Journal of Neurocytology, 6, 287-297 (1977) iii
The effects of postganglionic axotomy and nerve growth factor on the superior cervical ganglia of developing mice BARBARA
E. C. B A N K S
and S. J. W A L T E R *
Department of Pbysiology, University College London, Gower Street, London WC1E 6BT
Received 23 November 1976; revised 26 January 1977; accepted 28 January 1977
Summary Sectioning of the two major outflows from the superior cervical ganglia in two week mice results in a marked drop in the number of neurons within one week of operation and a smaller drop over the following two weeks. In animals receiving daily injections of nerve growth factor (NGF), the effect of axotomy is modified. One week after axotomy, the number of neurons in the axotomized ganglia is approxima the same in NGF treated animals as in the control, sham operated ganglia. Over the next two weeks, however, the cell death that results from axotomy is no longer prevented by treatment with NGF. The normal, hyperplastic response to NGF appears to occur independently of the cell reaction caused by axotomy.
Introduction
It has long been known (Levi-Montalcini and Booker, 1960) that members of the group of proteins called Nerve growth factor (NGF) cause enlargement of the sympathetic chain ganglia when injected into neonatal mammals. Mice are particularly sensitive in their response, treatment for six days giving superior cervical ganglia that are four to six times larger than the ganglia from control animals. Kittens and rats were reported by Zaimis (1972) to give a less marked response but there may be some differences in the response of different strains of rats since, in recent reports, (Aloe, Mugnaini and Levi-Montalcini, 1975; Hendry and Campbell, 1976), three to four-fold increases in the volume of rat superior cervical ganglia are recorded. The increase in ganglion size induced by NGF in mice has been shown (Banks et al., 1975) to be due to an increase in the rate at which neurons are produced from less differentiated cells, as first suggested by Zaimis (1972). The hyperplastic response resulted, in a 6 day old, treated mouse, in more neurons than were present in the *Present address: Department of Physiology, Charing Cross Hospital Medical School, London 9 1977 Chapman and Hall Ltd. Printed in Great Britain.
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adult. The excess neurons rapidly disappeared when NGF treatment ceased but were maintained when the NGF treatment was prolonged. This is consistent with the maintenance role played by NGF in vitro (Banthrope et al., 1974). Both Levi-Montalcini and Zaimis with their coworkers had concluded from the qualitative studies that NGF treatment induced both hyperplasia and hypertrophy of sympathetic neurons. This impression was confirmed by Black, Hendry and Iversen (1972) who showed that treatment of 4 day mice with NGF for 10 days approximately doubled the number of neurons in the superior cervical ganglia. The cell counts on which this conclusion was based were corrected for the split cell error by the method of Abercrombie (1946). More recently, Hendry (1976) has described a more accurate method of assessing cell numbers, applicable where a given treatment results in a significant change in cell size. Using this method, Hendry has shown that in neonatal rats, NGF causes a much smaller increase in cell number (ca. 25% in 5 days) even when the treatment is started immediately postpartum. The increase in ganglion volume in rats was shown (Hendry and Campbell, 1976) to be due largely to proliferation of non-neuronal cells. This finding contrasts with the report (Aloe, et al., 1975), that NGF treatment for 4 days from birth doubles the number of neurons in the rat superior cervical ganglion. The estimates of cell number in this work are, however, only semi-quantitative. No standard corrections were applied, nor were the data analysed statistically. It does seem that the responses of rats and of mice to NGF are quantitatively rather different. Experiments in vitro (Charlwood et al., 1972; Chamley et al., 1973) have shown that NGF may influence the orientation of sympathetic fibres to their end organ and, furthermore, it has been suggested (Hendry and Iversen, 1973) that the end organ may maintain sympathetic neurons by production of NGF which is transported up the axon to the cell body. Retrograde axonal transport of NGF has since been confirmed (Hendry et al., 1974; Stoeckel et al., 1974; Paravicini et al., 1975). If such transport were physiologically significant, it might be expected that interruption of the supply of NGF from the end organ by postganglionic axotomy would result in neuron death and that such death might be prevented by treatment of axotomized animals with NGF. We wish to report the morphological results obtained from total postganglionic axotomy of the superior cervical ganglia of two week mice and the effect of treatment of the axotomized animals with NGF. A preliminary account of these results has been given (Banks and Walter, 1975). Meanwhile, the results of a similar study on rats (Hendry and Campbell, 1976), and a related study on guinea pigs have also been published (Purves and Nj~, 1976). Materials and methods Nerve growth factor NGF was purified from adult male mouse salivary glands according to the method described by Varon et al., (1967). A dose level of 20 mg/Kg/day was used for all experiments involving NGF.
