striatal cultures (substance P/growth and development)

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 82, pp , November 1985 Neurobiology Nerve growth factor promotes cholinergic development in brain striatal cultures (substance P/growth and development) HUMBERTO J. MARTINEZ, CHERYL F. DREYFUS, G. MILLER JONAKAIT, AND IRA B. BLACK Department of Neurology, Division of Developmental Neurology, Cornell University Medical College, 515 East 71st Street, New York, NY Communicated by George B. Koelle, July 22, 1985 ABSTRACT We have examined the effect of the trophic protein, nerve growth factor (NGF), on organotypic cultures of fetal rat striatum. Treatment of cultures with NGF for days resulted in a 5- to 12-fold increase in the specific activity of the cholinergic enzyme chqline acetyltransferase (CAT; EC ). in a dose-dependent fashion. This effect was not elicited by insulin, ferritin, or cytochrome c, proteins similar in structure or physicochemical properties to NGF. The effect of NGF on CAT activity Was specifically blocked by anti-ngf antiserum, whereas treatment with the antiserum alone did not have a significant effect on the enzyme. Immunocytochemical studies of the treated cultures, using a monoclonal antibody directed against CAT, revealed positively stained neurons exhibiting dendritic and axonal processes. NGF did not have an effect on total protein content of the striatal cultures, suggesting a highly specific effect. Moreover, levels of substance P, a peptide localized to other, noncholinergic neurons, were not altered by NGF. Substance P remained unchanged after treatment with NGF for 12 days, whereas CAT activity increased 12-fold in sister cultures. Although the mechanisms of action of NGF on striatal cholinergic interneurons remain to be determined, the marked, specific response of CAT suggests that this well-defined trophic protein may play a critical role in normal brain development. 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 solely to indicate this fact. Although abundant evidence indicates that the protein nerve growth factor (NGF) is critical for normal survival and development of peripheral sympathetic and sensory neurons (for reviews, see refs. 1-3), little is known about the role of trophic agents in the brain. In the peripheral nervous system, NGF enhances neuronal survival (4), elicits neurite outgrowth (5), and may be active in the directional guidance of neurites (6). Moreover, NGF treatment increases the levels of catecholaminergic and peptidergic transmitters in receptive peripheral neurons (7-10). Despite these clearly delineated actions on the peripheral nervous system, actions of NGF on the central nervous system have resisted detection. Treatment with NGF or anti-ngf antiserum does not affect the pattern of locus coeruleus catecholamine histofluorescence (11). Further, NGF is not selectively transported by dopaminergic nigral neurons or by noradrenergic locus coeruleus neurons (12). Finally, NGF treatment does not increase tyrosine hydroxylase activity, the rate-limiting enzyme in catecholamine biosynthesis, in the above-mentioned brain nuclei in vivo (12) nor does it affect norepinephrine uptake in vitro (13). Nevertheless, more recent evidence has suggested that NGF may play a role in brain development and function. For example, NGF increases the activity of choline acetyltransferase (CAT; EC ), the enzyme which synthesizes acetylcholine (AcCho), in cell cultures of fetal rat telencephalon (14) and septal areas (15), in cortex (16) and several brain nuclei (17) of neonatal rats, and in hippocampus of adult rats with septohippocampal lesions (18). We now report that NGF treatment specifically and selectively increases the cholinergic phenotype of cells cultured from the fetal rat striatum. NGF treatment appears to exert specific and selective effects by a number of criteria. MATERIALS AND METHODS Experimental Animals. Pregnant Sprague-Dawley rats (Hilltop Labs, Philadelphia) were housed in clear plastic cages and were exposed to lux of cool white fluorescent illumination between 5 a.m. and 7 p.m. daily. Purina Lab Chow and water were offered ad lib. Embryonic age in days was computed from the day of discovery of the vaginal plug, which was considered day 1. Dissecting Procedures. On day 19 or 20, pregnant rats were sacrificed by exposure to ether vapor and were soaked in 80% (vol/vol) ethanol for 10 min. Fetuses were rapidly removed under sterile conditions and placed over ice in a Petri dish containing Hanks' balanced salt solution (HBSS). The brain