Visualization of Growth Factor in Rat Forebrain

SYNAPSE 2:212-218 (1988)

Receptor Sites

KkMI QUIRION, DALIA ARAUJO, N.P.V. NAIR, AND JEAN-GUY CHABOT Douglas Hospital Research Centre and Department of Psychiatry, Faculty of Medicine, McGill University,

Verdun, Quebec, Canada H4H 1R3

KEY WORDS Epidermal growth factor, Insulin-like growth factor, Somatomedin C, Ontogeny

ABSTRACT It is now known that various growth factors may also act in the central nervous system. Among them, it has recently been shown that epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) may possess trophic effects in the mamma- lian brain. We report here on the respective autoradiographic distribution of [ 1251]EGF and [1251]IGF-I receptor binding sites in the rat brain, both during ontogeny and in adulthood. It appears that [1251]EGF sites are mostly found in the rat forebrain during brain development. On the other hand, [125]IGF-I sites are more widely distributed both during ontogeny and in adulthood. These results reveal the plasticity of the expression of EGF and IGF-I receptor sites in the mammalian brain. This could be relevant for the respective role of these two growth factors in the development and maintenance of neuronal function.

INTRODUCTION The last two decades have witnessed a veritable explo-

sion in the discovery of polypeptide growth factors (GF). Up to date, GFs have been mostly classified either by their tissues of origin or their site of action. For exam- ple, the term “epidermal growth factor” (EGF) has been given to a trophic factor isolated from the mouse sub- mandibular gland that promotes the proliferation and differentiation of epidermal tissues, but recent data have shown that EGF may also have trophic effects in various other tissues (for reviews, see Carpenter and Cohen, 1979; Daughaday and Heath, 1984; Gospodarowicz, 1983; Hollenberg, 1979; Walker, 1982). Moreover, it has re- cently been shown that many GFs can act in brain tissues and can be responsible for the growth, develop- ment, and maintenance of various neuronal functions (Crutcher, 1986; Daughaday and Heath, 1984; Hefti and Weiner, 1986; Herschmann, 1986; Levi-Montalcini and Calissano, 1986; Thoenen and Edgar, 1985; Walker, 1982). Among them, nerve growth factor (NGF) (Fischer et al., 1987; Hefti and Weiner, 1986; Herschmann, 19861, EGF (Herschmann, 1986; Morrison et al., 1987), acidic and basic fibroblast growth factor (Herschmann, 1986; Pruss et al., 1982; Walicke et al., 1986), platelet-derived growth factor (Herschmann, 19861, and insulin-like growth factors (IGF) -I and -11 (Herschmann, 1986) have been recognized as putative neuronal and glial GFs. However, except for NGF, relatively little is currently known on the exact roles and functions of these factors in the brain. We report here on the localization of spe- cific receptor binding sites for [1251]EGF and [1251]IGF-I in the rat brain during ontogeny and in adulthood using in vitro autoradiography.

EGF was studied since it has been shown that EGF- like immunoreactivity is present in the central nervous

@ 1988 ALAN R. LISS, INC.

system (CNS) (Fallon et al., 1984; Hirata et al., 1982; Lakshmanan et al., 1986) and because it has been sug- gested that EGF may act as a neurotrophic and/or neu- romodulator substance in the brain. It is known that EGF acts as a mitogen on non-neuronal cells of brain origin and stimulates the activity of various enzymes in glial cells (Guentert-Lauber and Honegger, 1983; Honeg- ger and Guentert-Lauber, 1983; Leutz and Schachner, 1981; Simpson et al., 1982); it inhibits acetylcholine re- lease from cholinergic nerves (Takayanagi, 1980); and it induces tyrosine hydroxylase in rat sympathetic supe- rior cervical ganglion (Herschman et al., 1983) and pro- motes neurite outgrowth in rat postnatal neurons (Morrison et al., 1987). Moreover, the existence of spe- cific EGF receptor sites has been shown in the mouse brain (Adamson and Meek, 1984; Adamson and War- shaw, 1982; Adamson et al., 1981; Nexo et al., 1980), although their precise localization remains to be es- tablished.

Similarly, there is evidence indicating that IGFs or somatomedins act as GFs and/or neuromodulators in the CNS. It has been demonstrated that brain cells in cul- ture are capable of synthesizing IGF-like materials (Bin- oux et al., 1981; Birch et al., 1984), and a variant form of IGF-I has been isolated from human fetal brain tis- sues (Sara et al., 1986). Moreover, IGF-I reportedly re- duces growth hormone secretion, body weight, and food intake when directly administered into the brain (Tan- nenbaum et al., 1983) and increases somatostatin re- lease from hypothalamic cell cultures (Berelowitz et al.,

Received August 15, 1987; accepted October 15, 1987.

