem- mic hybridization signal for their mRNA were characterized icon) by means of the avidin-biotin-peroxidase complex with respect to the presence or absence of discrete concen- method with 3,3'-diaminobenzidine as the chromagen trations ofsilver grains in the emulsion underlying cell nuclei, (Vectastain ABC Elite kit, Vector Laboratories). Both anti- indicating binding of the iodinated estrogenic ligand. bodies have been extensively characterized and both anti- [12-I]MIE2 binding was considered specific, using a 5x back- gens survive autoradiographic development [D170, 2.5% ground labeling criterion, where the probability of false sodium sulfite/0.1% potassium bromide/0.45% 2,4- labeling is <0.001 (21). diaminophenol dihydrochloride (Kodak), pH =7 (9, 14)]. In Situ Hybridization Histochemistry. Nonisotopic (digox- RESULTS igenin) in situ hybridization histochemistry was carried out Distribution of Estrogen and Low-Affinity NGF Receptors. after steroid autoradiography for p75NG'R and ChAT mRNA Estrogen receptor mRNA expression and estrogen binding as described (9). Briefly, digoxigenin-labeled oligonucleo- sites (Fig. 1A) in the basal forebrain are distributed in a tides were synthesized as described (16, 17). The oligonu- pattern that clearly overlaps the expression of p75NGFR cleotides were 3'-end-labeled with digoxigenin-labeled de- mRNA and its encoded immunoreactive protein (Fig. 1B). As oxyuridine triphosphate (dUTP) (Boehringer Mannheim) by will be shown elsewhere, virtually all neurons of the medial terminal deoxynucleotidyl transferase (BRL). The developed septum/diagonal band coexpress estrogen receptor and autoradiograms were hybridized overnight at 35°C (NTB-3 p75NGFR mRNA by double-label (35S/digoxigenin) in situ emulsion melts off the slide at temperatures >37°C) with 50 hybridization (R.C.M., F.S., and C.D.T.-A., unpublished ng of probe per ml as well as with 2 pmol of a random data). Analysis of autoradiograms of the developing rat composition, unlabeled, 36-base oligonucleotide per ml con- medial septum, nuclei of the horizontal and vertical limbs of taining >1020 sequence combinations (DuPont; NEP 550) the diagonal band, the ventral pallidum, substantia innomi- added to decrease nonspecific hybridization. After hybrid- nata, and the nucleus basalis that had been reacted with ization to the target cDNA, the hybrids were detected by antibodies to the p75NGFR protein revealed numerous estro- enzyme-linked immunohistochemistry with anti-digoxigenin gen-concentrating neurons colocalizing p75NGFR immunore- antibodies conjugated to alkaline phosphatase (Fab fragment, activity. Neurons in these regions, however, were heteroge- 1:500; 48 hr; 4°C) (Boehringer Mannheim) and an enzyme- neous with respect to colocalization of estrogen and NGF catalyzed, blue-color reaction with 5-bromo-4-chloro-3- receptors. Many neurons that exhibited p75NGFR immunore- indolyl phosphate and nitroblue tetrazolium salt (Boehringer activity colocalized estrogen binding sites as well (Fig. 2 A Mannheim). After washing at 35°C and at relatively high and B). A smaller number ofNGF receptor-positive neurons, stringency, sections were cleared in Histo-Clear (National in contrast, were estrogen receptor negative but were found Diagnostics, Manville, NJ) (to avoid fading of the color admixed with those colocalizing NGF and estrogen receptors product) and mounted in Permount. (Fig. 2C). Some NGF receptor-positive but estrogen recep- p75NGFR mRNA. Because monoclonal antibody IgG 192 tor-negative neurons were also interspersed with still other recognizes the low-affinity NGF receptor ofthe rat only (15), neurons that exhibited either estrogen binding only (Fig. 2D) the distribution of immunoreactive p75NGIF could be studied or neither phenotype (data not shown). only in autoradiograms of developing rats. To minimize the To address the potential for p75NGFR expression in the amounts of [125I]MIE2 needed and to address this question in adult rodent, steroid autoradiography was combined with the adult rodent, nonisotopic in situ hybridization histochem- nonisotopic in situ hybridization histochemistry for p75NGFR istry was carried out for p75NGFR mRNA in the much smaller mRNA expression in