264
Brain Research, 148 (1978) 264 268 ~') Elsevier/North-Holland Biomedical Press
Biochemical effects of antibodies against nerve growth factor on developing and differentiated sympathetic ganglia
M. GOEDERT, U. OTTEN and H. THOENEN Dept. of Pharmacology, Biocenter of the University, Basel (Switzerland)
(Accepted December 8th, 1977)
Nerve growth factor (NGF) is a protein which is required for the normal development and maintenance of function of sympathetic neuronslL A major approach to the elucidation of its physiological role represents the analysis of the effects of exogenously administered N G F and the characterization of the consequences of the neutralization of endogenous N G F by administration of monospecific anti NGF-antibodies. Although it is well known that the administration of NGF-antiserum to newborn animals leads to an almost complete and irreversible destruction of the peripheral sympathetic nervous system 14, much less is known about the effects of anti NGFantibodies on fully differentiated sympathetic neurons2,L It was the aim of the present study to investigate the effects of a single dose of purified anti NGF-antibodies on the catecholamine-synthesizing enzymes in the superior cervical ganglion (SCG) of the rat at different stages of the development. Moreover, we investigated how the changes effected by anti NGF-antibodies in the postganglionic neurons are reflected by alterations in the preganglionic cholinergic fibers by measuring the level of choline acetyltransferase (CAT), an enzyme which is known to be localized to more than 95 ~ in the preganglionic cholinergic nerve terminals 9 and which represents a reliable marker for cholinergic neurons. N G F was prepared as the 2.5 S subunit from submaxillary glands of adult male mice according to Bocchini and AngelettP. The purity of N G F was controlled by SDS gel electrophoresis and its biological activity was determined according to Fenton 6 in a tissue culture assay with dorsal root ganglia of 7-9-day-old chicken embryos. The biological activity of our preparation was 250 biological units per #g of protein. Antibodies against 2.5 S N G F were raised in goats and purified by affinity chromatography according to StSckel et al. 24. Rats 2, 6, 12, 16 and 30 days old received a single subcutaneous injection of 200 mg/kg of anti NGF-antibodies. Control animals received the same volume of goat serum. The animals were killed at various time intervals ranging between 2 and 60 days after the injection. The SCG were rapidly removed and each pair was homogenized in 0,5 ml of 5 m M Tris • HCl buffer, pH 7.4, containing 0.1 ~o Triton X-100. The homogenates were spun at 15,000 × g f o r
265 20 min and the supcrnatant fraction was used for the assays of tyrosine hydroxylase (TH), dopamine fl-hydroxylase (DBH) and dopa decarboxylase (DDC)activities, whereas CAT activity was determined in the whole homogenate. The activity of TH was assayed according to Levitt et al. 17 with modifications described in detail by Mueller et al. 21. DBH activity was measured by the method of Molinoff et al. 19 with modifications described by Oesch et al. 22. DDC activity was determined according to H~ikanson and Owman s with modifications described by Oesch et al.2L CAT activity was assayed according to Fonnum 7. Protein concentrations were determined by the method of Lowry et al} 8, using bovine serum albumin as the standard. The administration of a single dose of anti NGF-antibodies to newborn rats resulted in a very marked drop in the activities of all the catecholamine-synthesizing enzymes (Fig. 1). There was a sharp decrease during the first two days after the injection, while the maximal reduction was obtained after one week. The reduction of the enzyme levels was irreversible, reflecting the morphologically detectable extensive destruction of the adrenergic neurons. In 30-day-old animals, the general enzyme reduction was much smaller and, most importantly, completely reversible. Control levels were reached again four weeks after the anti NGF-antibody injection (Fig. 1)
.g
DBH
TH
.g
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. .......
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15 ~" 10"
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,
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. .......
50
:
ib
60
~
~6o.
....
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.....
.
.oll...! "°" o °
. . . . .
10 Age in days
~0
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~ . - - - r 4"::'~'I.
i0
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Age in days
CAT
.g
DDC
'~,,,,~t,/,,,"
2@
........
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.g
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,_..~/]
.
.
.