This manuscript is dedicated to the memory of Thomas L. O’Donohur

GROWTHFACTORRECEPTORS 213

Fig. 1. Photomicrographs of the autoradiographic distribution of 200 pM ['251]EGF/100 nM EGF (E) to evaluate the s $ ecificity of bind- ['251]EGF binding sites in rat brain at the level of the striatum in ing. Note the apparent disappearance of selective [' 'IIEGF labeling animals at 1 day before birth (A), 3 days after birth (B,C,), and in adult brain tissues. Abbreviations used: C, cortex; CC, corpus cal- adulthood (3 months) (D,E). Coronal brain sections were incubated, as losum; CP, caudate-putamen; LS, lateral septum; NA, nucleus described in the text, in the presence of 200 pM ['251]EGF (A-D) and accumbens.

1981a,b). IGFs also act as mitogens in cultured fetal and neonatal rat brain astroglial and oligodendroglial cells

MATERIALS AND METHODS Materials

(Han et al., 1987; McMoGis et al., 1986; Shemer et al., 1987). Finally, IGF receptors have been identified in various fetal, neonatal, and adult mammalian brain regions such as the cerebral cortex, olfactory bulb, hip- pocampus, medulla oblongata, and amygdala (Gammel- toft et al., 1985; Goodyer et al., 1984; Han et al., 1987; Hill et al., 1986a; Lenoir and Honegger, 1983; McMorris et al., 1986; Sara et al., 1982, 1983; Shemer et al., 1987; Van Schravendijk et al., 1986). Using in vitro autora- diography, a recent report has identified the presence of IGF-I receptor binding sites in the external palisade of the median eminence (Bohannon et al., 1986). Thus, the aim of this studv was to characterize the comparative

(3-[ 1251] iodotyrosyl) [Thr5'] IGF-I (2,000 Ci/mmol) was purchased from Amersham (Arlington Heights, L). Non- radioactive IGF-I, EGF, bovine serum albumin (BSA), and [1251]EGF (1,040 Ci/mmol) were obtained from ICN Biomedicals Canada Ltd. (Montreal). The following chemicals were obtained from Sigma Chemical Co. (St. Louis): N-2 hydroxylethylpiperazine-N'-2-ethanesul- fonic acid (HEPES), bacitracin, aprotinin, and N-ethyl- maleimide (NEM). All other reagents were of analytical grade and were obtained locally. Adult Sprague-Dawley rats (200-250 g) were bought from Charles River Labo- ratories (St. Constant, Quebec).

In vitro autoradiography For receptor autoradiography, slide-mounted sections

of fetal, neonatal, and adult rat brains were prepared as described previously (Quirion et al., 1981). Briefly, rats were decapitated, and their entire brains were rapidly

distribution of siecific receptor sites for EGF and IGF-I in the rat brain during brain development. The results clearly show the differential localization, expression, and plasticity of ['251]EGF and [1251]IGF-I binding sites in the rat brain during postnatal ontogeny and adult- hood.

214 R. QUIRION ET AL

Fig. 2. Photomicrographs of the autoradiographic distribution of C, cortex; CC, corpus callosum; CP, caudate-putamen; DG, dentate ['251]IGF-I binding sites in rat brain of 1-day-old (A,B,E) and S-month- gyrus; EN, endopiriform nucleus; GP, globus pallidus; HI, hippocam- old (C,D,F) animals. Coronal sections were incubated, as described in pus; HY, hypothalamus; INF, infundibular stem; LS, lateral septum; the text, in the presence of 50 pM 1'25111GF-I (A-D) and 50 pM L'25111GF- NA, nucleus accumbens; OR, oriens layer of the hippocampus; PMCO, Ill00 nM IGF-I (E,F) to elevate the specificity of binding. Note the posteromedial cortical amygdaloid nucleus; PO, primary olfactory cor- differential distribution of labeling between neonatal and adult brain tex; PSC, preoptic suprachiasmatic nucleus; PV, posteroventral tha- tissues. Abbreviations used: BM, basomedial nucleus of the amygdala; lamic nuclear group.

frozen in 2-methyl butane at -4O"C, mounted on cryo- stat chucks, and cut into 20-~m-thick sections at -14°C. Sections were thaw-mounted on precleaned gelatin- coated slides and dessicated overnight a t 4°C prior to storing at -80°C until used.