the smaller, adult mouse as well as in adult mouse, as well as in developing rats. The p75NGFR probe developing rats. Colocalization of the blue/purple cytoplas- was a 46-base synthetic oligonucleotide sequence from the mic hybridization signal of p75NGFR mRNA and concentra- membrane-spanning region of chicken low-affinity NGF re- tion of silver grains underlying cell nuclei were seen in ceptor cDNA (18), which has been extensively characterized estrogen target neurons of the same adult murine and devel- and which shares extensive homology with the low-affinity oping rat basal forebrain regions where NGF receptor im- rat NGF receptor. munoreactivity was found in the developing rat (Fig. 2E). ChAT mRNA. Two synthetic oligonucleotides, 39 and 48 To identify the phenotype of these estrogen-binding neu- bases, were synthesized from opposite ends of the cDNA rons, the autoradiograms were also processed for ChAT sequence that encodes active rat ChAT (19). The first cor- mRNA or its immunoreactive protein. Our studies demon- responds to bases 202-243, which is comparable to that used strate that many estrogen-binding neurons of the medial in molecular biological studies of the enzyme (20). The septum, nuclei of the diagonal band, substantia innominata second spans bases 1120-1168, which corresponds to the and ventral pallidum, like the p75NGFRl~containing cells they porcine ChAT sequence used in enzyme induction studies may represent, at least in part, are cholinergic and coexpress (3). The p75NGFR and two ChAT probes have no significant ChAT mRNA (Fig. 2F) or ChAT immunoreactivity (Fig. homology with any ofthe known nucleotide sequences in the 2G). What other neurotransmitter phenotype(s) the estrogen GenBank/EMBL data bases. targets may also represent remains to be determined. Downloaded by guest on September 23, 2021 4670 Neurobiology: Toran-Allerand et al. Proc. Nad. Acad. Sci. USA 89 (1992)

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FIG. 1. Spatial distribution of estrogen binding sites and low-affinity NGF receptor immunoreactivity clearly overlaps in the P10 rat medial septum and nuclei ofthe diagonal band. (A) Dark-field autoradiogram of [1251]MIE2-concentrating neurons in the vertical nucleus ofthe diagonal band (V, ventricle). (B) NGF receptor immunoreactivity in the medial septum and nuclei of the diagonal band. (A, x15; B, x5.) DISCUSSION expression of specific genes or gene networks to influence neuronal survival, differentiation, regeneration, and plastic- Our findings demonstrate that estrogen target neurons not ity. Conversely, loss ofthe stimulus mediated through either only have the potential for p75NGIR and ChAT expression (by receptor may result in neuronal atrophy or even cell death. localizing the mRNA) but do, in fact, synthesize the encoded Structural and functional analyses of some of the genes protein (by identifying immunoreactive NGF receptors and regulated by estrogen reveal a common 13-base-pair palin- ChAT). Thus, basal forebrain cholinergic neurons may be dromic sequence, [5'-GGTCANNNTGACC-3'] (34), which, influenced not only by the neurotrophins but by estrogen as while often imperfect, is sufficient to mediate hormonal well. Both ChAT and , which normally induction of transcription. The possibility that coexpression increase in the basal forebrain in response to NGF during of the estrogen and NGF receptors may be of biological development (4) and in the adult (3), have also been shown to significance is heightened by our preliminary findings be estrogen responsive and to exhibit a sex difference in their (R.C.M., F.S., and C.D.T.