Brain Research, 108 (1976) 199-204
199
© Elsevier ScientificPublishing Company,Amsterdam- Printed in The Netherlands
The interaction of nerve growth factor and trans-synaptic regulation in the development of target organ innervation by sympathetic neurons
IRA B. BLACKANOCATHERINE MYTILINEOU Department of Neurology, Cornell University Medical College, New York, N.Y. 10021 and Department of Neurology, Mr. Sinai School of Medicine, New York, N.Y. 10029 (U.S.A.)
(Accepted February 18th, 1976)
Recent work has demonstrated that trans-synaptic factors regulate the maturation of nerve terminals2 and perikarya 1,a-5 of sympathetic neurons within the superior cervical ganglion (SCG). A number of studies indicate that transection of the presynaptic cholinergic nerves innervating the SCG in neonatal rats prevents the normal development of target organ innervation. For example, the developmental increases in tyrosine hydroxylase (T-OH) activity, [aH]norepinephrine uptake, nerve terminal fluorescence intensity and innervation density all fail to occur normally in irides innervated by decentralized ganglion neurons 2. Thus trans-synaptic influences are apparently necessary for the normal ontogeny of target innervation by sympathetic nerves. On the other hand, a considerable body of evidence suggests that nerve growth factor (NGF) 11 also regulates the development of end organ innervation by adrenergic neurons in vivo 4,8,11,14-16. The present studies were designed to investigate the relationship between trans-synaptic factors and NGF in the regulation of nerve terminal maturation. The development of iris innervation was monitored both biochemically and morphologically. The activity of T-OH, the rate-limiting enzyme in norepinephrine biosynthesisxz which is restricted to adrenergic terminals in iris, was used as one index of the maturation of innervation. In parallel, the morphologic development of the adrenergic ground plexus was defined by histofluorescent methods 7 as previously reported 2. Our observations suggest that trans-synaptic influences and NGF govern target organ innervation through different mechanisms. Litters of Sprague-Dawley rats were housed with mothers which were fed Rockland Lab chow and water ad libitum. Unilateral ganglion decentralization was performed in 2-3-day-old rats as previously described5. In each rat the contralateral intact ganglion and the iris which it innervated served as control. At appropriate times animals were killed by exposure to ether vapor and ganglia and irides were removed under a dissection microscope. T-OH activity was assayed as previously described2 using tetrahydrobiopterin as the cofactor. The irides were stretched on glass slides 2, dried overnight, and then exposed to formaldehyde vapor as previously described2 for fluorescence histochemistry. The iris preparations were examined
200 in a double-blind fashion by two independent observers, and representative areas were chosen for reproduction for this c o m m u n i c a t i o n from the mid-portion of the dilator area o f each iris. N G F was obtained from Burroughs W e l l c o m e Co. as the preparation for in vivo use with a potency o f 50,000 U/1000/~g. To help assess the interrelationship o f trans-synaptic regulation and N G F in the development o f iris innervation, unilateral decentralization o f the SCG was performed in neonatal rats. After surgery one group o f animals was treated with
10.00 - IRIS
8.00-
6.00
>.
4.1111
~
2.1111
< ..J
0.I~ -
>X
""
2000-GANGLION
,.-r-
-
1500>!
i~i li l i
H
500
::~ i ~
Unoperated Decentralized CONTROL
! 1 : 7 •• i!i!L ~
H: ::
Unoperated Decentralized NGF
Fig. 1. Effect of NGF and decentralization on the development of tyrosine hydroxylase activity in superior cervical ganglion and iris. The superior cervical ganglion was unilaterally decentralized in 2-day-old rats. One group of 6 rats was injected with nerve growth factor, 1/zg/g body wt. s.c. every other day and killed on day 15 of life, and the other group was injected with vehicle at appropriate times. Results are expressed as either mean pmoles/iris/h ~ S.EM. (vertical bars) or mean pmoles/ ganglion/h ± S.E.M. For both iris and ganglion in the 'control' and 'NGF' groups 'decentralized' differs.from 'unoperated' at P < 0.05. In the ganglia 'NGF' groups differ from corresponding 'control' groups at P < 0.05. For iris there is no significant di[ference between 'control' and 'NGF' decentralized groups (P > 0.05).