For [1251]EGF binding experiments, frozen sections were allowed to thaw for 1 hour on ice and then prein- cubated in Tris (25 mM) buffer (pH 7.4) containing BSA (0.1%) for 30 minutes, at room temperature (22°C). In- cubation with [1251]EGF (200 pM) was carried out in the same buffer containing MgCl2 (10 mM) at room temper- ature for 2 hours. Nonspecific binding was defined in the presence of an excess of unlabeled EGF (100 nM). At the end of the incubation period, slides were washed four times for 2 minutes each, in ice-cold incubation buffer. Finally, slides were rinsed in cold deionised water, rapidly dried, and juxtaposed tightly against tritium- sensitive film (Hyperfilm, Amersham). The films were developed after 2 weeks and analyzed as described pre- viously (Quirion et al., 1981).

For [125IJIGF-I binding studies, sections were incu- bated for 2 hours at room temperature in HEPES (10 mM) buffer containing 0.5% BSA, 0.0125% NEM, 0.025% bacitracin, 100 KIU/ml of aprotinin (Bohannon et al., 1986), and 50 pM [1251]IGF-I. Nonspecific binding was defined in the presence of an excess of unlabeled IGF-I (100 nMf. At the end of the incubation period, slides were washed three times for 1 minute each in ice-cold Tris (50 mM) buffer. Finally, slides were rinsed in cold deionised water, rapidly dried, and exposed against tri- tium-sensitive film for 12-24 hours before development.

RESULTS Both [lZ5I]EGF (Fig. 1) and [1251]IGF-I (Fig. 2) binding

sites are discretely distributed in the rat brain. More- over the localization of these two populations of sites changed during brain development (Figs. 1, 2; Table I). However, only a single nonsaturating concentration of ligands has been used in our binding studies. Thus, it is not possible to determine if modifications in the appar-

GROWTH FACTOR RECEPTORS

TABLE I. Distribution of receptor sites for [IZ5I]EGF and [1251jIGF-I in rat brain tissues during postnatal ontogeny‘

2 15

Region First week postnatally Adult (3 months)

[1251]EGF [’251]IGF-I [1251]EGF [1251]IGF-I

Frontal cortex ++ ++ - ++ Parietal cortex ++ ++ Striatum ++ + Hippocampus ND +++ - +++ Hypothalamus + + ND ++ Thalamus ND - ND + + +

++ - - -

’At this time, we are not attempting to determine precisely the discrete distribution of binding sites in a given structure (e.g., various hypothalamic nuclei); all areas are then taken as a whole. ND, not determined.

ent localization of sites is related to changes in the affinity (Kd) and/or maximal capacity (Bmax) of these receptors. Further studies will be necessary to clarify A few years ago, interest in the role of GFs in brain this point. tissues was fairly limited, possibly because of the as-

In the late prenatal and early postnatal periods, sumption that brain plasticity was restricted after mat- [1251]EGF binding sites are mostly concentrated in cor- uration. It is now known that neuronal plasticity can be tical areas in the rat forebrain. (Figs. 1A-C). Lower observed even at later stages in life (Fischer et al., 1987). amounts of sites are seen in subcortical regions such as This has stimulated research on the potential role of the caudate-putamen, olfactory tubercule, and septum various GFs in the development and normal functioning (Fig. 1A-C). However, only very low amounts of specific of the CNS (Crutcher, 1986). [I2 IIEGF binding sites are found in all these areas in However, relatively little is currently known on the adult forebrain (Fig. 1D; Table I). Very little difference precise roles of GFs in brain. Most recent research has in optical density was observed between sections incu- focused on NGF, probably since this factor appears to be bated in the presence of [1251]EGF alone (Fig. 1D) or in closely associated with the maturation and maintenance combination with 100 nM nonradioactive EGF (Fig. 1E). of certain cholinergic neuronal projections that may be This clearly demonstrates the differential expression of involved in the etiology of Alzheimer’s disease (Hefti [1251]EGF binding sites at various stages of development and Weiner, 1986). For example, it has been shown that in the rat forebrain. NGF receptor sites are present in areas associated with