-A., unpublished data), which activity and regulation, with the location and direction of suggest that the human low-affinity p75NGFR gene contains an change dependent on sex (22). Recent studies have shown estrogen response element (ERE)-like consensus sequence that NGF promotes cholinergic neuron survival in the medial within septum/diagonal band, after partial or complete transections its promoter region (bases -341 through -352) (35). of the fimbria-fornix, the septo-hippocampal pathway (23, This sequence includes the exact spacing of 3 base pairs 24), and in cognitively impaired aged rats (25). Whether or not between the two pentameric halves ofthe palindrome, which estrogen exerts a modifying influence on this process is is essential for estrogen receptor action (34). Since the rat unknown. However, localization of aromatase, an enzyme low-affinity NGF receptor gene has not been cloned, partic- that converts testosterone to the active estrogenic metabolite ularly in the 5' and promoter regions, the presence of estradiol to these regions throughout life (26) provides the ERE-like sequences is unknown. However, since the extra- basal forebrain with a potential endogenous source of estro- cellular domain of p75NGlR is conserved throughout evolu- gen and thus a sex difference in estrogen availability. tion (36), an ERE may also be present in the rodent. The Gonadal steroid enhancement of neurite growth and of functionality of this ERE remains to be evaluated. neuronal survival has been demonstrated in vitro and in vivo It should be emphasized that NGF may not be the sole not only during development (ref. 27; for refs. see ref. 28), but ligand acting on neurons where estrogen receptors and in steroid receptor-containing regions (including the septum) p75NOGR colocalize. The low-affinity state of the NGF re- of the deafferented, axotomized, or steroid-deprived adult ceptor, which is the form recognized by monoclonal antibody central nervous system (CNS) as well (29-31). These findings IgG 192, has also been shown to bind with low affinity to suggest that the potential for steroid responsiveness may also other neurotrophins such as BDNF and NT-3 (7, 8, 37). Thus, extend into adulthood, but only after insult to the CNS. Such basal forebrain neurons coexpressing estrogen binding and a pattern is reminiscent of the injury-induced, up-regulation immunoreactivity for the low-affinity NGF receptor may of p75NGFR expression and the return of sensitivity to NGF reflect not only potential NGF, but BDNF and possibly NT-3 not only in the basal forebrain of the adult rat (3) but also in responsiveness as well. BDNF, like NGF, has been shown to the adult striatum (32), normally a target of NGF only during increase survival and acetylcholinesterase activity of septal development (33). cholinergic neurons in culture (38). Binding of the neurotro- Colocalization of the estrogen and NGF receptor systems phins by p75NGFR, however, does not alone induce cellular implies that their ligands may each act on the same neuron, responses. Neurotrophin signal transduction appears to re- perhaps synergistically. Such interactions may regulate (en- quire association of p75NGFR with members of the tyrosine hance or suppress) by autocrine and/or paracrine actions the kinase receptor family of protooncogene products-trk Downloaded by guest on September 23, 2021 Neurobiology: Toran-Allerand et al. Proc. Natl. Acad. Sci. USA 89 (1992) 4671

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FIG. 2. Colocalization ofestrogen binding sites with low-affinity NGF receptors and ChAT (mRNA and the encoded immunoreactive protein) in neurons ofthe developing postnatal days 10 and 12 (P10 and P12) female rat or adult female mouse basal forebrain. These results were obtained by combining (125I]MIE2 autoradiography (discrete concentration of silver grains in the emulsion underlying neuronal nuclei) with either immunocytochemistry (brown cytoplasmic reaction product) or nonisotopic (digoxigenin) in situ hybridization histochemistry (blue cytoplasmic hybridization signal). (A and B) Colocalization ofestrogen and NGF receptors in P12 neurons ofthe medial septum/diagonal band (A) and basal nucleus of Meynert (B). (C) NGF receptor-positive/estrogen receptor-positive P12 neuron adjacent to an NGF receptor-positive/estrogen receptor-negative neuron. (D) NGF receptor-positive/estrogen receptor-negative neuron (top left) in the same field as a NGF receptor-negative/ estrogen receptor-positive neuron (bottom right). (E) Colocalization of estrogen binding sites and NGF receptor mRNA in neurons of the horizontal nucleus of the diagonal band of the adult female mouse. (F and G) Colocalization of estrogen receptors with ChAT mRNA (F) or its encoded immunoreactive protein product (G) in the P10 rat. (A-D, x340; E-G, x270.)