was removed and was placed in a Petri dish for microscopic dissection. The anterior one-fourth of each cerebral hemisphere was removed by a coronal cut. The striata were exposed by another coronal section through the middle of the cerebrum, were dissected free of the surrounding cerebral cortex, and were cut into pieces of approximately 1 mm3. Culture Procedures. Two pieces of striatum were placed on photoreconstituted collagen-coated coverslips and were maintained in Maximov depression-slide chambers at 34.50C, as lying-drop preparations (19, 20). Nutrient medium was changed once on the 6th day in culture. It consisted of human placental serum [33% (vol/vol)], Eagle's minimum essential medium [50% (vol/vol)], chicken embryo extract [10% (vol/vol)], glucose (600 mg/dl) and achromycin (5 g/ml), in HBSS. CAT Activity. After 10 days, cultures were rinsed well in cold HBSS and removed from the collagen-coated coverslips. Eight pieces of striatum were transferred to a 6 x 75 mm glass tube and homogenized in 10 mm EDTA, ph 7.4. CAT activity was measured as described (21). CAT Immunohistochemistry. Immunocytochemical detection of CAT was by a modification ofthe procedure of Houser et al. (22). Briefly, cultures were fixed for 3 hr in 4% (wt/vol) paraformaldehyde in 0.12 M sodium phosphate buffer (ph 7.3). After extensive rinsing and 48 hr in 30% (wt/vol) phosphate-buffered sucrose, frozen sections of 40,um were cut and transferred to 15-ml conical tubes. Floating sections were washed in 5% (vol/vol) normal goat serum and then were incubated with an anti-cat monoclonal antibody (23) first for 2 hr at room temperature, then at 4 C for hr. Abbreviations: CAT, choline acetyltransferase; NGF, nerve growth factor; SP, substance P; AcCho, acetylcholine; NVP, naphthylvinylpyridine. 7777

2 7778 Neurobiology: Martinez et al. The antibody was graciously supplied by P. Salvaterra at the City of Hope Research Institute (Duarte, CA). The antigenantibody complex was visualized using the double-bridge peroxidase-antiperoxidase (PAP) method of Sternberger (24) as modified by Ordronneau et al. (25). Species-specific goat anti-mouse IgG (American Qualex, La Mirada, CA) and mouse PAP (Sternberger-Meyer Immunochemicals, Jarretsville, MD) were used as reagents. After reaction with diaminobenzidine, sections were washed, placed'on gelatincoated slides, and covered with a coverslip mounted with glycerin/bicarbonate (ph 8.5). Substance P Radioimmuqoassay. The procedure of Powell et al. (26) was used with minor modifications (8). Preparation of NGF. Type P3NGF was prepared from adult male mouse salivary glands as described (8). Preparation of Antiserum to N(yF. Anti-NGF antiserum was prepared in rabbits as described (8). Protein Determination. Total protein was measured by the method of Bonting and Jones, using total protein extracted from fetal rat brain as a standard (27). Statistics. Data were analyzed by the Student's t test and where appropriate, by the single factor analysis of variance and Newman-Keuls tests. P values >0.05 were considered not significant. RESULTS Strigtal Cultures. To establish baseline developmental profiles for different striatal neuronal populations, we monitored the activity of CAT, which is localized to striatal interneurons (28, 29), and levels of substance P (SP), which is localized to striatonigral neurons (30, 31). Over the course of 2 weeks in culture CAT activity increased approximately 4-fold (Fig. 1). During the same period, total protein of the cultures decreased by 35%, rendering the increase in enzyme specific activity highly significant. In contrast, SP, localized to neurons that normally project out of the striatum, decreased to barely detectable levels, suggesting that these two types of neurons may have different growth requirements. Effect of NGF on CAT Activity. Striatal cultures were exposed to 100 units' of exogenous NGF. The protein had no apparent. effect on neurite outgrowth. Control and NGFtreated cultures exhibited similar neurite fascicles and a similar pattern of support cells radiating from the explant } 4.8 % / e 2.4 /r " () Proc. Natl. Acad Sci. USA 82 (1985) However, NGF exposure resulted in a striking, 5-fold increase in CAT activity over the 10- to 11-day culture period (Fig. 2). There was no similar change in total protein (control cultures = 83.6 ± 5.88 jig of protein per culture and NGF-treated cultures = 67.7 ± 6.31 Aug of protein per culture [mean + SEMI), indicating that NGF treatment had markedly increased CAT specific activity. Although explants were washed