20 3o Age m doys
,
40
,
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,
60
Fig. 1. Effects of a single subcutaneous injection of 200 mg/kg anti NGF-antibodies on the normal developmental pattern of tyrosine hydroxylase (TH), dopamine fl-hydroxylase (DBH), dopa decarboxylase (DDC) and choline acetyltransferase (CAT) in the superior cervical ganglion of the rat. The injection was performed at 2, 6, 12, 16 and 30 days of age. Values from animals treated with anti NGF-antibodies are shown by the dotted lines and from corresponding controls by the solid line. Each value represents the mean -I- S.E.M. (n = 8).
266 and the protein concentrations remained unchanged over the whole period studied. While T H and D D C activities were affected to a similar extent, the maximal reduction of DBH was distinctly larger (75 vs. 25 ~). Twelve and 16-day-old animals showed an intermediate effect between newborn and 30 day old rats (Fig. 1). The enzyme levels and the protein concentrations were still markedly reduced 4 weeks after the injection in animals injected 12 days after birth, whereas in animals injected 16 days after birth the effects seen were nearly completely reversible after 4 weeks. Here also, the DBH activity was the most affected, both in 12 and 16 day old animals. In contrast to the effects seen on the catecholamine-synthesizing enzymes, the changes in the CAT activity did not result in a decrease but in a marked delay of their normal developmental increase at all ages studied (Fig. 1). The present results support the view that N G F is not only an absolute prerequisite for the normal development of major parts of the peripheral sympathetic nervous system but also for the maintenance of its function after complete development, This is reflected by a decrease of all the enzymes involved in the synthesis of the adrenergic transmitter norepinephrine. In contrast to previous findings obtained in the mousO, 10 where no decrease in T H could be noted in the adult animals after NGF-antiserum administration, we found that the T H levels were reduced at all ages studied. This discrepancy could be due to the quantity of anti NGF-antibodies injected. Thus, it seems that a relatively large antibody dose is required in order to achieve an effect on the catecholamine-synthesizing enzymes and on CAT in the adult animals. The decrease of DBH activity was much larger than that of T H and DDC in developing and adult rats. This difference could result from the more rapid turnover of DBH as compared to T H and DDC z°,2~. The time-course of the DBH changes reflects also the reduction of the norepinephrine levels in sympathetic ganglia of adult mice after NGF-antiserum treatment as described by Bjerre et al. 3. This suggests that DBH is becoming the rate-limiting enzyme for norepinephrine synthesis in this particular situation. The only delayed developmental increase in CAT activity as compared to the marked decrease of TH, DBH and DDC suggests that there is a retrograde transsynaptic influence from the postganglionic adrenergic neuron to the preganglionic cholinergic nerve terminals at all ages studied. A direct effect of anti NGF-antibodies on preganglionic nerve fibers is very unlikely, as discussed extensively before 26. Moreover, it has been shown that the destruction of ganglionic cells by NGF-antiserum causes a retrograde loss of preganglionic fibers in the SCG of the newborn rat a. This phenomenon could account for the effects seen on CAT. There is a progressive shift from irreversible to reversible effects if one compares animals of different ages, indicating that the period of highest sensitivity to an interruption of endogenous N G F supply is the immediate postnatal phase. The timecourse observed in the present experiments is virtually identical to that resulting from the transection of the postganglionic nerve fibers 11,12, indicating that anti N G F antibodies can mimic the effects of axotomy. Since the effects of axotomy and of
67 selective a d r e n e r g i c nerve t e r m i n a l d e s t r u c t i o n b y 6 - h y d r o x y d o p a m i n e c a n b e prevented b y l a r g e ' d o s e s o f N G F , it has been c o n c l u d e d t h a t the deleterious effects o f a x o t o m y la a n d o f 6 - h y d r o x y d o p a m i n e le on the cell b o d y in the early p o s t n a t a l phase results f r o m the i n t e r r u p t i o n o f the n o r m a l N G F supply f r o m the periphery. The present results s u p p o r t this i n t e r p r e t a t i o n , suggesting a d d i t i o n a l l y t h a t the interr u p t i o n o f the a x o p l a s m i c t r a n s p o r t a n d the n e u t r a l i z a t i o n o f e n d o g e n o u s N G F by anti N G F - a n t i b o d i e s are p r o d u c i n g the same effect. Interestingly, Purves a n d NjA 23 have shown t h a t exogenous N G F largely prevents the synaptic depression following a x o t o m y in a d u l t guinea pigs. The present e x p e r i m e n t s which have s h o w n t h a t anti N G F - a n t i b o d i e s lead to a g r a d u a l a n d irreversible d e s t r u c t i o n o f the adrenergic n e u r o n s in the n e w b o r n a n d to an only t r a n s i e n t effect in the a d u l t rat favours the a s s u m p t i o n that i m m u n o s y m p a t h e c t o m y results from N G F d e p r i v a t i o n r a t h e r than f r o m an acute c y t o t o x i c effect. This w o r k was s u p p o r t e d by the Swiss N a t i o n a l F o u n d a t i o n for Soientific R e s e a r c h ( G r a n t 3.432.74).