201 vehicle, while the other g r o u p received N G F , 1 p g / g b o d y wt. every other day for 14 days. Consequently, this p r o t o c o l resulted in 4 sets o f ganglia for each experiment (Fig. 1). Decentralization prevented the n o r m a l developmental increase in T - O H activity in ganglion cell bodies and iris nerve terminals as previously reported2, 3 (Fig. 1). N G F treatment, on the other hand, significantly increased T - O H activity in cell bodies o f b o t h unoperated and contralateral, decentralized ganglia (Fig. 1). In contradistinction to its effect on ganglion cell bodies, N G F increased T - O H activity only in those iris terminals emanating from unoperated ganglia. N G F had no significant effect on enzyme activity in terminals o f decentralized neurons (Fig. 1). 10.00 - IRIS 8.00
m
-V
6.00
iiiiiiiiiiiiii!!i i:ii! ?~
>I--
4.00
0.05).
202 Since the lack of effect of N G F on decentralized nerve terminals may possibly have been dose-related, additional studies were performed using twice the dose: rats received N G F every day instead of every other day. As expected this regimen was associated with a greater increase in T-OH activity in ganglion perikarya (Fig. 2). Activity increased approximately 2-fold in the unoperated ganglia, and 3.5-fold in decentralized ganglia. However, once again decentralization prevented the N G F induced increase in iris terminals, while the rise on the contralateral side was highly significant (P < 0.02) (Fig. 2). The interaction of trans-synaptic regulation and N G F was further characterized by examining the morphologic development of iris nerve terminals. Fluorescence microscopy was performed in parallel with the biochemical studies, on the 4 groups of irides resulting from the aforementioned studies. To obtain an estimate of the effects of N G F on development of innervation in control and decentralized irides, a number of photomicrographs taken from the dilator area of irides from the 4 groups were examined in a 'blind' fashion by two independent observers. In total, 186 such photo-
Fig. 3. Fluorescence photomicrographs of the dilator muscle from rat irides after treatment for catecholamine fluorescence. A and B are irides from an animal decentralized at 3 days of age and injected with 1 /~g/g NGF daily for 9 days. A: normal iris; B: decentralized iris. C and D are irides from an animal treated as above but injected with vehicle alone. C: normal iris; D: decentralized iris. × 450.
203 TABLE I EFFECTS OF
NGF
AND DECENTRALIZATION ON THE DEVELOPMENT OF IRIS INNERVATION DENSITY
Photomicrographs taken from the iris dilator area were examined for the 4 groups of irides described in Fig. 2. Two independent observers scored the innervation density as 'very poor', 'poor', 'rich', or 'very rich', without knowing the source of the micrographs. A total of 186 micrographs were examined and results represent averages obtained by the two viewers. See text for details. Experimental group
Decentralized Control NGF Unoperated Control NGF
Per cent o f total in group
Total
Very poor
Poor
Rich
Very rich
51.8 43.8
37.0 39.6
11.2 16.6
0 0
100 100
0 0
9.4 20.9
67.6 47.1
23 32
100 100