extensively prior to assay, we sought to determine whether NGF treatment itself could have affected the CAT catalytic assay. Incubation of assay tubes with up to 20% (wt/vol) of the total NGF concentration used in the cultures had no effect on CAT activity, excluding the remote possibility that NGF could directly affect the assay. To ensure that true CAT, and not carnitine acetyltransferase, was being assayed, the specific CAT 'inhibitor, naphthylvinylpyridine (NVP), was employed. We used a concentration of NVP, 0.1 mm, that does not affect carnitine acetyltransferase but does inhibit CAT (32). Addition ofnvp to the assay incubation medium decreased measured activity by 87% and 85%'in control and NGF culture' groups, respectively, indicating that true CAT activity was, indeed, being measured. To more fully characterize the NGF effects, dose-response experiments were performed. At 10 units per culture, the lowest dose, a 4-fold increase in CAT was produced, whereas at 50 units per culture, the optimal dose, nearly a 10-fold rise in enzyme activity was elicited (Fig. 3). Doses above 100 units per culture were less effective. To ensure that CAT was, in fact, localized to neurons in the striatal cultures, as assumed, explants were stained with a monoclonal antibody directed against the enzyme. Immunoreactivity was localized primarily to large neurons occurring in clusters (Fig. 4). In many instances dendrites and aspiny axonal processes could be identified. Specificity of Effects. The marked increase in CAT elicited by NGF treatment and the absence of altered striatal total protein (see above) suggested that effects were specific. Thus, wecompared CAT activity with SP levels. Over a 12-day culture period in NGF-treated cultures CAT activity increased 12-fold, whereas the amount of SP was unchanged (Fig. 5). Next we sought to determine whether effects on CAT were specific to NGF treatment. Cultures were exposed to a 200 CAT SP ' 125 a C, Days in Culture FIG. 1. Development of striatal CAT activity and SP levels in culture. Explants of fetal rat striatum were cultured and harvested at the indicated times. CAT activity is expressed as nmol of AcCho synthesized per mg of protein per hr. SP is expressed as pg per pair of caudates. Values represent mean ± SEM. n = 6.

3 Neurobiology: Martinez et A 7 :.z. ': Hi is FE at Re> was ens,.s w Proc. Natl. Acad. Sci. USA 82 (1985) * 9 *!&.. hi i... 8.t :, :4.:..4 i;. Control NGF FIG. 2. Effect of NGF on CAT activity. Explants of fetal rat striatum were grown for 11 days in the presence of 100 biological units of NGF or a similar volume of 0.05 M sodium acetate. An equivalent volume of NaOH was added to the nutrient medium of both groups to neutralize its acidity. CAT activity is expressed as nmol of AcCho synthesized per culture per hr. Values represent mean ± SEM. n = 6. *Differs from control group at P <0.01 (two-tailed t test). There was no difference in total protein content (control cultures = ,Ag protein per culture and NGF-treated cultures = Etg of protein per culture). number of proteins similar in structure or physicochemical properties to the trophic agent (Fig. 6). While NGF increased CAT activity as expected, insulin, ferritin, or heat-inactivated NGF had no discernible effects. 14 q&4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~j,~~~~~~~~~~~~~~~~a.'.;.mu FIG. 4. Cultures grown for 10 days in the presence of NGF were examined immunocytochemically to localize CAT. (A) A network of CAT-positive cells is evident. (Bar = 50 Am.) (B) These large oval cells (25-35 ;Lm x 10-12,um) have long aspiny processes. (Bar = 20 pm.) Effects of Anti-NGF Antiserum. To further characterize specificity of effects, cultures were treated with NGF and/or anti-ngf antiserum (Fig. 7). NGF treatment alone significantly increased CAT activity, whereas the antiserum blocked this effect, suggesting that enzyme elevation was due to the presence of NGF itself. Addition of anti-ngf alone did not significantly alter CAT activity U,_) C- U Iz C._ 0a AL 0~ 10 y a0 6 en Heat Inactivated FIG. 3. NGF dose-response curve. Cultures were grown for 11 days in the presence of various concentrations of NGF. Numbers on the horizontal axis represent biological units of NGF. CAT activity is expressed as nmol AcCho synthesized per culture per hr. Values represent the mean ± SEM. n = 5. *Differs from control at P <0.05. tdoes not differ from control group (one-way analysis of Control NGF Control NG F FIG. 5. Effect of NGF on striatal SP content. Cultures were grown in the presence of 50 biological units of NGF. SP is expressed as pg per culture. CAT activity is expressed as nmol AcCho synthesized per culture per hr. Values represent the mean ± SEM. n = 6. *P <0.02 (two-tailed t test). 2