1 Aguayo, A. J., Peyronnard, J. M., Terry, L. C., Romine, J. S. and Bray, G. M., Neonatal neuronal loss in rat superior cervical ganglia: retrograde effects on developing preganglionic axons and Schwann cells, J. NeurocytoL, 5 (1976) 137-155. 2 Angeletti, P. U., Levi-Montalcini, R. and Caramia, F., Analysis of the effects of the antiserum to nerve growth factor in adult mice, Brain Research, 27 (1971) 343-355. 3 Bjerre, B., Wiklund, L. and Edwards, C. D., A study of the de- and regenerative changes in the sympathetic nervous system of the adult mouse after treatment with the antiserum to nerve growth factor, Brain Research, 92 (1975) 257-278. 4 Black, I. B., Hendry, I. A. and Iversen, L. L., The role of post-synaptic neurones in the biochemical maturation of presynaptic ganglion, J. Physiol. (Lond.), 221 (1972) 149-159. 5 Bocchini, V. and Angeletti, P. U., The nerve growth factor: purification as a 30000 molecular weight protein, Proc. nat. Acad. Sci. (Wash.), 64 (1969) 787-794. 6 Fenton, E. L., Tissue culture assay of nerve growth factor and the specific antiserum, Exp. Cell Res., 59 (1970) 383-392. 7 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409. 8 H~tkanson, R. and Owman, Ch., Pineal dopa decarboxylase and monoaminoxidase activities as related to the monoamine stores, J. Neurochem., 13 (1966) 597-605. 9 Hebb, C. O. and Waites, G. M. H., Choline acetylase in antero- and retrograde degeneration of a cholinergic nerve, J. Physiol. (Lond.), 132 (1956) 667-671. 10 Hendry, I. A. and Iversen, L. L., Effect of nerve growth factor and its antiserum on tyrosine hydroxylase activity in mouse superior cervical sympathetic ganglion, Brain Research, 29 (1971) 159-162.
11 Hendry, I. A., The retrograde trans-synaptic control of the development of cholinergic terminals in sympathetic ganglia, Brain Research, 86 (1975) 783-787. 12 Hendry, I. Ao, The effects of axotomy on the development of the rat superior cervical ganglion, Brain Research, 90 (1975) 235-244. 13 Hendry, I. A., The response of adrenergic neurons to axotomy and nerve growth factor, Brain Research, 94 (1975) 87-97. 14 Levi-Montalcini, R. and Angeletti, P. U., Immunosympathectomy, Pharmacol. Rev., 18 (1966) 619-628. 15 Levi-Montalcini, R. and Angeletti, P. U., Nerve growth factor, PhysioL Rev., 48 (1968) 534-569.