micrographs were examined. The density of innervation for each photograph was characterized as 'very poor', 'poor', 'rich', or 'very rich' (Fig. 3; Table I). The great majority of irides innerv~tted by intact adrenergic neurons fell within the 'rich' and 'very rich' groups whether the rats had been treated with vehicle or NGF; 90.6~/o of the vehicle-treated rats, and 79.1 ~ of the NGF-treated animals were judged to have either 'rich' or 'very rich' iris innervation. Moreover, none of the irides invested by intact neurons had 'very poor' innervation as judged by either observer. Conversely, of the irides innervated by decentralized adrenergic neurons, terminal density was either 'very poor', or 'poor' in 88.8% of rats treated with vehicle, and 83.4% of rats treated with NGF. No irides innervated by decentralized adrenergic neurons exhibited 'very rich' innervation (Fig. 3; Table I). These biochemical and morphological studies suggest that NGF administration in the doses employed cannot prevent the inhibitory effect of decentralization on cell body and nerve terminal maturation. Apparently, NGF cannot replace transsynaptic influences in the regulation of nerve terminal maturation and development of iris innervation. These observations imply that orthograde trans-synaptic factors and NGF regulate target organ innervation through different mechanisms. Such a conclusion is consistent with recent work which has demonstrated that orthograde transsynaptic regulation may be exerted via the mediation of acetylcholine1, while NGF may play a role through retrograde axonal transport to the perikaryon9,15 from endorgan nerve terminals. In the present studies NGF appeared to exert different effects on cell body and nerve terminal. Although NGF treatment increased T-OH activity both in control and decentralizedperikarya, it caused no significant change in terminals of decentralized neurons (Figs. 1 and 2). Neither the low nor the high dose of NGF altered T-OH activity in decentralized neuron terminals. Consequently, the effects of NGF on nerve terminal maturation and the development of iris innervation in vivo appear to require
204 intact cholinergic innervation in the ganglion. However, in organ culture 100 units/ml o f N G F a p p e a r s to stimulate terminal a r b o r i z a t i o n a n d iris innervation by s y m p a t h e tic ganglia a p p a r e n t l y lacking cholinergic i n n e r v a t i o n t0. In o u r studies, injection of 50 units/g b o d y wt. in n e o n a t a l rats m a y have resulted in a significantly lower concentration o f N G F than t h a t o b t a i n e d in vitro. A l t h o u g h it is possible t h a t even higher doses o f N G F m i g h t have increased decentralized t e r m i n a l ramification, the present studies clearly d e m o n s t r a t e a difference between p e r i k a r y o n and t e r m i n a l response to N G F . These o b s e r v a t i o n s suggest t h a t the d e v e l o p m e n t o f different segments o f the symp a t h e t i c n e u r o n m a y be subject to different mechanisms o f regulation. This w o r k was s u p p o r t e d by N I H G r a n t s N S 10259, NS 11666, N S 05184, a i d e d by a g r a n t f r o m the N a t i o n a l F o u n d a t i o n M a r c h o f D i m e s , a n d m a d e possible by a g r a n t f r o m the D y s a u t o n o m i a F o u n d a t i o n Inc. I.B.B. is the recipient o f the T e a c h e r - I n v e s t i g a t o r A w a r d o f N I N D S 11032. We t h a n k Ms. Susan G e e n a n d Ms. Irene T a r r for excellent technical assistance.
1 BLACK, I. B., AND GLEN, S. C., Trans-synaptic regulation of adrenergic neuron development: inhibition by ganglionic blockade, Brain Research, 63 (1973) 291-302. 2 BLACK, I. B., AND MYTILINEOU, C., Trans-synaptic regulation of the development of end organ innervation by sympathetic neurons, Brain Research, 101 (1976) 503-521.