E f f e c t s o f a x o t o m y a n d N G F o n m o u s e s u p e r i o r cervical ganglia
289
Axotomy 14 day Porton albino mice were anaesthetized with halothane and the right superior cervical ganglion was exposed. Both major postganglionic nerves were sectioned close to the ganglion using a pair of sharp scissors. The ganglion itself was manipulated as little as possible. The left superior cervical ganglion served as an unoperated control. Sham operations were also performed in which the ganglion was exposed and manipulated minimally as before but the postganglionic nerves were left untouched. After the operations the mice were divided into two groups. The mice of one group were each given an injection of 20 mg/Kg of NGF while the control animals of the second group received saline injections. These injections were repeated daily until the day before the mice were killed.
Histology The ganglia were dissected out immediately after the mice were killed and weighed individually on a Kahn balance. They were then fixed in a mixture of 80% ethanol, glacial acetic Acid and 40% formaldehyde (18:1:1 by volume), dehydrated in 90% and absolute alcohol, cleared in benzene and embedded in paraffin wax (m.pt. 56 ~ C). 5/lm sections were cut and stained using Cresyl Fast Violet. To estimate the cell density, the sections were projected on to paper at a final magnification of 2400, using a drawing-mirror attached to a microscope. Neurons were recognized as large cells with a darkly-stained cytoplasm and a lightly-stained round or oval nucleus. The number of neuron nuclei reflected on to a given area of paper (624 cm 2) from a randomly chosen area of each section was determined together with the nuclear diameters. This procedure was carried out for every section from each ganglion. All scores were carried out blind. The apparent density of neurons (NA) in the projected areas was corrected for the inclusion of nuclei of average diameter D in more than one section of thickness T by the method of Abercrombie (1946). The number of units that should be counted, NV, is given by:
T was in all cases 5 ym.
Relative cell n u m b e r per ganglion To obtain a measure of the total number of cells present in each ganglion it was assumed that the specific gravity of the ganglia was unaltered by either growth or postganglionic denervation. Under these conditions the ganglion weights are proportional to the ganglion volumes, and therefore the products of the ganglion weights and the mean cell densities will be proportional to the total number of cells present in the different ganglia. These products yield a series of values that give a measure of any change that occurs in the average number of cells per ganglion as a result of postganglionic denervation or the action of NGF. Results T h e values o b t a i n e d f o r t h e m e a n g a n g l i o n w e i g h t s , t h e a p p a r e n t n e u r o n d e n s i t i e s ( a r b i t r a r y u n i t s ) a n d m e a n n u c l e a r d i a m e t e r s are given w i t h t h e s t a n d a r d e r r o r s o f Table 1. Mice killed at 14 days postpartum
Total no. of ganglia 9
Mean ganglion weight (rag) 156 + 9
No. of fields counted 310
Apparent neuron density NA
Mean nuclear diameter D (#m)
True neuron density Nv
Ganglion weight x neuron density
2 3 . 0 -+ 0.4
7.6 + 0.2
9.1
1.41
8 2 6 8 3 7
Control Sham Operated
Control Sham Operated
199 • 14 217 • 41 152 -+ 22
157 • 10 190 • 20 137 + 18
(mg)
Mean ganglion weight
419 184 322
106 169
176
No. o f fields counted
20.6 • 0.26 14.2 + 0.46 4.8 + 0.19
20.3 + 0.4 16.2 • 0.6 7.1 + 0.4
Apparent neuron density, NA
8.2• 9.1• 8.4•
7.1• 8.9• 6.8•
D (/am)
Mean nuclear diameter
8 2 6 8 3 5
Control Sham Operated
Control Sham Operated
* Identical values for the two ganglia.