4 7780 Neurobiology: Martfnez et al. 4 0) * %; Insulin Ferritin CytochromeC FIG. 6. Effect of different proteins on striatal CAT activity. Cultures were grown for 10 days in the presence of 50 biological units of NGF or equivalent concentrations of related proteins (18 jg/ml). CAT activity is expressed as nmol AcCho synthesized per culture per hr. Values represent means ± SEM. n = 5. *Differs from all groups at P <0.01 (one-way analysis of DISCUSSION Our observations indicate that NGF, selectively and specifically, increased the activity of CAT, a marker for cholinergic interneurons in cultured rat striatum. Total protein in these cultures actually decreased slightly during the 2-week period, resulting in a greater than 10-fold increase in CAT specific activity in response to optimal NGF concentrations. Moreover, specific inhibition of acetyltransferase activity by NVP ensured that we were measuring true CAT activity and not a 1.4 I. I >% I.V I 4 I <: 0. 8 o NGF+ Anti-NGF FIG. 7. Effect of NGF and anti-ngf antiserum on striatal CAT activity. Cultures were grown in the presence of 3 biological units of NGF per culture and/or 3% (vol/vol) rabbit anti-ngf antiserum. Both control and NGF group nutrient medium contained 3% (vol/vol) preimmune serum. CAT activity is expressed as nmol AcCho synthesized per culture per hr. Values represent means + SEM. n.4. *Differs from all groups at P <0.05. tdoes not differ from the control group (one-way analysis of Proc. Natl. Acad Sci. USA 82 (1985) potentially confounding enzyme, such as carnitine acetyltransferase. Finally, CAT was indeed localized to specific intemeurons immunocytochemically by using a monoclonal antibody. We conclude, consequently, that NGF treatment specifically increased at least one index of cholinergic intemeuron function in cultured fetal rat striatum. Increasing concentrations of NGF evoked a progressive increase in CAT activity over the range of 0 to 50 units with an apparent plateau at 50 to 100 units. Although definitive conclusions are not warranted at this point, the lack of an all-or-none response suggests that NGF elicits a progressive increase in CAT activity in a stable, responsive population of neurons. Nevertheless, it is possible that increasing the concentration of NGF in the medium caused a progressive increase in cholinergic neuron survival or even stimulated neuronal mitosis. These alternatives can certainly be tested experimentally. While the precise mechanism of action of NGF on these cholinergic neurons remains to be defined, NGF treatment exhibited striking specificity for CAT. SP, localized to a distinct population, the striatonigral projection neurons, was not responsive to NGF. These observations of specificity for central cholinergic neurons are consistent with reports indicating that NGF administration does not affect glutamic acid decarboxylase, tyrosine hydroxylase, or other transmitterrelated enzymes in brain regions where CAT increases (16, 17). Nevertheless, abundant evidence indicates that the actions of NGF are neuron-specific and not transmitter-specific in the peripheral nervous system. That is, NGF appears to increase any transmitter characters expressed by responsive neurons. Although NGF selectively stimulated cholinergic neurons in our cultures to increase CAT activity, these neurons, conversely, responded selectively to NGF. Proteins closely related to NGF, either structurally or physicochemicallysuch as ferritin, insulin and heat-inactivated NGF-failed to alter CAT activity. Further, specific anti-ngf antiserum blocked the increase in CAT elicited by NGF. Finally, it is unlikely that the increase in CAT activity was due to a contaminant such as renin, since previous work has indicated that angiotensin I and II do not increase CAT activity in cholinergic telencephalic neurons in culture (14) and that treatment with renin-free NGF does increase brain CAT activity in vivo (16, 17) and in vitro (15). While our studies indicate that NGF treatment stimulates brain cholinergic neurons in culture, several lines of evidence suggest that NGF may play a physiological role in brain in vivo. 125I-labeled NGF is specifically taken up by rat cholinergic hippocampal neurons and is transported retrogradely to the nucleus of the diagonal band of Broca (12), and it is transported from frontal and occipital cortex to presumptive