3 BLACK,I. B., HENDRY, I. A., AND ]VERSES, L. L., Transsynaptic regulation of growth and development of adrenergic neurons in a mouse sympathetic ganglion, Brain Research, 34 (1971) 229-240. 4 BLACK,I. B., HENDRY, I. A., AND IVERSEN,L. L., Effects of surgical decentralization and nerve growth factor on the maturation of adrenergic neurons in a mouse sympathetic ganglion, J. Neurochem., 19 (1972) 1367-1377. 5 BLACK, I. B., JOH, T. H., AND RE]S, D. J., Accumulation of tyrosine hydroxytase molecules during growth and development of the superior cervical ganglion, Brain Research, 75 (1974) 133-144. 6 CHARLWOOD, K. A., LAMONT,D. M., AND BANKS,B. C., Apparent orientating effects produced by nerve growth factor. In E. ZAIMIS(Ed.), Nerve Growth Factor and its Antiserum, Athlone Press, London, 1972, pp. 102-107. 7 FALCK,B., HILLARP,N. A., THIEME, G., AND TORP, A., Fluorescence of catecholamines and related compounds condensed with formaldehyde, J. Histochem. Cytochem., 10 (1962) 348-354. 8 HENDRY, I. A., AND IVERSEN,L. L., Effect of nerve growth factor and its antiserum on tyrosine hydroxylase activity in mouse superior cervical ganglion, Brain Research, 29 (1971) 159-162. 9 HENDRY, I. A., AND ]VERSEN, L. L., Reduction in the concentration of nerve growth factor in mice after sialectomy and castration, Nature (Lond.), 243 (1973) 500-504. 10 JOHNSON, D. G., SILBERSTEIN,S. D., HANBAUER,I., AND KOPIN, L, The role of nerve growth factor in the ramification of sympathetic nerve fibers into the rat iris in organ culture, J. Neurochem., 19 (1972) 2025-2029. l l LEvI-MONTALCINI,R., AND ANGELETTI, P. U., Nerve growth factor, Physiol. Rev., 48 (1968) 534--569. 12 LEVn'T, M., SaEcrOg, S., SJOERDSMA,A., AND UOENF~END, S., Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea pig heart, J. PharmacoL exp. Ther., 148 (1965) 1-8. 13 LOWRY, O. H., ROSENBROUGH,N. J., FARR, A. L., AND RANDALL,R. J., Protein measurement with Folin phenol reagent, J, biol. Chem., 193 (1951) 265-275. 14 OLSON, L., Outgrowth of sympathetic adrenergic neurons in mice treated with a nerve-growth factor (NGF), Z. Zellforsch., 81 (1967) 155-173. 15 PARAVACINI, U., STOECKEL, K., AND THOENEN, H., Biological importance of retrograde axonal transport of nerve growth factor in adrenergic neurons, Brains Research, 84 (1975) 279-291. 16 THOENEN, H., SANER, A., KET'tLER, R., AND ANOELETTI,P. U,, Nerve growth factor and preganglionic eholinergic nerves; their relative importance to the development of the terminal adrenergic neurone, Brain Research, 44 (1972) 593-602.
199
© Elsevier ScientificPublishing Company,Amsterdam- Printed in The Netherlands
The interaction of nerve growth factor and trans-synaptic regulation in the development of target organ innervation by sympathetic neurons
IRA B. BLACKANOCATHERINE MYTILINEOU Department of Neurology, Cornell University Medical College, New York, N.Y. 10021 and Department of Neurology, Mr. Sinai School of Medicine, New York, N.Y. 10029 (U.S.A.)
(Accepted February 18th, 1976)
Recent work has demonstrated that trans-synaptic factors regulate the maturation of nerve terminals2 and perikarya 1,a-5 of sympathetic neurons within the superior cervical ganglion (SCG). A number of studies indicate that transection of the presynaptic cholinergic nerves innervating the SCG in neonatal rats prevents the normal development of target organ innervation. For example, the developmental increases in tyrosine hydroxylase (T-OH) activity, [aH]norepinephrine uptake, nerve terminal fluorescence intensity and innervation density all fail to occur normally in irides innervated by decentralized ganglion neurons 2. Thus trans-synaptic influences are apparently necessary for the normal ontogeny of target innervation by sympathetic nerves. On the other hand, a considerable body of evidence suggests that nerve growth factor (NGF) 11 also regulates the development of end organ innervation by adrenergic neurons in vivo 4,8,11,14-16. The present studies were designed to investigate the relationship between trans-synaptic factors and NGF in the regulation of nerve terminal maturation. The development of iris innervation was monitored both biochemically and morphologically. The activity of T-OH, the rate-limiting enzyme in norepinephrine biosynthesisxz which is restricted to adrenergic terminals in iris, was used as one index of the maturation of innervation. In parallel, the morphologic development of the adrenergic ground plexus was defined by histofluorescent methods 7 as previously reported 2. Our observations suggest that trans-synaptic influences and NGF govern target organ innervation through different mechanisms. Litters of Sprague-Dawley rats were housed with mothers which were fed Rockland Lab chow and water ad libitum. Unilateral ganglion decentralization was performed in 2-3-day-old rats as previously described5. In each rat the contralateral intact ganglion and the iris which it innervated served as control. At appropriate times animals were killed by exposure to ether vapor and ganglia and irides were removed under a dissection microscope. T-OH activity was assayed as previously described2 using tetrahydrobiopterin as the cofactor. The irides were stretched on glass slides 2, dried overnight, and then exposed to formaldehyde vapor as previously described2 for fluorescence histochemistry. The iris preparations were examined
200 in a double-blind fashion by two independent observers, and representative areas were chosen for reproduction for this c o m m u n i c a t i o n from the mid-portion of the dilator area o f each iris. N G F was obtained from Burroughs W e l l c o m e Co. as the preparation for in vivo use with a potency o f 50,000 U/1000/~g. To help assess the interrelationship o f trans-synaptic regulation and N G F in the development o f iris innervation, unilateral decentralization o f the SCG was performed in neonatal rats. After surgery one group o f animals was treated with
10.00 - IRIS
8.00-
6.00
>.