35 days
Killed at
21 days
Killed at
Treatment
Total no. o f ganglia
530 • 25 370 + 28 310-+ 43
330-+ 24 390* 310 -+ 38
(mg)
Mean ganglion weight
502 210 343
225 114 277
No. o f fields counted
15.6 + 0.16 15.7 -+ 0.26 12.3 • 0.29
17.3 -+ 0.3 12.4 -+ 0.4 14.0 + 0.4
Apparent neuron density, NA
10.4• 9.4• 9.1•
9.4• 9.0• 8.1•
D (gin)
nuclear diameter
Mean
Table 3, Response to a x o t o m y and NGF. All animals received NGF (20 mg/Kg) postoperatively.
35 days
Killed at
21 days
Killed at
Treatment
Total no. o f ganglia
Table 2. Response to axotomy. All animals received saline postoperatively.
neuron
5.05 5.45 4.36
5.99 4.42 5.36
density, NV
neuron
True
7.8 5.0 1.8
8.0 5.8 3.0
2.67 NC 2.01 NS 1.35 NE
1.97 NC 1.72 NS 1.66 NE
density
neuron
Ganglion weight x
1.56 CC 1.09 CS 0.27 CE
1.25 CC 1.11 CS 0.41 CE
density
density,
Nv
G a nglio n weight x
True neuron
291
Effects of a x o t o m y and NGF on mouse superior cervical ganglia 3.0
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Fig. 1. Effects of a x o t o m y and N G F o n n e u r o n n u m b e r in m o u s e superior cervical ganglion. N, - - - - N G F t r e a t e d animals, C, - - saline i n j e c t e d controls. C, c o n t r o l ganglia, S, s h a m o p e r a t e d ganglia, E, a x o t o m i z e d ganglia. A x o t o m y was carried o u t at t w o weeks.
the mean in the three tables. In Table 1, the results refer to animals killed at 14 days, that is at the same age at which a x o t o m y was carried out on littermates. In Table 2, results are for animals that had been subjected to unilateral axotomy or to sham operations at 14 days and then allowed to survive to 21 days or to 35 days, during which time they received daily injections of saline. In Table 3, the results are T a b l e 4. S u m m a r y o f t h e effects of a x o t o m y a n d t r e a t m e n t w i t h N G F o n relative n u m b e r s of n e u r o n s in m i c e s u p e r i o r cervical ganglia
Ganglion specification
21 days
35 days
Effect of NGF treatment
NC - CC NS - CS NE -- CE
0.69 0.61 1.24
1.11 0.91 1.09
Effect of sham operation
CC - CS NC - NS
0.17 0.31
0.46 0.66
E f f e c t o f a x o t o m y plus manipulation
CC -- CE
0.86
1.30
E f f e c t of a x o t o m y , m a n i p u l a t i o n and NGF treatment
NC - NE
0.31
1.32
E f f e c t of a x o t o m y a l o n e
CS -- CE
0.69
0.84
E f f e c t of a x o t o m y a n d N G F treatment
NS - NE
0.06
0.66
E x p e c t e d value*
NE -- CE
1.34
1.85
*Value e x p e c t e d if N G F p r e v e n t s t h e cell d e a t h d u e t o a x o t o m y (CS - CE) a n d also increases t h e n u m b e r of n e u r o n s ( M e a n of NC - CC, NC - CS a n d NE -- CE)
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BANKS and WALTER
given for animals treated in the same way except that the post-operative injections were of NGF in place of saline. The results are summarized in Fig. 1. The terminology is as follows. The two post-operative treatments are designated C for saline (control) and N for treatment with NGF. The ganglia are designated E for axotomized, S for sham and C for the control (contralateral) ganglion. Each ganglion is therefore specified by two letters, one for the treatment of the animal and the other for the treatment of the ganglion. The problem under investigation is that of cell death. The results, as summarized in Table 4, are given as arbitrary measures of the change in neuron number that results from given treatments and operations at the two post-operative stages of seven and 21 days. It is not possible to give the statistical significance of these figures because groups of ganglia from a given treatment were pooled prior to sectioning. Discussion