cholinergic perikarya in the nucleus basalis region (33). Further, NGF receptors have been described in embryonic chicken brain (34). Administration of NGF increases CAT activity in a number of brain areas in vivo (16, 17). Moreover, NGF mrna has recently been detected in rat brain (35), suggesting that the trophic molecule is synthesized locally. These studies strongly suggest that NGF does play a neurotrophic role in the brain. These observations may be directly relevant to a number of human developmental and degenerative diseases. For example, it is well-documented that Alzheimer disease is associated with degeneration of cholinergic neurons in the nucleus basalis complex, with a marked decrease in CAT activity (36). On the other hand, Huntington disease is associated with profound striatal degeneration (37). It may now be possible to determine whether NGF synthesis, NGF processing, or NGF receptors are defective in patients with these or related degenerative disorders. We thank Dr. Patricia Phelps for technical advice and Marlene Gillies and Bettye Mayer for excellent technical assistance. This

5 Neurobiology: Martinez et al. work was supported by National Institutes of Health Grants NS 10259, NS 20788, and HD 12108; National Science Foundation Grant BNS ; and by a grant from the American Parkinson Disease Association. H.J.M. is the recipient of a fellowship from the Instituto de Investigaciones Clinicas, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela. C.F.D. is the recipient of a Teacher- Scientist Award from the Andrew W. Mellon Foundation. 1. Greene, L. A. & Shooter, E. M. (1980) Annu. Rev. Neurosci. 3, Thoenen, H. & Barde, Y.-A. (1980) Physiol. Rev. 60, Levi-Montalcini, R. (1982) Annu. Rev. Neurosci. 5, Levi-Montalcini, R. (1968) Physiol. Rev. 48, Varon, S. (1975) Exp. Neurol. 48, Gundersen, R. W. & Barret, J. N. (1979) Science 206, Thoenen, H., Angeletti, P. U., Levi-Montalcini, R. & Kettler, R. (1971) Proc. Nati. Acad. Sci. USA 68, Kessler, J. A. & Black, I. B. (1980) Proc. Nati. Acad. Sci. USA 77, Kessler, J. A. & Black, I. B. (1981) Proc. Nad. Acad. Sci. USA 78, Adler, J. E., Kessler, J. A. & Black, I. B. (1984) Dev. Biol. 102, Konkol, R. J., Mailman, R. B., Bendeich, E. G., Garrison, A. M., Mueller, R. A. & Breese, G. R. (1978) Brain Res. 144, Schwab, M. E., Otten, U., Agid, Y. & Thoenen, H. (1979) Brain Res. 168, Dreyfus, C. F., Peterson, E. R. & Crain, S. M. (1980) Brain Res. 194, Honegger, P. & Lenoir, D. (1982) Dev. Brain Res. 3, Hefti, F., Hartikka, J., Eckenstein, F., Gnahn, H., Heumann, R. & Schwab, M. (1985) Neuroscience 14, Gnahn, H., Hefti, F., Heumann, R., Schwab, M. E. & Thoenen, H. (1983) Dev. Brain Res. 9, Mobley, W. C., Tennekoon, G., Buchanan, K. & Johnston, M. V. (1984) Soc. Neurosci. Abstr. 10, 367 (abstr.). 18. Hefti, F., Dravid, A. & Hartikka, J. (1984) Brain Res. 293, Proc. Natl. Acad. Sci. USA 82 (1985) Bornstein, M. B. (1973) in Tissue Culture Methods and Applications, eds. Kruse, P. F. & Patterson, M. K. (Academic, New York), pp Dreyfus, C. F., Gershon, M. D. & Crain, S. M. (1979) Brain Res. 161, Fonnum, F. (1975) J. Neurochem. 24, Houser, C. R., Crawford, G. D., Barber, R. P., Salvaterra, P. M. & Vaughn, J. E. (1983) Brain Res. 266, Crawford, G. D., Correa, L. & Salvaterra, P. M. (1982) Proc. Natl. Acad. Sci. USA 79, Sternberger, L. A. (1974) Immunocytochemistry (Prentice- Hall, Englewood Cliffs, NJ). 25. Ordronneau, P., Lindstrom, P. B.-M. & Petrusz, P. (1981) J. Histochem. Cytochem. 29, Powell, D., Leeman, S., Tregear, G., Niall, H. & Potts, J. (1973) Nature (London) New Biol. 241, Bonting, S. L. & Jones, M. (1957) Arch. Biochem. Biophys. 66, McGeer, P. L., Bagchi, S. P. & McGeer, E. G. (1971) Brain Res. 35, Hattori, T., Singh, V. K., McGeer, E. G. & McGeer, P. L. (1976) Brain Res. 102, Brownstein, M. J., Mroz, E. A., Tappaz, M. L. & Leeman, S. E. (1977) Brain Res. 135, Cuello, A. C., Priestley, J. V. & Matthews, M. R. (1982) in Substance P in the Nervous System (Pitman, London), pp White, H. L. & Wu, J. C. (1973) Biochemistry 12, Seiler, M. & Schwab, M. E. (1984) Brain Res. 300, Frazier, W. A., Boyd, L. F., Pulliam, M. W., Szutowicz, A. & Bradshaw, R. A. (1974) J. Biol. Chem. 249, Shelton, D. L. & Reichardt, L. F. (1984) Proc. Natl. Acad. Sci. USA 81, Coyle, J. T., Price, D. L. & DeLong, M. R. (1983) Science 219, Gusella, J. F., Wexler, N. S., Conneally, P. M., Naylor, S. L., Anderson, M. A., Tanzi, R. E., Watkins, P. C., Ottina, K., Wallace, M.R., Sakaguchi, A. Y., Young, A. B., Shoulson, I., Bonilla, E. & Martin, J. B. (1983) Nature (London) 306,