Selective Induction by Nerve Growth Factor of Tyrosine Hydroxylase and Dopamine-S-Hydroxylase in the Rat Superior Cervical Ganglia

Proc. Nat. Acad. Sci. USA Vol. 68, No. 7, pp. 1598-1602, July 1971

Selective Induction by Nerve Growth Factor of Tyrosine Hydroxylase and Dopamine-s-Hydroxylase in the Rat Superior Cervical Ganglia

(dopa decarboxylase/monoamine oxidase)

H. THOENEN*, P. U. ANGELETTIt, R. LEVI-MIONTALCINIt, AND R. KETTLER* *Department of Experimental Pharmacology, Hoffman-La Roche, Basel, Switzerland; and tLaboratorio di Biologia Cellulare, C.N.R., Via Romagnosi 18A, 00196 Rome, Italy Contributed by R. Levi-Montalcini, May 6, 1971

ABSTRACT Treatment of newborn rats with 10 gg/g of microscope, fixed in Bouin's solution, and stained with tolu- nerve growth factor for 10 days enhanced not only the idine blue. Ganglia of 5- and 10-day-old experimental and growth but also the differentiation of neuroblasts in superior cervical ganglia. These morphological changes control littermates were then sectioned serially at 10 ,m. Cell were accompanied by selective induction of tyrosine counts were performed by inserting a micrometer disk into the hydroxylase and dopamine #-hydroxylase (EC 1.14.2.1), ocular and counting all nerve cells in every other section of the whereas the total and specific activities of other enzymes experimental and control ganglia. For ultrastructural studies, involved in biosynthesis or metabolic degradation of hr in 0.1 norepinephrine rose only in proportion to the increase in the ganglia were fixed for 2 in 3% glutaraldehyde MI volume of the sympathetic ganglia. There are remarkable phosphate buffer (pH 7.4) with added CaCl2; the specimens similarities between this effect of nerve growth factor and were washed for 2 hr in the same buffer and postfixed in osmic the induction of trans-synaptic enzymes by increased hydroxide (1.33%) for 2 hr. Sections were cut with a Porter- activity of the sympathetic nervous system. Blum-Sorvall MT-2 microtome, stained with uranyl acetate in Ever since the discovery that a specific nerve growth factor 50% ethanol or in lead hydroxide, and examined in a Philips (NGF) enhances growth and differentiative processes of 300 electron microscope. For the determination of tyrosine sympathetic neurons (1, 2), the question has been raised hydroxylase activity, each pair of ganglia was homogenized in whether this factor selectively stimulates metabolic pathways 0.5 ml of ice-cold 0.25 M sucrose; for dopamine 0-hydroxylase, characteristic of this particular nerve cell type. Sympathetic in 0.5 ml of 0.005 M phosphate buffer (pH 7.5) containing 0.1% neurons offer an almost ideal system to test this hypothesis, Triton X-100; for dopa decarboxylase, in 0.5 ml of 0.1 1\1 since the biochemical correlates of their function are rather phosphate buffer (pH 7.0); and for monoamine oxidase in 0.1 well known. However, it was not until recently that suffi- M phosphate buffer pH 7.2. ciently sensitive methods became available to measure the The activity of tyrosine hydroxylase was determined by the activity of enzymes involved in the synthesis and metabolic method of Levitt et al. (3), dopamine 0-hydroxylase according degradation of the adrenergic neurotransmitter in small tissue to Duch et al. (4), dopa decarboxylase according to Hakanson samples (3-6). It thus became possible to study whether the and Owman (5), with modifications described in detail by hyperplastic and hypertrophic ganglia of newborn rats treated Thoenen et al. (9), and monoamine oxidase according to with NGF also differ from controls in the specific activity of Wurtman and Axelrod (6). these enzymes. Enzyme activities are all expressed both in terms of product The results to be reported here show that NGF, besides its formed per hr per pair of ganglia (total activity) and in terms characteristic morphological effects, also produces a selective of product formed per hr per mg protein. The proteins were induction of tyrosine hydroxylase and dopamine ,3-hydroxyl- determined by the method of Lowry et al. (10). ase (EC 1.14.2.1), enzymes located in adrenergic neurons (7). Enzyme kinetic parameters were determined by the method In contrast, the activities of Dopa decarboxylase (EC 4.1.1.26) of Wilkinson (11) with a digital computer according to a and monoamine oxidase (EC 1.4.3.4), enzymes which are pres- program by Cleland (12). ent also in other cell types (7), increase only in proportion to RESULTS the rise in volume of the adrenergic neurons in the superior Morphological effects of NGF on sympathetic ganglia in cervical ganglia. newborn rats METHODS The effects of daily injections of NGF in newborn rats for periods of 5 and 10 days are illustrated in Figs. 1-7. The Newborn rats (Wistar descent) of either sex were injected subcutaneously daily for 5 and 10 days with 10 ug/g of NGF TABLE 1. Cell number in superior cervical ganglia of 5-day-old dissolved in 0.9% NaCl. The NGF was prepared according to NGF-treated and control rats the method of Bocchini and Angeletti (8). The animals were killed by transection of the thorax above the heart. The su- Number of sections Cell number perior cervical ganglia were removed under the dissecting Control 141 29,800 NGF-treated 216 74,000 Abbreviation: NGF, nerve growth factor. 1598 Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Enzyme Induction by Nerve Growth Factor 1599