1251 IGF-I binding sites are more widely distributed the septohippocampal and the nucleus basalis of Mey- thlan $“I]EGF sites in the rat forebrain (Fig. 2; Table nert-cortical cholinergic projections (Hefti et al., 1986; I). High amounts of [1251]IGF-I sites are found in various Raivich and Kreutzberg, 1987; Richardson et al., 1986; brain regions during postnatal ontogeny (Fig. 2A,B) and Springer et al., 1987). It appears that NGF is most likely in adulthood (Fig. 2C,D). Coincubation with excess non- synthesized in the terminal areas (Korsching et al., 1985; radioactive IGF-I (100 nM) resulted in a large diminu- Large et al., 1986., 1986; Rennert and Heinrich, 1986; tion of the labeling and demonstrated the specificity of Whittemore et al., 1986) where it binds to specific recep- the binding of ligand (Fig. 2E, F). One day after birth, tors; this complex is then retrogradely transported to high levels of [125I]IGF-I binding sites are present in the cell bodies where it induces its effects (Schwab et al., superficial cortical layers (Fig. 2A,B), nucleus accum- 1979; Seiler and Schwab, 1984). Among these effects is bens (Fig. 2A), and hippocampus (Fig. 2B). Lower the enhancement of choline acetyltransferase activity, amounts are seen in most thalamic and hypothalamic the key enzyme responsible for the synthesis of acetyl- areas (Fig. 2B). In the adult rat forebrain,, high amounts choline (Gahwiler et al., 1987; Haroutunian et al., 1986; of specific [1251]IGF-I binding sites are found in the olfac- Martinez et al., 1985). Moreover, treatment with NGF tory bulb (not shown), endopiriform nucleus (Fig. 2C), decreases the effects of chemical and/or mechanical le- primary olfactory cortex (Fig. 2C), certain layers of the sions of cholinergic neurons (Hefti, 1986; Kromer, 1987), hippocampus (Fig. 2D), median eminence (not shown), facilitates implantation of transplanted cholinergic neu- and amygdaloid nuclei (Fig. 2D). Moderate levels of sites rons in appropiate regions (Toniolo et al., 19851, and are present in the septum (Fig. 2C), preoptic suprachias- partly restores learning ability in spatial memory tasks matic nucleus of the hypothalamus (Fig. 2C), striatum in septa1 lesioned animals (will and Hefti, 1985). Thus, (Fig. ZC), and postero-ventral nuclear group of the thal- there is great interest in the potential uses of NGF in amus (Fig. 2D). Lower amounts are seen in other areas the treatment of certain aspects of Alzheimer’s disease. such as the globus pallidus (Fig. 2C) and various hypo- Much less is known on the roles of other GFs in brain thalamic nuclei (Fig. 2C, D). In the cortex, the highest tissues. We have shown here that two of these GFs, EGF degree of labeling is observed in superficial and deep and IGF-Usomatomedin C, interact with specific and layers, while only low levels are seen in midlayers (Fig. selective receptor sites in the rat brain. In the forebrain, 2C,D). Thus, the distribution of [1251]IGF-I binding sites the existence of [ lZ5I]EGF binding sites is especially undergoes certain modifications during brain matura- evident in the early postnatal period, when high tion (Table I). amounts of sites are present in cortical areas. Interest-

DISCUSSION

2 16 R. QUIRION ET AL.

ingly, it seems that the expression of [1251]EGF binding sites is markedly reduced in the adult brain cortex, which suggests that EGF in the forebrain could be mostly involved in the early stages of development. However, the presence and expression of EGF receptor sites during ontogeny in other brain regions would have to be studied in order to determine if a similar pattern is seen throughout the CNS.

The presence of EGF-like immunoreactivity in the fore- and midbrain structures has recently been re- ported, both in neonatal and adult rat brain (Fallon et al., 1984), although other groups have not been able to confrm this finding (Hirata and Orth, 1979; Poulsen et al., 1986; Probstemeier and Schachner, 1986). This clearly demonstrates the need for further studies on the presence of EGF-like material in mammalian brain. On the other hand, EGF receptor binding sites have been identified in mouse embyronic brain (Adamson et al., 1981; Nexo et al., 1980), rodent astrocytes, and oligoden- drocytes (Leutz and Schachner, 1982; Simpson et al., 1982), in several glial cell lines derived from human brain (Fabricant et al., 1977), and in cells obtained from human brain tumors (Libermann et al., 1984), and we have reported here on the presence of specific [1251]EGF binding sites in neonatal rat forebrain. This suggests some relevance for EGF in the CNS, and various studies have already shown that EGF possesses biological ef- fects in nervous tissues. For example, EGF has been shown to induce enzymatic activities in glial cells and to act as a mitogen in neuronal and non-neuronal cells (Guentert-Lauber and Honegger, 1983; Honegger and Guentert-Lauber, 1983; Leutz and Schachner, 1981; Simpson et al., 1982), to stimulate growth of cultured neurons from neonatal rat brain (Morrison et al., 1987), to inhibit acetylcholine release from cholinergic nerves (Takayanagi, 1980), and to activate tyrosine hydroxy- lase in rat sympathetic superior cervical ganglion (Herschman et al., 1983). Thus, further studies on the characterization and roles of EGF-like factors in brain tissues are certainly warranted.