4.1111
~
2.1111
< ..J
0.I~ -
>X
""
2000-GANGLION
,.-r-
-
1500>!
i~i li l i
H
500
::~ i ~
Unoperated Decentralized CONTROL
! 1 : 7 •• i!i!L ~
H: ::
Unoperated Decentralized NGF
Fig. 1. Effect of NGF and decentralization on the development of tyrosine hydroxylase activity in superior cervical ganglion and iris. The superior cervical ganglion was unilaterally decentralized in 2-day-old rats. One group of 6 rats was injected with nerve growth factor, 1/zg/g body wt. s.c. every other day and killed on day 15 of life, and the other group was injected with vehicle at appropriate times. Results are expressed as either mean pmoles/iris/h ~ S.EM. (vertical bars) or mean pmoles/ ganglion/h ± S.E.M. For both iris and ganglion in the 'control' and 'NGF' groups 'decentralized' differs.from 'unoperated' at P < 0.05. In the ganglia 'NGF' groups differ from corresponding 'control' groups at P < 0.05. For iris there is no significant di[ference between 'control' and 'NGF' decentralized groups (P > 0.05).
201 vehicle, while the other g r o u p received N G F , 1 p g / g b o d y wt. every other day for 14 days. Consequently, this p r o t o c o l resulted in 4 sets o f ganglia for each experiment (Fig. 1). Decentralization prevented the n o r m a l developmental increase in T - O H activity in ganglion cell bodies and iris nerve terminals as previously reported2, 3 (Fig. 1). N G F treatment, on the other hand, significantly increased T - O H activity in cell bodies o f b o t h unoperated and contralateral, decentralized ganglia (Fig. 1). In contradistinction to its effect on ganglion cell bodies, N G F increased T - O H activity only in those iris terminals emanating from unoperated ganglia. N G F had no significant effect on enzyme activity in terminals o f decentralized neurons (Fig. 1). 10.00 - IRIS 8.00
m
-V
6.00
iiiiiiiiiiiiii!!i i:ii! ?~
>I--
4.00
0.05).
202 Since the lack of effect of N G F on decentralized nerve terminals may possibly have been dose-related, additional studies were performed using twice the dose: rats received N G F every day instead of every other day. As expected this regimen was associated with a greater increase in T-OH activity in ganglion perikarya (Fig. 2). Activity increased approximately 2-fold in the unoperated ganglia, and 3.5-fold in decentralized ganglia. However, once again decentralization prevented the N G F induced increase in iris terminals, while the rise on the contralateral side was highly significant (P < 0.02) (Fig. 2). The interaction of trans-synaptic regulation and N G F was further characterized by examining the morphologic development of iris nerve terminals. Fluorescence microscopy was performed in parallel with the biochemical studies, on the 4 groups of irides resulting from the aforementioned studies. To obtain an estimate of the effects of N G F on development of innervation in control and decentralized irides, a number of photomicrographs taken from the dilator area of irides from the 4 groups were examined in a 'blind' fashion by two independent observers. In total, 186 such photo-
Fig. 3. Fluorescence photomicrographs of the dilator muscle from rat irides after treatment for catecholamine fluorescence. A and B are irides from an animal decentralized at 3 days of age and injected with 1 /~g/g NGF daily for 9 days. A: normal iris; B: decentralized iris. C and D are irides from an animal treated as above but injected with vehicle alone. C: normal iris; D: decentralized iris. × 450.