"t. .1 X I ,.

#Ij. A

E 41

FIGS. 1-7. Transverse sections through superior cervical ganglia of control (Fig. 1, X 114) and experimental (Fig. 2, X 114) animals (3-day-old rats), shown in whole mounts in Fig. 5 (X 13). Arrows in Fig. 5 indicate the level of sections. Figs. 3 and 4 (X399) show size difference in sympathetic nerve cells of control (Fig. 3) and experimental (Fig. 4) ganglia reproduced in Figs. 1, 2, and 5. Note large fiber bundles among cells in NGF-treated ganglion. Figs. 6 and 7 (X855) show difference in size of sympathetic nerve cells of superior cervical ganglia of 10-day-old control (Fig. 6) and NGF-treated (Fig. 7) rats.

volume difference of superior cervical ganglia of NGF-treated and high (Figs. 3 and 4) magnification give evidence for the and control rats sacrificed at 5 days is shown in Fig. 5. size increase of sympathetic neurons in the ganglion of the Transverse sections of the same ganglia at low (Figs. 1 and 2) experimental rat and for a marked difference in the texture of Downloaded by guest on September 24, 2021 1600 Zoology: Thoenen et al. Proc. Nat. Acad. Sci. USA 68 (1971)

In:

FIG.L8.iKS'Electron micrograph of cytoplasmic area of sympathetic neuron of 10-day-old rat treated since birth with NGF. Large neuro- filament bundles fill part of the field. (X 13,940)

the ganglia. While in controls (Figs. 1 and 3) the nerve cell and of nerve cells in these ganglia are given in Table 1. Figs. 6 population is compact and evenly distributed, large fiber and 7 show transverse sections of superior cervical ganglia of bundles segregate the population in the experimental ganglion 10-day-old NGF-treated and control rats. The size increase of into cell groups (Figs. 2 and 4). The total numbers of sections individual neurons is even more marked than in younger Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Enzyme Induction by Nerve Growth Factor 1601 TABLE 2. Effect of NGF on specific and total activities of enzymes in superior cervical ganglia of newborn rats Controls NGF-treated Specific Total Specific Total Tyrosine hydroxylase* 1.9 i±0.06 0.10 ± 0.004 8.4 i 0.9 1.9 ±0.02i DopamineB-hydroxylaset 11.8 ± 0.1 1.1 ± 0.04 42.0 i 2.6 13.0 ± 0.6 Dopa decarboxylaset 0.32 ± 0.03 0.02 ±- 0.001 0.51 ± 0.03 0.13 ± 0.07 Monoamineoxidase§ 160- ± 8 23.5 ± 2.2 189 ±t 12 57.8 ± 8.5