Forebrain [ 1251]IGF-Usomatomedin C receptor binding sites are differentially distributed and expressed in rat forebrain. Early postnatally, the highest number of sites is concentrated in cortical and hippocampal areas. How- ever, in contrast ot [1251]EGF binding, high levels of specific [ 1251]IGF-I binding sites are present in various cortical and subcortical regions in the adult rat fore- brain. Thus, [ 1251]IGF-I binding sites are apparently expressed during brain development as well as later during adulthood. This may suggest that IGF-I-like GF may act as a trophic substance as well as a neuromodu- lator of brain functions. Already, it has been demon- strated that IGF-I and IGF-I1 are mitogens in cultured fetal and neontal rat brain cells (Han et al., 1987; Mc- Morris et al., 1986; Shemer et al., 1987). IGF-I has also been shown to reduce growth hormone secretion, body weight, and food intake when directly administered into brain (Tannenbaum et al., 1983 and to increase somato- statin release in hypothalamic cell cultures (Berelowitz et al., 1981a,b).

The precise nature of [1251]IGF-I binding sites charac- terized in this study remains to be determined using related and unrelated GFs. However, there is much evi- dence demonstrating that IGF-I and IGF-I1 receptor sites are clearly distinguishable from insulin receptors in the

brain and in peripheral tissues (Bohannon et al., 1986; Czech, 1982; Goodyer et al., 1984; Gammeltoft et al., 1985; Han et al., 1987; Hill et al., 1986a,b; Sara et al., 1982; Shemer et al., 1987; Van Schravendijk et al., 1986). Moreover, recent data, including this study, suggest that IGF-I and IGF-I1 are acting through their respective population of receptors (Bohannon et al., 1986; Goodyer et al., 1984; Gammeltoft et al., 1985; Han et al., 1987; Hill et al., 1986a; Lenoir and Honnegger, 1983; Mc- Morris et al., 1986; Sara et al., 1982; Shemer et al., 1987; Van Schravendijk et al., 1986). For example, Bohannon et al. (1986) have reported that [1251]IGF-I sites are con- centrated in the rat median eminence, whereas [1251] IGF-I1 sites are probably not found in this area (Gam- meltoft et al., 1985; Hill et al., 1986a; Sara et al., 1982). Interestingly, the existence of a neuronal subtype of [1251]IGF-I receptor has recently been suggested (Bur- gess et al., 1987). Further studies will be necessary to determine if [1251]IGF-I binds to this receptor subtype under our incubation conditions.

In summary, [1251]EGF and [1251]IGF-I receptor bin- dign sites are differentially distributed and expressed in the rat forebrain during postnatal ontogeny and in the mature brain. This suggests that these two GFs proba- bly possess different trophic effects on selected popula- tions of neuronal and non-neuronal cells in brain. Moreover, the presence of high amounts of [lZ5I]IGF-I binding sites in the adult forebrain indicates that this GF may be involved in the maintenance of brain plastic- ity in mature tissues as well as in the neuromodulation of certain brain functions.

ACKNOWLEDGMENTS This research was supported by the Medical Research

Council of Canada (MA-8580) and the Aging Funds of the Douglas Hospital Research Center. R. Quirion is a “Chercheur-Boursier” of the “Fonds de la Recherche en Sante du Qubbec (FRSQ).” D. Araujo is currently holder of a postdoctoral fellowship from the FRSQ.

REFERENCES Adamson, E.D., and Meek, J. (1984) The ontogeny of epidermal growth

factor receptors during mouse development. Dev. Biol., 103:62-70. Adamson, E.D., and Warshaw, J.B. (1982) Down-regulation of epider-

mal growth factor receptors in mouse embryos. Dev. Biol., 90:430- 434.

Adamson, E.D., Deller, M.J., and Warshaw, J.B. (1981) Functional EGF receptors are present on mouse embryo tissues. Nature, 291:656- 659.

Berelowitz, M., Firestone, S.L., and Frohman, L.A. (1981a) Effects of growth hormone excess and deficiency on hypothalamic somatostatin content and release and on tissue somatostatin distribution. Endocri- nology, 109:714-720.

Berelowitz, M., Szabo, M., Frohman, L.A., Firestone, S., Chu, L., and Hintz, R.L. (1981b) Somatomedin-C mediates growth hormone nega- tive feed-back by effects on both the hypothalamus and the pituatary. Science, 212:1279-1281.

Binoux, M., Hossenlopp, C., Lassare, C., and Hardouin, N. (1981) Pro- duction of insulin-like growth factors and their carrier by rat pitu- itary gland and brain explants in culture. FEBS Lett., 124:178-184.

Birch, N.P., Christie, D.L., and Renwick, A.G.C. (1984) Proinsulin-like material in mouse foetal brain cell culture. FEBS Lett., 169:299- 302.

Bohannon, N.J., Figlewicz, D.P., Corp, E.S., Wilcox, B.J., Porte, D. Jr., and Baskin, D.G. (1986) Identification of binding sites for an insulin- like growth factor (IGF-I) in the median eminence of the rat brain by quantitative autoradiography. Endocrinology, 119:943-945.