203 TABLE I EFFECTS OF
NGF
AND DECENTRALIZATION ON THE DEVELOPMENT OF IRIS INNERVATION DENSITY
Photomicrographs taken from the iris dilator area were examined for the 4 groups of irides described in Fig. 2. Two independent observers scored the innervation density as 'very poor', 'poor', 'rich', or 'very rich', without knowing the source of the micrographs. A total of 186 micrographs were examined and results represent averages obtained by the two viewers. See text for details. Experimental group
Decentralized Control NGF Unoperated Control NGF
Per cent o f total in group
Total
Very poor
Poor
Rich
Very rich
51.8 43.8
37.0 39.6
11.2 16.6
0 0
100 100
0 0
9.4 20.9
67.6 47.1
23 32
100 100
micrographs were examined. The density of innervation for each photograph was characterized as 'very poor', 'poor', 'rich', or 'very rich' (Fig. 3; Table I). The great majority of irides innerv~tted by intact adrenergic neurons fell within the 'rich' and 'very rich' groups whether the rats had been treated with vehicle or NGF; 90.6~/o of the vehicle-treated rats, and 79.1 ~ of the NGF-treated animals were judged to have either 'rich' or 'very rich' iris innervation. Moreover, none of the irides invested by intact neurons had 'very poor' innervation as judged by either observer. Conversely, of the irides innervated by decentralized adrenergic neurons, terminal density was either 'very poor', or 'poor' in 88.8% of rats treated with vehicle, and 83.4% of rats treated with NGF. No irides innervated by decentralized adrenergic neurons exhibited 'very rich' innervation (Fig. 3; Table I). These biochemical and morphological studies suggest that NGF administration in the doses employed cannot prevent the inhibitory effect of decentralization on cell body and nerve terminal maturation. Apparently, NGF cannot replace transsynaptic influences in the regulation of nerve terminal maturation and development of iris innervation. These observations imply that orthograde trans-synaptic factors and NGF regulate target organ innervation through different mechanisms. Such a conclusion is consistent with recent work which has demonstrated that orthograde transsynaptic regulation may be exerted via the mediation of acetylcholine1, while NGF may play a role through retrograde axonal transport to the perikaryon9,15 from endorgan nerve terminals. In the present studies NGF appeared to exert different effects on cell body and nerve terminal. Although NGF treatment increased T-OH activity both in control and decentralizedperikarya, it caused no significant change in terminals of decentralized neurons (Figs. 1 and 2). Neither the low nor the high dose of NGF altered T-OH activity in decentralized neuron terminals. Consequently, the effects of NGF on nerve terminal maturation and the development of iris innervation in vivo appear to require
204 intact cholinergic innervation in the ganglion. However, in organ culture 100 units/ml o f N G F a p p e a r s to stimulate terminal a r b o r i z a t i o n a n d iris innervation by s y m p a t h e tic ganglia a p p a r e n t l y lacking cholinergic i n n e r v a t i o n t0. In o u r studies, injection of 50 units/g b o d y wt. in n e o n a t a l rats m a y have resulted in a significantly lower concentration o f N G F than t h a t o b t a i n e d in vitro. A l t h o u g h it is possible t h a t even higher doses o f N G F m i g h t have increased decentralized t e r m i n a l ramification, the present studies clearly d e m o n s t r a t e a difference between p e r i k a r y o n and t e r m i n a l response to N G F . These o b s e r v a t i o n s suggest t h a t the d e v e l o p m e n t o f different segments o f the symp a t h e t i c n e u r o n m a y be subject to different mechanisms o f regulation. This w o r k was s u p p o r t e d by N I H G r a n t s N S 10259, NS 11666, N S 05184, a i d e d by a g r a n t f r o m the N a t i o n a l F o u n d a t i o n M a r c h o f D i m e s , a n d m a d e possible by a g r a n t f r o m the D y s a u t o n o m i a F o u n d a t i o n Inc. I.B.B. is the recipient o f the T e a c h e r - I n v e s t i g a t o r A w a r d o f N I N D S 11032. We t h a n k Ms. Susan G e e n a n d Ms. Irene T a r r for excellent technical assistance.