Cell death due to axotomy Postganglionic axotomy of sympathetic ganglia has long been known to result in morphological changes in the cell body and axon degeneration distal to the lesion (see Lieberman, 1971). Matthews and Nelson (1975) have reported a 30% decrease in the numbers of neuronal nuclei in sections of rat superior cervical ganglia at some 40 days after axotomy while Purves (1975) has shown that in adult guinea pigs, postganglionic axotomy of the same ganglion results in the death of roughly half the neurons in 30 days. Hendry and Campbell (1976) have recently shown that the numbers of neurons in rat superior cervical ganglia decrease by 30% between 6 and 28 days postpartum in the absence of axotomy and by as much as 90% in animals in which the postganglionic fibres are crushed at 6 days. In the present work on mice, no cell death was found to occur in unoperated animals between 14 and 35 days but section of the major outflows close to the superior cervical ganglia of 14 day mice resulted in a 50% reduction in the number of neurons at 7 days post operation and a 60% drop over three weeks post operation. Comparison of the results of different workers is complicated because the severity of the cell reaction depends on the age of the animal (Lieberman, 1971) the type of injury, i.e. crush, section or ligation (Watson, 1965; Cragg, 1970; Matthews and Raisman, 1972) the proximity of the lesion to the cell bodies (Watson, 1968; Cragg, 1970) and the presence or absence of intact axon collaterals (Fry and Cowan, 1972). The cell death reported here is that due to axotomy alone. A considerable amount of cell death was found to result from sham operation (c.a. 30% in 3 weeks postoperation) and this has been subtracted from the total cell death resulting from axotomy. Treatment of animals with NGF did not effectively reduce the cell death resulting from sham operation (Table 4). The effect of NGF in tbe absence of axotorny A hyperplastic response to NGF was first reported by Levi-Montalcini and Booker (1960) working with mice and confirmed by Zaimis (1972) in rats and kittens. Both
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293
studies were qualitative. In rats, two to three-fold increases in neuron number have been reported to result from the treatment of neonatal animals with NGF (Thoenen et al., 1971, Aloe et al., 1975) but this degree of hyperplasia has not been found by Hendry (1976) when cell counts have been carefully corrected for the split cell error (Banks et al., 1977). In neonatal rats, NGF has been found by Hendry (1976) to give only a 25% increase in neuron number whereas in neonatal mice, the hyperplastic response is considerably greater (Black et al., 1972; Banks et al., 1975). In the latter reference, no corrections for the split cell error were made but application of a simple correction procedure (Abercrombie, 1946) does not alter the conclusions regarding the reported hyperplasia under the influence of NGF. The choice of the type of correction procedure to be used is determined by the effect of a given treatment on the size of the cell or nucleus being counted. In the case of rats, NGF produces a considerable increase in nuclear and cell size (Thoenen e t al., 1971; Aloe et al., 1975; Hendry, 1976), necessitating the more complex correction procedure of Hendry (1976). In mice, on the other hand, the increase in nuclear size is minimal (see Table 5) and therefore the simple correction procedure of Abercrombie (1946) is adequate.
Table 5. Effect of NGF treatment on neuronal nuclear size. Rats*
Micet Average nuclear diameter (/~m)
Average nuclear diameter (/~m) Treatment
Treatment Control Treated
10 mg/Kg. NGF 8 days from 6 days 15 days
10 10
14.5 15.5
Control Treated
20 mg/Kg NGF from 14 days
7 days 21 days
7.6 8.6
8.8 9.6
*Data from Hendry and Campbell, 1976 tData from present work
In mice, the effect of NGF on neuron number is roughly the same in the control and sham operated ganglia both at one week and at three weeks postoperation, amounting to increases of 50% and 70% respectively. It therefore appears that 14 day mice are more responsive to NGF, with respect to neuron number, than are 6 day rats (Hendry and Campbell, 1976). The higher sensitivity of mice than rats may well be due to the presence in the former of a larger number of primitive or pluripotential cells as first suggested by Zaimis (1972). The increase in neuron number between 14 and 35 days under the influence of NGF is not due to mitosis since mitotic figures are not seen beyond 9 days p o s t p a r t u m (Levi-Montalcini and Angeletti, 1968).