268 16 Levi-Montalcini, R., Aloe, L., Mugnaini, E., Oesch, F. and Thoenen, H., Nerve growth tactor induces volume increase and enhances tyrosine hydroxylase synthesis in chemically axotomized sympathetic ganglia of newborn rats, Proc. nat. Acad. Sci. (Wash.), 72 (1975) 595-599. 17 Levitt, M., Gibb, J. W., Daly, J., Lipton, M. and Udenfriend, S., A new class of tyrosine hydroxylase inhibitors and a simple assay of inhibitionin vivo, Biochem. PharmacoL, 16 (1967) 1313-1321. 18 Lowry, O. H., Rosebrough, N. H., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 19 Molinoff, P. B., Weinshilboum, R. and Axelrod, J., A sensitive enzyme assay for dopamine /~hydroxylase, J. Pharmacol. exp. Ther., 178 (1971) 425-431. 20 Mueller, R. A., Thoenen, H. and Axelrod, J., Inhibition of trans-synaptically increased tyrosine hydroxylase activity by cycloheximide and actinomycin, Molec. PharmacoL, 5 (1969) 463-469. 21 Mueller, R.A., Thoenen, H. and Axelrod, J., Increase in tyrosine hydroxylase activity after reserpine administration, J. PharmacoL exp. Ther., 169 (1969) 74-79. 22 Oesch, F., Otten, U. and Thoenen, H., Relationship between the rate of axoplasmic transport and subcellular distribution of enzymes involved in the synthesis of norepinephrine, J. Neurochem., 20 (1973) 1691-1706. 23 Purves, D. and Nj~, A., Effects of nerve growth factor on synaptic depression after axotomy, Nature (Lond.), 260 (1976) 535-536. 24 Stoeckel, K., Gagnon, C., Guroff, G. and Thoenen, H., Purification of NGF-anlibodies by affinity chromatography, J. Neurochem., 26 (1976) 1207-1211. 25 Thoenen, H., Kettler, R., Burkhard, W. and Saner, A., Neuronally mediated control of enzymes involved in the synthesis of norepinephrine; are they regulated as an operational unit?, NaunynSchmiedeber gs Arch. exp. Path. PharmacoL, 27 (1971) 146--160. 26 Thoenen, H., Comparison between the effect of neuronal activity and nerve growth factor on the enzymes involved in the synthesis of norepinephrine, Pharmacol. Rev., 24 (1972) 255-267.
Brain Research, 148 (1978) 264 268 ~') Elsevier/North-Holland Biomedical Press
Biochemical effects of antibodies against nerve growth factor on developing and differentiated sympathetic ganglia
M. GOEDERT, U. OTTEN and H. THOENEN Dept. of Pharmacology, Biocenter of the University, Basel (Switzerland)
(Accepted December 8th, 1977)
Nerve growth factor (NGF) is a protein which is required for the normal development and maintenance of function of sympathetic neuronslL A major approach to the elucidation of its physiological role represents the analysis of the effects of exogenously administered N G F and the characterization of the consequences of the neutralization of endogenous N G F by administration of monospecific anti NGF-antibodies. Although it is well known that the administration of NGF-antiserum to newborn animals leads to an almost complete and irreversible destruction of the peripheral sympathetic nervous system 14, much less is known about the effects of anti NGFantibodies on fully differentiated sympathetic neurons2,L It was the aim of the present study to investigate the effects of a single dose of purified anti NGF-antibodies on the catecholamine-synthesizing enzymes in the superior cervical ganglion (SCG) of the rat at different stages of the development. Moreover, we investigated how the changes effected by anti NGF-antibodies in the postganglionic neurons are reflected by alterations in the preganglionic cholinergic fibers by measuring the level of choline acetyltransferase (CAT), an enzyme which is known to be localized to more than 95 ~ in the preganglionic cholinergic nerve terminals 9 and which represents a reliable marker for cholinergic neurons. N G F was prepared as the 2.5 S subunit from submaxillary glands of adult male mice according to Bocchini and AngelettP. The purity of N G F was controlled by SDS gel electrophoresis and its biological activity was determined according to Fenton 6 in a tissue culture assay with dorsal root ganglia of 7-9-day-old chicken embryos. The biological activity of our preparation was 250 biological units per #g of protein. Antibodies against 2.5 S N G F were raised in goats and purified by affinity chromatography according to StSckel et al. 24. Rats 2, 6, 12, 16 and 30 days old received a single subcutaneous injection of 200 mg/kg of anti NGF-antibodies. Control animals received the same volume of goat serum. The animals were killed at various time intervals ranging between 2 and 60 days after the injection. The SCG were rapidly removed and each pair was homogenized in 0,5 ml of 5 m M Tris • HCl buffer, pH 7.4, containing 0.1 ~o Triton X-100. The homogenates were spun at 15,000 × g f o r