1 BLACK, I. B., AND GLEN, S. C., Trans-synaptic regulation of adrenergic neuron development: inhibition by ganglionic blockade, Brain Research, 63 (1973) 291-302. 2 BLACK, I. B., AND MYTILINEOU, C., Trans-synaptic regulation of the development of end organ innervation by sympathetic neurons, Brain Research, 101 (1976) 503-521.
3 BLACK,I. B., HENDRY, I. A., AND ]VERSES, L. L., Transsynaptic regulation of growth and development of adrenergic neurons in a mouse sympathetic ganglion, Brain Research, 34 (1971) 229-240. 4 BLACK,I. B., HENDRY, I. A., AND IVERSEN,L. L., Effects of surgical decentralization and nerve growth factor on the maturation of adrenergic neurons in a mouse sympathetic ganglion, J. Neurochem., 19 (1972) 1367-1377. 5 BLACK, I. B., JOH, T. H., AND RE]S, D. J., Accumulation of tyrosine hydroxytase molecules during growth and development of the superior cervical ganglion, Brain Research, 75 (1974) 133-144. 6 CHARLWOOD, K. A., LAMONT,D. M., AND BANKS,B. C., Apparent orientating effects produced by nerve growth factor. In E. ZAIMIS(Ed.), Nerve Growth Factor and its Antiserum, Athlone Press, London, 1972, pp. 102-107. 7 FALCK,B., HILLARP,N. A., THIEME, G., AND TORP, A., Fluorescence of catecholamines and related compounds condensed with formaldehyde, J. Histochem. Cytochem., 10 (1962) 348-354. 8 HENDRY, I. A., AND IVERSEN,L. L., Effect of nerve growth factor and its antiserum on tyrosine hydroxylase activity in mouse superior cervical ganglion, Brain Research, 29 (1971) 159-162. 9 HENDRY, I. A., AND ]VERSEN, L. L., Reduction in the concentration of nerve growth factor in mice after sialectomy and castration, Nature (Lond.), 243 (1973) 500-504. 10 JOHNSON, D. G., SILBERSTEIN,S. D., HANBAUER,I., AND KOPIN, L, The role of nerve growth factor in the ramification of sympathetic nerve fibers into the rat iris in organ culture, J. Neurochem., 19 (1972) 2025-2029. l l LEvI-MONTALCINI,R., AND ANGELETTI, P. U., Nerve growth factor, Physiol. Rev., 48 (1968) 534--569. 12 LEVn'T, M., SaEcrOg, S., SJOERDSMA,A., AND UOENF~END, S., Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea pig heart, J. PharmacoL exp. Ther., 148 (1965) 1-8. 13 LOWRY, O. H., ROSENBROUGH,N. J., FARR, A. L., AND RANDALL,R. J., Protein measurement with Folin phenol reagent, J, biol. Chem., 193 (1951) 265-275. 14 OLSON, L., Outgrowth of sympathetic adrenergic neurons in mice treated with a nerve-growth factor (NGF), Z. Zellforsch., 81 (1967) 155-173. 15 PARAVACINI, U., STOECKEL, K., AND THOENEN, H., Biological importance of retrograde axonal transport of nerve growth factor in adrenergic neurons, Brains Research, 84 (1975) 279-291. 16 THOENEN, H., SANER, A., KET'tLER, R., AND ANOELETTI,P. U,, Nerve growth factor and preganglionic eholinergic nerves; their relative importance to the development of the terminal adrenergic neurone, Brain Research, 44 (1972) 593-602.