Burgess, S.K., Jacobs, S., Cuatrecasas, P., and Sahyoun, N. (1987) Characterization of a neuronal subtype of insulin-like growth factor factor I receptor. J. Biol. Chem., 262:1618-1622.

GROWTH FACTOR RECEPTORS 217

Carpenter, G., and Cohen, S. (1979) Epidermal growth factor. Annu. Rev. Biochem., 48193-216.

Crutcher, K.A. (1986) The role of growth factors in neuronal develop- ment and plasticity. CRC Crit. Rev. Clin. Neurobiol., 2:297-333.

Czech, M.P. (1982) Structural and functional homologies in the recep- tors for insulin and the insulin-like growth factors. Cell, 312-10.

Daughaday, W.D., and Heath, E. (1984) Physiological and possible clinical significance of epidermal and nerve growth factors. Clin. Endocrinol. Metab., 13:207-226.

Fabricant, R.N., DeLarco, J.E., and Todaro, G. (1977) Nerve growth factor receptors on human melanoma cells in culture. Proc. Natl. Acad. Sci. USA, 74:565-569.

Fallon, J.H., Seroogy, K.B., Loughlin, S.E., Morrison, R.S., Bradshaw, R.A., Knauer D.J., and Cunningham D.D. (1984) Epidermal growth factor immunoreactive material in the central nervous system: Lo- cation and development. Science, 224:1107-1109.

Fischer, W., Wictorin, K., Bjorklund, A., Williams, L.R, Varon, S., and Gage, F.H. (1987) Amelioration of cholinergic neuron atrophy and spatial memory impairment in aged rats by nerve growth factor. Nature, 329:65-68.

Gahwiler, B.H., Enz, A., and Hefti F. (1987) Nerve growth factor pro- motes development of the rat septo-hippocampal cholinergic projec- tion in uitro. Neurosci. Lett., 75:6-10.

Gammeltoft, S, Haselbacher, G.K., Humbel, R.E., Fehlmann, M., and Van Obberghen, E. (1985) Two types of receptor for insulin-like growth factors in mammalian brain. EMBO J., 4:3407-3412.

Goodyer, C., De Stephano, L. Lai, W.H., Guyda, H.J., and Posner, B.I. (1984) Characterization of insulin-like growth factor receptors in rat anterior pituitary, hypothalamus, and brain. Endocrinology, 114: 1187-1195.

Gospodarowicz, D. (1983) Growth factors and their action in uiuo and in uitro. J. Pathol., 141:201-233.

Guenter-Lauber, B., and Honegger, P. (1983) Epidermal growth factor (EGF) stimulation of cultured brain cells. 11. Increased production of extracellular soluble proteins. Dev. Brain Res., 11:253-260.

Han, V.K.M., Lauder, J.M., and D’Ercole, A.J. (1987) Characterization of somatomedidinsulin-like growth factor receptors and correlation with biologic action in cultured neonatal rat astroglial cells. J. Neu- rosci., 7:501-511.

Haroutunian, V., Kanof, P.D., and Davis, K.L. (1986) Partial reversal of lesion-induced deficits in cortical cholinergic markers by nerve growth factor. Brain Res., 386:397-399.

Hefti, F.J. (1986) Nerve growth factor (NGF) promotes survival of septa1 cholinergic neurons after fimbrial transsections. J. Neurosci., 6:2155-2162.

Hefti, F., Hartikka, J., Salvatierrra, A., Weiner, W.J., and Mash, D.C. (1986) Localization of nerve growth factor receptors in cholinergic neurons of the human basal forebrain. Neurosci. Lett., 69:37-41.

Hefti, F., and Weiner, W.J. (1986) Nerve growth factor and Alzheimer’s disease. Ann. Neurol., 20:275-281.

Herschmann, H.R. (1986) Polypeptide growth factors and the CNS. TINBS, 9:53-57.

Herschman, H.R., Goodman, R., Chandler, C., Simpson, D., Cawley, D., Cole, R., and de Vellis J. (1983) Is epidermal growth factor a modulator of nervous system function? In: Nervous System Regen- eration. B. Haber, J.R. Perez, A. Hashim, and A.M. Giuffrida, eds. New York, Alan R. Liss, Inc., New York, pp. 79-94.Hil1, J.M., Les- niak, M.A., and Pert, C.B. (1986a) IGF-11, II-I receptors and THY 1.1 antigen have the same pattern of distribution in rat brain. Proc. SOC. Neurosci., 12:1387.

Hill, J.M., Lesniak, M.A., Pert, C.B., and Roth, J. (1986b) Autoradio- graphic localization of insulin receptors in rat brain: Prominence in olfactory and limbic areas. Neuroscience, 17:1127-1138.