265 20 min and the supcrnatant fraction was used for the assays of tyrosine hydroxylase (TH), dopamine fl-hydroxylase (DBH) and dopa decarboxylase (DDC)activities, whereas CAT activity was determined in the whole homogenate. The activity of TH was assayed according to Levitt et al. 17 with modifications described in detail by Mueller et al. 21. DBH activity was measured by the method of Molinoff et al. 19 with modifications described by Oesch et al. 22. DDC activity was determined according to H~ikanson and Owman s with modifications described by Oesch et al.2L CAT activity was assayed according to Fonnum 7. Protein concentrations were determined by the method of Lowry et al} 8, using bovine serum albumin as the standard. The administration of a single dose of anti NGF-antibodies to newborn rats resulted in a very marked drop in the activities of all the catecholamine-synthesizing enzymes (Fig. 1). There was a sharp decrease during the first two days after the injection, while the maximal reduction was obtained after one week. The reduction of the enzyme levels was irreversible, reflecting the morphologically detectable extensive destruction of the adrenergic neurons. In 30-day-old animals, the general enzyme reduction was much smaller and, most importantly, completely reversible. Control levels were reached again four weeks after the anti NGF-antibody injection (Fig. 1)
.g
DBH
TH
.g
.~ 30o ~2oo
1
/.,,
~.,--'-i:---, .............. , " .....
. .......
2o 30 Age in clays
"'t. it,>._;,," "-r'"
15 ~" 10"
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ti~..
,
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. .......
50
:
ib
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....
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.
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. . . . .
10 Age in days
~0
:i:iii!i ............ ..,...iS.
~ . - - - r 4"::'~'I.
i0
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Age in days
CAT
.g
DDC
'~,,,,~t,/,,,"
2@
........
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.g
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,_..~/]
.
.
.
20 3o Age m doys
,
40
,
5O
,
60
Fig. 1. Effects of a single subcutaneous injection of 200 mg/kg anti NGF-antibodies on the normal developmental pattern of tyrosine hydroxylase (TH), dopamine fl-hydroxylase (DBH), dopa decarboxylase (DDC) and choline acetyltransferase (CAT) in the superior cervical ganglion of the rat. The injection was performed at 2, 6, 12, 16 and 30 days of age. Values from animals treated with anti NGF-antibodies are shown by the dotted lines and from corresponding controls by the solid line. Each value represents the mean -I- S.E.M. (n = 8).
266 and the protein concentrations remained unchanged over the whole period studied. While T H and D D C activities were affected to a similar extent, the maximal reduction of DBH was distinctly larger (75 vs. 25 ~). Twelve and 16-day-old animals showed an intermediate effect between newborn and 30 day old rats (Fig. 1). The enzyme levels and the protein concentrations were still markedly reduced 4 weeks after the injection in animals injected 12 days after birth, whereas in animals injected 16 days after birth the effects seen were nearly completely reversible after 4 weeks. Here also, the DBH activity was the most affected, both in 12 and 16 day old animals. In contrast to the effects seen on the catecholamine-synthesizing enzymes, the changes in the CAT activity did not result in a decrease but in a marked delay of their normal developmental increase at all ages studied (Fig. 1). The present results support the view that N G F is not only an absolute prerequisite for the normal development of major parts of the peripheral sympathetic nervous system but also for the maintenance of its function after complete development, This is reflected by a decrease of all the enzymes involved in the synthesis of the adrenergic transmitter norepinephrine. In contrast to previous findings obtained in the mousO, 10 where no decrease in T H could be noted in the adult animals after NGF-antiserum administration, we found that the T H levels were reduced at all ages studied. This discrepancy could be due to the quantity of anti NGF-antibodies injected. Thus, it seems that a relatively large antibody dose is required in order to achieve an effect on the catecholamine-synthesizing enzymes and on CAT in the adult animals. The decrease of DBH activity was much larger than that of T H and DDC in developing and adult rats. This difference could result from the more rapid turnover of DBH as compared to T H and DDC z°,2~. The