Activities (mean i SE, n = 6-8) are amounts of product formed per hr per mg protein (specific) and amounts of product formed per hr per pair of ganglia (total): * nanomoles of dopa, t picomoles of octopamine, t micromoles of dopamine, § nanomoles of indoleacetic acid. TABLE 3. Kinetic characterization of tyrosine hydroxylase and pair of ganglia amounts to 225 ± 10 ,g in the NGF-treated rat dopamine jp-hydroxylase in superior cervical ganglia of NGF- as compared to 65 i 2 ug in controls. treated rats and controls Fig. 8 shows an electron micrograph of sympathetic nerve cells of the superior cervical ganglion of a rat treated with Tyrosine hydroxylase Dopamine 0-hydroxylase NGF from birth to the tenth day of age. The most impressive Km (MM tyrosine) Km (uM tyramine) effect is the massive production of neurotubules and neurofila- Controls 20.5 ±4 3 277 ± 34 ments. NGF-treated 23.0 ± 2 256 ±: 32 This effect is in all respects similar to those elicited by NGF on sensory neurons in chick embryos in vitro and on sym- Newborn animals were treated for 10 days with 10 ug/g of NGF pathetic neurons in newborn mice (13, 14). daily. Effect of NGF on total and specific enzyme activity in superior cervical ganglia specimens. The results of these studies, in agreement with Fig. 9 and Table 2 show that treatment of newborn rats for 10 those reported in mice (1, 2), indicate that the NGF calls forth days with NGF produced an 18-fold increase in total activity hyperplastic and hypertrophic effects. Volume measurement of tyrosine hydroxylase (product formed per hr per pair of of the superior cervical ganglia of 10-day-old experimental and ganglia) and a 13-fold increase in that of dopamine #-hy- control rats showed that the former is five times larger than droxylase. The activities of dopa decarboxylase and mono- the latter. On a protein basis, the average protein content per amine oxidase rose only 4.5-fold and 2.5-fold, respectively. The