Hirata, Y., and Orth, D.N. (1979) Epidermal growth factor (urogas- trone) in human tissues. J. Clin. Endocrinol. Metab., 48:667-672.

Hirata, Y., Uchihashi, M., Nakajima, H., Fujita, T., and Matsukura, S. (1982) Presence of epidermal growth factor in human cerebrospinal fluid. J. Clin. Endocrinol. Metab., 55:1174-1177.

Hollenberg, M.D. (1979) Epidermal growth factor-urogastrone. a poly- peptide acquiring hormonal status. Vitamin. Horm., 37:69- 110.

Honegger, P., and Guentert-Lauber, B. (1983) Epidermal growth factor (EGF) stimulation of cultured brain cells. I. Enhancement of the developmental increase in glial enzymatic activity. Dev. Brain Res., 11:245-251.

Korsching, S., Auberger, G., Heumann, R. Scott, J., and Thoenen, H. (1985) Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation. EMBO J., 4:1389-1393.

Kromer, L.F. (1987) Nerve growth factor treatment after brain injury prevents neuronal death. Science, 235:214-216.

Lakshmanan, J., Weischel, M.E. Jr., and Fisher, D.A. (1986) Epidermal

growth factor in synaptosomal fractions of mouse cerebral cortex. J. Neurochem., 461081-1085.

Large, T.H., Bodary, S.C., Clegg, D.O., Weskamp, G., Otten, U., and Reichardt, L.F. (1986) Nerve growth factor gene expression in the developing rat brain. Science, 234:352-355.

Lenoir, D., and Honenger, P. (1983) Insulin-like mowth factor I (IGF I) stimulates DNA &;thesis in fetal rat brain-cultures. Dev. Brain Res.. 7:205-213.

Leutz,’ A., and Ghachner, M. (1981) Epidermal growth factor stimu- lates DNA synthesis of astrocytes in primary cerebellar cultures. Cell Tissue Res., 220~393-400.

Leutz, A., and Schachner, M. (1982) Cell type-specificity of epidermal growth factor (EGF) binding in primary cultures of early postnatal mouse cerebellum. Neurosci. Lett., 30:179-182.

Levi-Montalcini, R., and Calissano, P. (1986) Nerve growth factor as a paradigm for other polypeptide growth factor. TINS., 9:473-477.

Libermann, T.A., Razon, N., Bartal, A.D., Yarden, Y., Schiessinger, J., and Soreq, H. (1984) Expression of epidermal growth factor receptors in human brain tumors. Cancer Res., 44:753-760.

Martinez, H.J., Dreyfus, C.F., Jonakeit, M., and Black, I.B. (1985) Nerve growth factor promotes cholinergic development in brain striatal cultures. Proc. Natl. Acad. Sci USA, 82:7777-7781.

McMorris, F.A., Smith, T.M., DeSalvo, S., and Furlanetto R.W. (1986) IGF-YSomatomedin-C: A potent inducer of oligodendrocyte develop- ment. Proc. Natl. Acad. Sci. USA, 83:822-826.

Morrison, R.S., Kornblum, H.I., Leslie, F.M. and Bradshaw, R.A. (1987) Trophic stimulation of cultured neurons from neonatal rat brain by epidermal growth factor. Science, 238:72-75.

Nexo, E., Hollenberg, M.D., Figueroa, A,, and Pratt, R.M. (1980) Detec- tion of epidermal growth factor-urogastrone and its receptor during fetal mouse development. Proc. Natl. Acad. Sci. USA, 77:2782-2785.

Poulsen, S.S., Nexo, F., Skov Olsen, P., Hess, J., and Kirkegaard, P. (1986) Immunohistcchemical localization of epidermal growth factor in rat and man. Histochemistry, 85:389-394.

Probstmeier, R., and Schachner, M. (1986) Epidermal growth factor is not detectable in developing and adult rodent brain by a sensitive double-site enzyme immunoassay. Neurosci. Lett., 63:290-294.

Pruss, R.M., Bartlett, P.F., Gavriloc, J., Lisak, P.P., and Rattray, S. (1982) Mitogens for glial cells: A comparison of the response of cul- tured astrocytes, oligodendrocytes and Schwann cells. Dev. Brain Res., 2:19-35.

Quirion, R., Hammer, R.P. Jr , Herkenham, M., and Pert, C.B. (1981) Phenylcyclidine (angel dustliu “opiate” receptor: Visualization bu tritium sensitive film. Proc. Natl. Acad. Sci. USA, 78:5881-5885.