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The effect of NGF following axotomy The cell death that follows axotomy of six day rats has been reported (Hendry and Campbell, 1976) to be reversed during a fifteen day treatment with NGF, although it appears from the data given that the number of neurons in the axotomized ganglia of NGF treated animals was consistently lower than in the control ganglia of similarly treated animals. The result was consistent with earlier findings relating to the amounts of the enzymes tyrosine hydroxylase and dopamine decarboxylase in the axotomized ganglia of NGF treated and control animals, (Hendry, 1975). The activities of both of these key enzymes in the synthesis of adrenaline were somewhat decreased by axotomy though the drop was not as dramatic as in the case of the number of neurons. NGF treatment increased the activities of the two enzymes but, in axotomized ganglia, the enzyme levels were never as high as in the control ganglia from NGF treated animals. Both the biochemical and morphological findings have been taken as support for the earlier suggestion (Hendry and Iverson, 1973) that contact with the end organ is necessary for the survival of neurons in sympathetic ganglia. In the present work on 14 day mice, NGF has been shown to prevent the cell death that occurs in the first week after axotomy but at 3 weeks postoperation, the cell death was the same in the axotomized ganglia of mice treated daily with NGF as in the axotomized ganglia of animals receiving only saline. The number of neurons in the axotomized ganglia from NGF treated animals at 3 weeks exceeds that in the axotomized ganglia from saline treated animals but only to the same extent as the difference in cell number induced by NGF in unoperated or sham operated ganglia. If NGF had prevented the cell death due to axotomy as well as causing the normal increase in neuron number, the difference between the neuron numbers in the axotomized ganglia from NGF treated and control animals would have been 70% higher than the difference observed at 35 days. The contrast between the results of treatment of axotomized animals for 7 days and for 21 days is of interest. It suggests that, as in vitro, NGF does play a maintenance role, assisting in the survival of sympathetic neurons or rather in slowing the rate of neuronal death (Levi-Montalcini and Angeletti, 1968; Banthorpe et al., 1974; Banks et aI., 1975). However, we can find no evidence that prolonged treatment with exogenous NGF can, in the end, prevent the effects of axotomy on the adrenergic neurons in mice. This is not to say that retrograde axonal transport of NGF from the end organ is not an essential feature of the long term maintenance of sympathetic neurons. It does rather suggest that this route of supply of NGF is vital and cannot be replaced by exogenous NGF reaching the neurons by other means. Although both blood-borne NGF and NGF transported axonally have been shown to affect sympathetic ganglion cells, this does not necessarily mean that the mechanisms of action are the same in both cases. Indeed, it is difficult to envisage a common mechanism in view of the lack of evidence for the appearance of axonally transported NGF on the external surface of the cell (Paravicini et al., 1975) while exogenous NGF appears to act specifically on the cell surface. Bannerjee et al., (1973) showed that NGF bound specifically to receptors on the surface of
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sympathetic neurons while Frazier et al. (1973) have demonstrated that NGF bound to Sepharose beads maintains its biological activity and, in this form, the factor must certainly be extracellular. It is therefore possible that axonally transported NGF and blood-borne NGF have two distinct effects on sympathetic neurons. The blood-borne factor appears, at least in mice, to stimulate the production of neurons from more primitive or pluripotential cells and to maintain these newly matured cells throughout the period of treatment. It also maintains, for limited periods only, those mature neurons which, having been deprived of an intracellular source of NGF, will eventually die. These results are consistent with the report by Johnson and Aloe (1974) that NGF treatment can suppress for a limited period only the death of sympathetic neurons that results from treatment of neonatal rats with guanethidine. On the other hand, the effects of 6-hydroxydopamine on the same nerve cells has been found to be prevented over prolonged periods (21 days) by simultaneous administration of NGF, (Aloe et al., 1975), a result that leads to the conclusion that exogenous NGF gained access to the cell bodies through the plasma membrane. The chemical axotomy produced by 6-hydroxydopamine may be presumed to have blocked axonal transport of NGF from the end organ. The prolonged survival of the neurons in spite of deprivation of this possible source of NGF is in conflict with the results reported here on the effects of surgical axotomy in mice.