[tropomyosin receptor kinase (39)] and the structurally re- elsewhere, recent evidence from our laboratory by double- lated genes trkB (40) and trkC (41). Thus, some forebrain label in situ hybridization indicates that the estrogen receptor regions known to express p75NGFR have also been shown to mRNA-containing neurons of the basal forebrain also colo- localize trk and trkB mRNA (40, 42, 43). As will be shown calize the mRNAs for trk and trkB (R.C.M., F.S., and Downloaded by guest on September 23, 2021 4672 Neurobiology: Toran-Allerand et al. Proc. Natl. Acad Sci. USA 89 (1992) C.D.T.-A., unpublished data). Colocalization of estrogen 4. Gnahn, H., Hefti, F., Heumann, R., Schwab, M. & Thoenen, H. receptor mRNA with trk, trkB, and mRNAs in the (1983) Dev. Brain Res. 9, 45-52. p75NGFR 5. Koh, S. & Loy, R. (1989) J. Neurosci. 9, 2999-3018. basal forebrain increases the importance of the findings 6. Schatteman, G. L., Gibbs, L., Lanahan, A. A., Claude, P. & presented here by suggesting that those estrogen target Bothwell, M. (1988) J. Neurosci. 8, 860-873. neurons that coexpress p75NGFR mRNA and protein have the 7. Rodriguez-Tdbar, A., Dechant, G. & Barde, Y.-A. (1990) Neuron 4, potential for expressing functional neurotrophin receptors 487-492. 8. Ernfors, P., Wetmore, C., Olson, L. & Persson, H. (1990) Neuron and thus for being regulated by the neurotrophins. Moreover, 5, 511-526. these findings also imply the potential for reciprocal regula- 9. Toran-Allerand, C. D., Miranda, R. C., Hochberg, R. B. & Mac- tion of the estrogen and neurotrophin receptor systems by Lusky, N. J. (1992) Brain Res. 576, 25-41. their ligands by documenting the substrate for estrogen 10. Toran-Allerand, C. D., Pfenninger, K. & Ellis, L. (1988) Dev. Brain Res. 41, 87-100. regulation of the neurotrophin receptors as well. 11. Mukku, V. R. & Stancel, G. M. (1985) J. Biol. Chem. 260, 9820- Whether or not all the estrogen receptor-containing neu- 9824. rons that express ChAT or p75NGFR represent the same 12. Nelson, K. G., Takahashi, T., Bossert, N. L., Walmer, D. K. & subset is unknown at this time. Recent studies have docu- McLachlan, J. A. (1991) Proc. Natl. Acad. Sci. USA 88, 21-25. 13. Zielinski, J., Yabuki, H., Pahuja, S., Larner, J. & Hochberg, R. mented that the majority of the NGF receptor immunoreac- (1986) Endocrinology 119, 130-139. tive neurons in the rodent (44) medial septum, diagonal band, 14. Brown, T. J., MacLusky, N. J., Toran-Allerand, C. D., Zielinski, and nucleus basalis are cholinergic, as determined by colo- J. E. & Hochberg, R. B. (1989) Endocrinology 24, 2074-2088. calization of markers such as ChAT. However, although 15. Chandler, C. E., Parsons, L. M. & Shooter, E. M. (1984) J. Biol. most of the estrogen receptor-containing cholinergic neurons Chem. 78, 6882-6889. 16. Baldino, F., Jr., & Lewis, M. E. (1989) in Methods in Neuroscience, may be targets of NGF or of the other neurotrophins, not all ed. Conn, P. M. (Academic, New York), Vol. 1, pp. 282-292. of the estrogen- and NGF-receptor-containing neurons are 17. Miranda, R. C. & Toran-Allerand, C. D. (1992) Cereb. Cortex 2, likely to be cholinergic. Preliminary observations (C.D.T.- 1-15. A., R.C.M., T.J.B., R.B.H., and N.J.M., unpublished data) 18. Ernfors, P., Halib66k, F., Ebendal, T., Shooter, E. M., Radeke, Neuron 983-996. some the neurons M. J., Misko, T. P. & Persson, H. (1988) 1, suggest that of 125I-estrogen-concentrating 19. Brice, A., Berrard, S., Raymoud, B., Ansieau, S., Coppola, T., of the medial septum/diagonal band may be -aminobutyric Weber, M. J. & Mallet, J. (1989) J. Neurosci. Res. 23, 266-273. acid (GABA)-ergic in that they also contain immunoreactive 20. Berrard, S., Brice, A. & Mallet, J. (1989) Brain Res. Bull. 22, parvalbumin, which has been shown to colocalize with 147-153. GABA in neurons of the medial septum/diagonal band, 21. Arnold, A. P. (1980) J. Comp. Neurol. 189, 421-436. 22. Luine, V. N. & McEwen, B. S. (1983) Neuroendocrinology 36, , and (45). Whether this neu- 475-482. ronal population is p75NGFR containing as well is not known. 23. Hefti, F. (1986) J. Neurosci. 