Raivich, G., and Kreutzberg, G.W. (1987) The localization and distri- bution of high affinity &nerve growth factor binding sites in the central nerous system of the adult rat. A light microscopic autoradio- graphic study using [’251]p-nerve growth factor. Neuroscience, 20:23- 36.

Rennert, P.D., and Heinrich, G. (1986) Nerve growth factor mRNA in brain: Localization by in situ hybridization. Biochem. Biophys. Res. Commun., 138:813-818.

Richardson, P.M., Verge Issa, V.M.K., and Riopelle, R.J. (1986) Distri- bution of neuronal receptors for nerve growth factor in the rat. J. Neurosci., 6:2312-2321.

Sara, V.R., Carlsson-Skwirut, C., Anderson, C., Hall, E., Sjogren, B., Holmgren, A., and Jornvall, H. (1986) Characterization of somato- medins from human fetal brain: Identification of a variant form of insulin-like growth factor. I. Proc. Natl. Acad. Sci. USA, 83:4904- 4907.

Sara, V.R., Hall, K., Von Holtz, H., Misaki, M., Pryklund, L., Christen- sen, N., and Wetterberg, L. (1983) Ontogenesis of somatomedin and insulin receptors in the human fetus. J. Clin Invest., 71:1084-1094.

Sara, V.R., Hall, K., Von Holtz, H., Humbel, R., Sjorgen, B., and Wetterberg, L. (1982) Evidence for the presence of specific receptors for insulin-like growth factors I CIGf-I) and I1 (IGF-11) and insulin throughout the adult human brain. Neurosci. Lett., 34:39-44.

Schwab, M., Otten, U., Agid, Y., and Thoenen, H. (1979) Nerve growth factor (NGF) in the rat C N S Absence of specific retrograde axonal transport and tyrosine hydroxylase induction in locus coeruleus and substantia nigra. Brain Res., 168:473-483.

Seiler, M., and Schwab, M.E. (1984) Specific retrograde transport of nerve growth factor (NGF) from neocortex to nucleus basalis in the rat. Brain Res., 300:33-36.

Shemer. J.. Raizada. M.K.. Masters. B.A.. Ota. A. and LeRoith. D. I I ,

(1987j Insulin-like ’growth factor I’ receptors in neuronal and glial cells. J. Biol. Chem., 262:7693-7699.

Simpson, D.L., Morrison, R., de Vellis, J., and Herschman, H.R. (1982) Epidermal growth factor binding and mitogenic activity on purified populations of cells from the central nervous system. J. Neurosci., 8:453-462.

218 R. QUIRION ET AL.

Springer, J.E., Koh, S., Tayrien, M.W., and Loy, R. (1987) Basal fore- brain magnocellular neurons stain for nerve growth factor receptor: Correlation with cholinergic cell bodies and effects of axotomy. J. Neurosci. Res., 17:lll-118.

Takayanagi, I. (1980) Effects of urogastrone on mechanical activities of the stomach and intestine of guinea pig. J. Pharm. Pharmacol., 32:228-230.

Tannenbaum, G.S., Guyda, H.J., and Posner, B.I. (1983) Insulin-like growth factors: A role in growth hormone negative feedback and body weight regulation. Science, 220:77-79.

Thoenen, H., and Edgar, D. (1985) Neurotrophic factors. Science, 229:238-242.

Toniolo, G., Dunnett, S.B., Hefti, F., and Will, B. (1985) Acetylcholine- rich transplants in the hippocampus: Influence of intrinsic growth factors and application of NGF on choline acetyltransferase activity. Brain Res., 345:141-146.

Van Schravendijk, C.F.H., Hooghe-Peters, E.L., Van den Brande, J.L., and Pipeleers, D.G. (1986) Receptors for insulin-like growth factors

and insuiin on murine fetal cortical brain cells. Biochem Biophys. Res. Commun., 135:228-238.

Walicke, P., Cowan, W.M., Ueno, H., Baird, A,, and Guillemin, R. (1986) Fibroblast growth factor promotes survival of dissociated hip- pocampal neurons and enhances neurite extension. Proc. Natl. Acid. Sci. USA, 83:3012-3016.

Walker, P. (1982) The mouse submaxillary gland: A model for the study of hormonally dependent growth factors. J. Endocrinol. Invest., 5383- 195.

Whittemore, S.R., Ebendal, T., Larkfors, L., Olson, L., Seiger, A,, Stromberg, I., and Persson, H. (1986) Development and regional distribution of p nerve growth factor messenger RNA and protein in the rat central nervous system., Proc. Natl. Acad. Sci. USA, 83:817- 821.

Will, B., and Hefti, F. (1985) Behavioral and neurochemical effects of chronic intraventricular injections of nerve growth factor in adult rats with fimbria lesions. Behav. Brain Res., 17:17-24.