Physiological effects of axotomy and NGF Postganglionic axotomy in adult animals (rabbit and cat) has long been known to result in a temporary depression of synaptic transmission through the sympathetic ganglia (Brown and Pascoe, 1954; Acheson and Schwarzacher, 1956). A recent morphological study by Matthews and Nelson (1975) on the superior cervical ganglion of the adult rat has shown that the altered synaptic transmission may be due to detachment of otherwise normal presynaptic nerve endings presumably caused by changes in the postsynaptic membrane induced by axotomy. The study of Purves (1975) on the superior cervical ganglion of the adult guinea pig has confirmed that the depression of synaptic transmission in the first week following axotomy is paralleled by a decrease in the number of morphologically distinctive synapses. However, the transmission recovers in the third or fourth postoperative week whereas the loss of synapses is irreversible, as is the reduction in the number of neurons (50%) that occurs after the period of maximum synaptic depression. More recently, a brief report has appeared (Purves and Nj~, 1976) showing that NGF applied locally to the superior cervical ganglion of the adult guinea pig markedly reduces the synaptic depression otherwise occurring in the first seven days after axotomy. The authors suggest that NGF may normally regulate synaptic function in mature, sympathetic ganglia. Although this is an interesting possibility, it is also possible that, as in the mouse, the exogenous NGF may merely delay the onset of chromatolytic or other unrecognized changes that precede neuronal death.
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Acknowledgements We thank the SRC for a postdoctoral award to one of us (S.J.W.) and gratefully acknowledge the financial assistance of the Whitehall Foundation. We also wish to thank Dr D. C. Edwards and Professor C. A. Vernon for their continued interest in this work and for valuable discussions. References ABERCROMBIE,
M. (1946)
Estimation of nuclear population from microtome sections.
Anatomical Record 94, 2 7 4 - 3 2 9 . ACHESON, G. H. and SCHWARZACHER, H. G. (1956) Correlations between the physiological changes and the morphological changes resulting from axotomy in the inferior mesenteric ganglion of the cat. Journal of Comparative Neurology 106, 2 4 7 - 6 7 . ALOE, L., MUGNAINI, E. and LEVDMONTALCINI, R. (1975). Light and electron microscopic studies on the excessive growth of sympathetic ganglia in rats injected daily from birth with 6-OHDA and NGF. Archives italienne de Biologic 1 1 3 , 3 2 6 - 5 3 . BANERJEE, S. P., SNYDER, S. H., CUATRECASAS, P. and GREENE, L. A. (1973) Binding of nerve growth factor receptor in sympathetic ganglia. Proceedings of the National Academy of Sciences U.S.A. 70, 2519--23. BANKS, B. E. C. and WALTER, S. J. (1975) The effects of a x o t o m y and nerve growth factor on the neuronal population of the superior cervical ganglion of the mouse. Journal of Physiology 249, 6 1 - 2 P . BANKS, B. E. C., CHARLWOOD, K. A., EDWARDS, D. C., VERNON, C. A. and WALTER, S. J. (1975) Effects of nerve growth factors from salivary glands and snake venoms on the sympathetic ganglia of neonatal and developing mice. Journal of Physiology 247, 2 8 9 - 9 8 . BANKS, B. E. C., HENDRY, I. A. and KHAN, A. A. (1977) A program to compute the sizes and numbers of spherical bodies from observations made on tissue sections. Journal of