time-course of the DBH changes reflects also the reduction of the norepinephrine levels in sympathetic ganglia of adult mice after NGF-antiserum treatment as described by Bjerre et al. 3. This suggests that DBH is becoming the rate-limiting enzyme for norepinephrine synthesis in this particular situation. The only delayed developmental increase in CAT activity as compared to the marked decrease of TH, DBH and DDC suggests that there is a retrograde transsynaptic influence from the postganglionic adrenergic neuron to the preganglionic cholinergic nerve terminals at all ages studied. A direct effect of anti NGF-antibodies on preganglionic nerve fibers is very unlikely, as discussed extensively before 26. Moreover, it has been shown that the destruction of ganglionic cells by NGF-antiserum causes a retrograde loss of preganglionic fibers in the SCG of the newborn rat a. This phenomenon could account for the effects seen on CAT. There is a progressive shift from irreversible to reversible effects if one compares animals of different ages, indicating that the period of highest sensitivity to an interruption of endogenous N G F supply is the immediate postnatal phase. The timecourse observed in the present experiments is virtually identical to that resulting from the transection of the postganglionic nerve fibers 11,12, indicating that anti N G F antibodies can mimic the effects of axotomy. Since the effects of axotomy and of
67 selective a d r e n e r g i c nerve t e r m i n a l d e s t r u c t i o n b y 6 - h y d r o x y d o p a m i n e c a n b e prevented b y l a r g e ' d o s e s o f N G F , it has been c o n c l u d e d t h a t the deleterious effects o f a x o t o m y la a n d o f 6 - h y d r o x y d o p a m i n e le on the cell b o d y in the early p o s t n a t a l phase results f r o m the i n t e r r u p t i o n o f the n o r m a l N G F supply f r o m the periphery. The present results s u p p o r t this i n t e r p r e t a t i o n , suggesting a d d i t i o n a l l y t h a t the interr u p t i o n o f the a x o p l a s m i c t r a n s p o r t a n d the n e u t r a l i z a t i o n o f e n d o g e n o u s N G F by anti N G F - a n t i b o d i e s are p r o d u c i n g the same effect. Interestingly, Purves a n d NjA 23 have shown t h a t exogenous N G F largely prevents the synaptic depression following a x o t o m y in a d u l t guinea pigs. The present e x p e r i m e n t s which have s h o w n t h a t anti N G F - a n t i b o d i e s lead to a g r a d u a l a n d irreversible d e s t r u c t i o n o f the adrenergic n e u r o n s in the n e w b o r n a n d to an only t r a n s i e n t effect in the a d u l t rat favours the a s s u m p t i o n that i m m u n o s y m p a t h e c t o m y results from N G F d e p r i v a t i o n r a t h e r than f r o m an acute c y t o t o x i c effect. This w o r k was s u p p o r t e d by the Swiss N a t i o n a l F o u n d a t i o n for Soientific R e s e a r c h ( G r a n t 3.432.74).
1 Aguayo, A. J., Peyronnard, J. M., Terry, L. C., Romine, J. S. and Bray, G. M., Neonatal neuronal loss in rat superior cervical ganglia: retrograde effects on developing preganglionic axons and Schwann cells, J. NeurocytoL, 5 (1976) 137-155. 2 Angeletti, P. U., Levi-Montalcini, R. and Caramia, F., Analysis of the effects of the antiserum to nerve growth factor in adult mice, Brain Research, 27 (1971) 343-355. 3 Bjerre, B., Wiklund, L. and Edwards, C. D., A study of the de- and regenerative changes in the sympathetic nervous system of the adult mouse after treatment with the antiserum to nerve growth factor, Brain Research, 92 (1975) 257-278. 4 Black, I. B., Hendry, I. A. and Iversen, L. L., The role of post-synaptic neurones in the biochemical maturation of presynaptic ganglion, J. Physiol. (Lond.), 221 (1972) 149-159. 5 Bocchini, V. and Angeletti, P. U., The nerve growth factor: purification as a 30000 molecular weight protein, Proc. nat. Acad. Sci. (Wash.), 64 (1969) 787-794. 6 Fenton, E. L., Tissue culture assay of nerve growth factor and the specific antiserum, Exp. Cell Res., 59 (1970) 383-392. 7 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409. 8 H~tkanson, R. and Owman, Ch., Pineal dopa decarboxylase and monoaminoxidase activities as related to the monoamine stores, J. Neurochem., 13 (1966) 597-605. 9 Hebb, C. O. and Waites, G. M. H., Choline acetylase in antero- and retrograde degeneration of a cholinergic nerve, J. Physiol. (Lond.), 132 (1956) 667-671. 10 Hendry, I. A. and Iversen, L. L., Effect of nerve growth factor and its antiserum on tyrosine hydroxylase activity in mouse superior cervical sympathetic ganglion, Brain Research, 29 (1971) 159-162.