Total activity Specific activity

controls E I NGF 2000

300

F 00

FIG. 9. Effect of NGF on enzymes involved in synthesis and metabolic degradation of norepinephrine. Newborn rats were treated with 10 pg/g of NGF for 10 days. The activity of all enzymes studied in the superior cervical ganglia is expressed in % of controls both for total (product formed per hr per pair of ganglia) and specific activity (product formed per hr per mg protein). Downloaded by guest on September 24, 2021 1602 Zoology: Thoenen et al. Proc. Nat. Acad. Sci. USA 68 (1971) selectivity of the increase in enzyme activity becomes even The considerable rise in the activity of tyrosine hydroxylase clearer if one compares specific enzyme activity (product and of dopamine ,3-hydroxylase does not result from the formed per hr per mg protein) of NGF-treated-animals with formation of activators or disappearance of inhibitors, but that of untreated controls. The ratio between the specific rather from an increased synthesis of new enzyme protein activity of NGF-treated animals and controls amounts to 5.4 We are therefore dealing with the selective induction of tVo0 for tyrosine hydroxylase, 3.6 for dopamine 0-hydroxylase, 1.5 enzymes that are exclusively located in adrenergic neurons by for dopa decarboxylase, and 1.2 for monoamine oxidase a specific growth factor. (Fig. 4). The small increase in specific activity of the last two Enhanced activity of the peripheral sympathetic nervous enzymes could be explained by a relative increase in the system resulting from cold exposure, or from depletion of the volume of neuronal versus satellite cells. In superior cervical adrenergic transmitter stores by reserpine produces, similar ganglia of controls, the neuronal cells represent about 0.75 of changes in the pattern of enzymes (15-17) involved in the the total volume. synthesis of norepinephrine as does administration of NGp. This finding raises the question whether similar basic Mechanism of increased tyrosine hydroxylase and dop- mechanisms are amine #-hydroxylase activity responsible in the two instances. It remains also to be elucidated whether this specific growth factor and It could be assumed that the increased activity of these two the functional activity of neurons play similar roles in the enzymes results from a direct activation by NGF. To examine growth regulation of the target cells during ontogenesis and in this possibility, we added 1-30 jg of NGF to enzyme prepara- their adaptation to changed functional stages. tions (100 al) used for the determination of tyrosine hydroxylase and dopamine #-hydroxylase activity. In both cases the 1. Levi-Montalcini, R., Harvey Lect., 60, 217 (1966). 2. Levi-Montalcini, R., and P. U. Angeletti, Physiol. Rev., 48, enzyme activity remained unchanged after the in vitro addi- 534 (1968). tion of NGF. To determine whether the NGF promotes 3. Levitt, M., J. WV. Gibb, J. WV. Daly, M. Lipton, and S. the formation of an activator, inhibits the production of an Udenfriend, Biochern. Pharmacol., 16, 1313 (1967). inhibitor, or enhances selectively the synthesis of tyrosine 4. Duch, D. S., 0. H. Viveros, and N. Kirshner, Biochern. hydroxylase and dopamine we Pharmacol., 17, 255 (1968). ,3-hydroxylase, combined 5. HAkanson, R., and C. Owman, J. Neurochem., 13, 597 enzyme solutions from controls and NGF-treated animals (1966). in several proportions. Both for tyrosine hydroxylase and 6. Wurtman, R., and J. Axelrod, Biochem.. Pharmacol., 12, 1433 dopamine 0-hydroxylase, the activities of these combina- (1964). tions were always additive and therefore did not provide 7. Thoenen, H., "Catecholamines", in Handbook of Experi- mental Pharmacology (Springer, 1971), in press. evidence for the loss of an inhibitor or appearance of an acti- 8. Bocchini, V., and P. U. Angeletti, Proc. Nat. Acad. Sci. vator in the enzyme preparations of animals treated with USA, 64, 787 (1969). NGF. Also, the fact that no significant change in Km values 9. Thoenen, H., R. Kettler, W. Burkard, and A. Saner, (Table 3) occurred speaks in favor of an increase in the Naunyn-Schmiedebergs Arch. Pharmakol., in press. amount of enzyme 10. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. protein. Randall, J. Riot. Chem., 193, 265 (1951). 11. Wilkinson, G. N., Biochem. J., 80, 324 (1961). DISCUSSION 12. Cleland, W. W., Nature, 198, 463 (1963). Evidence is here presented that NGF produces a selective 13. Levi-Montalcini, R., F. Caramia, S. A. Luse, and P. U. 15- to 20-fold increase in Angeletti, Brain Res., 8, 347 (1968). the activity of tyrosine hydroxylase 14. Angeletti, P. U., R. Levi-Montalcini, and F. Caramia, J. and dopamine ,3-hydroxylase, enzymes that are exclusively Ultrastruct. Res., 1971, in press. located in adrenergic neurons (7). In contrast, the activity of 15. Mueller, R. A., H. Thoenen, and J. Axelrod, J. Pharvmacol. dopa decarboxylase and monoamine oxidase rose only about Exp. Ther. 169, 74 (1969). in proportion to the increase in volume of 16. Mueller, R. A., H. Thoenen, and J. Axelrod, Mol. Pharma- sympathetic col., 5, 463 (1969). ganglia, corresponding to a 4-fold increase in their protein 17. Molinoff, P. B., S. Brimijoin, R. Weinshilboum, and J. content. Axelrod, Proc. Nat. Acad. Sci. USA, 66, 453 (1970). Downloaded by guest on September 24, 2021