Neurocytology 6, 231-9. BANTHORPE, D. V., PEARCE, F. L. and VERNON, C. A. (1974) Effects of nerve growth factor from the venom of Vipera russelti on dispersed sensory ganglion cells from the embryonic chick. Journal of Embryology and Experimental Morphology 31,151--67. BLACK, I. B., HENDRY, I. A. and 1VERSEN, L. L., (1972) Effects of surgical decentralisation and nerve growth factor on the maturation of adrenergic neurons in a mouse sympathetic ganglion. Journal of Neurochemistry 19, 1 3 6 7 - 7 7 . BROWN, G. L. and PASCOE, J. E. (1954) The effect of degenerative section of ganglionic axons on transmission through the ganglion. Journal of Physiology 1 2 3 , 5 6 5 - 7 3 . CHARLWOOD, K. A., LAMONT, D. M. and BANKS, B. E. C. (1972) Apparent orientating effects produced by nerve growth factor. In Nerve Growth Factor and its Anti-Serum (edited by Z AM IS, E.), pp. 102--107. London: Athlone Press. CHAMLEY, J. H., GOLLER, 1. and BURNSTOCK, G. (1973) Selective growth of sympathetic fibres to explants of normally densely innervated autonomic effector organs in tissue culture. Developmental Biology 31, 3 6 2 - 7 9 . CRAGG, B. G. (1970) What is the signal for chromatolysis? Brain Research 23, 1--21. FRAZIER, W. A., BOYD, L. F. and BRADSHAW, R. A. (1973) Interaction of nerve growth factor with surface membranes: biological competence of insolubilised nerve growth factor. Proceedings of the National Academy of Sciences U.S.A. 70, 2931--5. FRY, F. J. and COWAN, W. M. (1972) A study of the retrograde cell degeneration in the lateral mammill~ry nucleus of the cat with special reference to the role of axonal branching in preservation of the cell. Journal of Comparative Neurology 144, 1 - 2 3 .
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HENDRY, 1. A. (1975) The response of adrenergic neurons to axotomy and nerve growth factor. Brain Researcb 94, 87--97. HENDRY, I. A. (1976) A method to correct adequately for the change in neuronal size when estimating neuronal numbers after nerve growth factor treatment. Journal of Neurocytology 5, 337-49. HENDRY, I.A. and CAMPBELL, J. (1976) Morphometric analysis of rat superior cervical ganglion after axotomy and nerve growth factor treatment. Journal of Neurocytology 5, 351-60. HENDRY, I. A. and IVERSEN, L. L. (1973) Changes in tissue and plasma concentrations of nerve growth factor following removal of the submaxillary glands in adult mice and their effects on the sympathetic nervous system. Nature 2 4 3 , 5 0 0 - 4 . HENDRY, I.A., STOECKEL, K., THOENEN, H. and IVERSEN, L. L. (1974) The retrograde axonal transport of nerve growth factor. Brain Research 68, 103--21. JOHNSON, E. M. and ALOE, L. (1974) Suppression of the in vitro and in vivo cytotoxic effects of guanethidine in sympathetic neurons by nerve growth factor. Brain Researcb 81, 5 1 9 - 3 2 . LEV1--MONTALCINI, R. and ANGELETTI, P. U. (1968) Nerve Growth Factor. Pbysiological Reviews 48, 534--69. LEVI--MONTALCINI, R. and BOOKER, B. (1960) Excessive growth of the sympathetic ganglia evoked by a protein isolated from mouse salivary glands. Proceedings oftbe National Academy of Sciences, U.S.A. 46, 373--84. LIEBERMAN, A. R. (1971) The axon reaction: a review of the principal features of perikaryal responses to axon injury. International Review of Neurobiology 14, 4 9 - 1 2 4 . MATTHEWS, M. R., and NELSON, V. H. (1975) Detachment of structurally intact nerve endings from chromatolytic neurones of rat superior cervical ganglion during the depression of synaptic transmission induced by post-ganglionic axotomy. Journal of Physiology 245, 9 1 - 1 3 6 . MATTHEWS, M. R. and RAISMAN, G. (1972) A light and electron microscopical study of the cellular response to axonal injury in the superior cervical ganglion of the rat. Proceedings of tbe Royal Society, Series B 181, 43--79. PARAVICINI, U., sTOECKEL, K and THOENEN, H. (1975)Biological importance of retrograde axonal transport of nerve growth factor in adrenergic neurones. Brain Researcb 84, 