6, 2158-2162. If interactions between neurotrophins and estrogen are of 24. Kromer, L. F. (1987) Science 235, 214-216. fundamental importance for development and survival of 25. Fischer, W., Bjdrklund, A., Chen, K. I. & Gage, F. H. (1991) J. target basal forebrain neurons in both sexes, then clinical Neurosci. 11, 1889-1906. 26. MacLusky, N. J., Clark, A. S., Naftolin, F. & Goldman-Rakic, conditions that are associated with gonadal steroid deficiency P. S. (1987) Steroids 50, 459-474. could contribute to the atrophy or death of these neurons in 27. Toran-Allerand, C. D. (1976) Brain Res. 106, 407-412. both sexes. Some studies suggest that gonadal steroid defi- 28. Toran-Allerand, C. D. (1991) Psychoneuroendocrinology 16, 7-24. ciency may have a profound impact on cognitive function (46, 29. Matsumoto, A. & Arai, Y. (1981) J. Comp. Neurol. 197, 197-206. 47). Similar considerations apply to the possible involvement 30. Nance, D. M., Shryne, J. & Gorski, R. A. (1975) Horm. Behav. 6, of steroids in neurodegenerative dis- 289-299. gonadal age-associated 31. Kurz, E. M., Sengelaub, D. R. & Arnold, A. P. (1986) Science 232, ease states, where a declining stimulus from circulating 395-398. estrogen or aromatizable androgen levels might make estro- 32. Gage, F. H., Batchelor, P., Chen, K. S., Chin, D., Higgins, G. A., gen target neurons in the brain less resistant to other age- or Koh, S., Deputy, S., Rosenberg, M. B., Fisher, W. & Bjdrklund, A. disease-related processes. In both sexes, therefore, the nat- (1989) Neuron 2, 177-184. ural decline in gonadal steroid levels in old age could con- 33. Mobley, W. C., Woo, J. E., Edwards, R. H., Riopelle, R. J., vital to Longo, F. M., Weskamp, G., Otten, U., Valletta, J. S. & Johnston, tribute to the loss of neuronal systems cognitive M. V. (1989) Neuron 3, 655-664. function, whether this occurs to the extreme observed in 34. Weisz, A. & Rosales, R. (1990) Nucleic Acids Res. 18, 5097-5106. Alzheimer disease or follows the less traumatic path associ- 35. Sehgal, A., Patil, N. & Chao, M. (1988) Mol. Cell. Biol. 8, 3160- ated with normal aging. 3167. 36. Radeke, M. J., Misko, T. P., Hsu, C., Herzenberg, L. A. & We thank Drs. Patricia Goldman-Rakic, Lloyd A. Greene, Carol Shooter, E. M. (1987) Nature (London) 325, 593-597. A. Mason, and Anne-Judith Silverman for their helpful comments 37. Barde, Y.-A. (1989) Neuron 2, 1525-1534. and E. M. Johnson, Jr., for the gift of monoclonal antibody IgG 192. 38. Alderson, R. F., Alterman, A. L., Barde, Y.-A. & Lindsay, R. M. This work (1990) Neuron 5, 297-306. We also thank Harriet Ayers for typing the manuscript. 39. Martin-Zanca, D., Barbacid, M. & Parada, L. F. (1990) Genes Dev. was supported by National Institutes of Health Grants HD-08364 and 4, 683-694. AG-08099 (C.D.T.-A.) and CA-3799 (R.B.H.); National Science 40. Klein, R., Parada, L. F., Coulier, F. & Barbacid, M. (1989) EMBO Foundation Grant BNS 87-00400 (C.D.T.-A.); grants from the Amer- J. 8, 3701-3709. ican Health Assistance Foundation (C.D.T.-A.), the Eppley Foun- 41. Lamballe, F., Klein, R. & Barbacid, M. (1991) Cell 66, 967-979. dation for Research (C.D.T.-A.), and MRC Canada (N.J.M.); and 42. Kaplan, D. R., Martin-Zanca, D. & Parada, L. P. (1991) Nature Alcohol, Drug Abuse, Mental Health Administration Research Sci- (London) 350, 158-160. entist Award MH-00192 (C.D.T.-A.). 43. Klein, R., Jing, S., Nanduri, V., O'Rourke, E. & Barbacid, M. (1991) Cell 65, 189-197. 1. Bartus, R. T., Dean, R. L., Beer, B. & Lippa, A. S. (1982) Science 44. Batchelor, P. E., Armstrong, D. M., Blaker, S. N. & Gage, F. H. 217, 408-417. (1989) J. Comp. Neurol. 284, 187-204. 2. Whitehouse, P. J., Price, D. L., Strubble, R. G., Clark, A. W., 45. Freund, T. F. (1989) Brain Res. 478, 375-381. Coyle, J. T. & DeLong, M. R. (1982) Science 215, 1237-1239. 46. Hier, D. & Crowley, W., Jr. (1982) N. Engl. J. Med. 306,1202-1227. 3. Higgins, G. A., Koh, S., Chen, K. S. & Gage, F. H. (1989) Neuron 47. Gabriefli, J. D. E., Corkin, S. & Crawford, J. D. (1985) Ann. N. Y. 3, 247-256. Acad. Sci. 444, 457-461. Downloaded by guest on September 23, 2021