11 Hendry, I. A., The retrograde trans-synaptic control of the development of cholinergic terminals in sympathetic ganglia, Brain Research, 86 (1975) 783-787. 12 Hendry, I. Ao, The effects of axotomy on the development of the rat superior cervical ganglion, Brain Research, 90 (1975) 235-244. 13 Hendry, I. A., The response of adrenergic neurons to axotomy and nerve growth factor, Brain Research, 94 (1975) 87-97. 14 Levi-Montalcini, R. and Angeletti, P. U., Immunosympathectomy, Pharmacol. Rev., 18 (1966) 619-628. 15 Levi-Montalcini, R. and Angeletti, P. U., Nerve growth factor, PhysioL Rev., 48 (1968) 534-569.
268 16 Levi-Montalcini, R., Aloe, L., Mugnaini, E., Oesch, F. and Thoenen, H., Nerve growth tactor induces volume increase and enhances tyrosine hydroxylase synthesis in chemically axotomized sympathetic ganglia of newborn rats, Proc. nat. Acad. Sci. (Wash.), 72 (1975) 595-599. 17 Levitt, M., Gibb, J. W., Daly, J., Lipton, M. and Udenfriend, S., A new class of tyrosine hydroxylase inhibitors and a simple assay of inhibitionin vivo, Biochem. PharmacoL, 16 (1967) 1313-1321. 18 Lowry, O. H., Rosebrough, N. H., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 19 Molinoff, P. B., Weinshilboum, R. and Axelrod, J., A sensitive enzyme assay for dopamine /~hydroxylase, J. Pharmacol. exp. Ther., 178 (1971) 425-431. 20 Mueller, R. A., Thoenen, H. and Axelrod, J., Inhibition of trans-synaptically increased tyrosine hydroxylase activity by cycloheximide and actinomycin, Molec. PharmacoL, 5 (1969) 463-469. 21 Mueller, R.A., Thoenen, H. and Axelrod, J., Increase in tyrosine hydroxylase activity after reserpine administration, J. PharmacoL exp. Ther., 169 (1969) 74-79. 22 Oesch, F., Otten, U. and Thoenen, H., Relationship between the rate of axoplasmic transport and subcellular distribution of enzymes involved in the synthesis of norepinephrine, J. Neurochem., 20 (1973) 1691-1706. 23 Purves, D. and Nj~, A., Effects of nerve growth factor on synaptic depression after axotomy, Nature (Lond.), 260 (1976) 535-536. 24 Stoeckel, K., Gagnon, C., Guroff, G. and Thoenen, H., Purification of NGF-anlibodies by affinity chromatography, J. Neurochem., 26 (1976) 1207-1211. 25 Thoenen, H., Kettler, R., Burkhard, W. and Saner, A., Neuronally mediated control of enzymes involved in the synthesis of norepinephrine; are they regulated as an operational unit?, NaunynSchmiedeber gs Arch. exp. Path. PharmacoL, 27 (1971) 146--160. 26 Thoenen, H., Comparison between the effect of neuronal activity and nerve growth factor on the enzymes involved in the synthesis of norepinephrine, Pharmacol. Rev., 24 (1972) 255-267.