279--91. PURVES, D. (1975) Functional and structural changes in mammalian sympathetic neurons following interruption of their axons. Journal of Physiology 2 5 2 , 4 2 9 - 6 3 . PURVES, D. and NJX, A. (1976) Effect of nerve growth factor on synaptic depression after axotomy. Nature 260, 5 3 5 - 6 . STOECKEL, K., PARAVICINI, U. and THOENEN, H. (1974) Specificity of the retrograde axonal transport of nerve growth factor. Brain Researcb 76, 413--21. THOENEN, H., ANGELETTI, P.H., LEVI-MONTALCINI, R. and KETTLER, R. (1971) Selective induction by nerve growth factor of tyrosine hydroxylase and dopamine-hydroxylase in the rat superior cervical ganglion. Proceedings of tbe National Academy of Sciences, U.S.A. 68, 1598--602. VARON, S., NOMURA, J. and SHOOTER, E. M. (1967). The isolation of the mouse nerve growth factor protein in a high molecular weight form. Bio chemistry 6, 2 2 0 2 - 9 . WATSON, W. E. (1965) An autoradiographic study of the incorporation of nucleic acid precursors by neurons and glia during nerve regeneration. Journal of Physiology 180, 7 4 1 - 5 3 . WATSON, W. E. (1968) Observations on the nucleolar and total cell body nucleic acid of injured nerve cells. Journal of Physiology 196, 6 5 5 - 7 6 . ZAIMIS, E. (1972). Nerve Growth Factor: the target cells. In Nerve Growth Factor and Its Antiserum (edited by ZAIMIS, E.) pp. 59--70, Athlone Press: London.
The effects of postganglionic axotomy and nerve growth factor on the superior cervical ganglia of developing mice BARBARA
E. C. B A N K S
and S. J. W A L T E R *
Department of Pbysiology, University College London, Gower Street, London WC1E 6BT
Received 23 November 1976; revised 26 January 1977; accepted 28 January 1977
Summary Sectioning of the two major outflows from the superior cervical ganglia in two week mice results in a marked drop in the number of neurons within one week of operation and a smaller drop over the following two weeks. In animals receiving daily injections of nerve growth factor (NGF), the effect of axotomy is modified. One week after axotomy, the number of neurons in the axotomized ganglia is approxima the same in NGF treated animals as in the control, sham operated ganglia. Over the next two weeks, however, the cell death that results from axotomy is no longer prevented by treatment with NGF. The normal, hyperplastic response to NGF appears to occur independently of the cell reaction caused by axotomy.
Introduction
It has long been known (Levi-Montalcini and Booker, 1960) that members of the group of proteins called Nerve growth factor (NGF) cause enlargement of the sympathetic chain ganglia when injected into neonatal mammals. Mice are particularly sensitive in their response, treatment for six days giving superior cervical ganglia that are four to six times larger than the ganglia from control animals. Kittens and rats were reported by Zaimis (1972) to give a less marked response but there may be some differences in the response of different strains of rats since, in recent reports, (Aloe, Mugnaini and Levi-Montalcini, 1975; Hendry and Campbell, 1976), three to four-fold increases in the volume of rat superior cervical ganglia are recorded. The increase in ganglion size induced by NGF in mice has been shown (Banks et al., 1975) to be due to an increase in the rate at which neurons are produced from less differentiated cells, as first suggested by Zaimis (1972). The hyperplastic response resulted, in a 6 day old, treated mouse, in more neurons than were present in the *Present address: Department of Physiology, Charing Cross Hospital Medical School, London 9 1977 Chapman and Hall Ltd. Printed in Great Britain.
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adult. The excess neurons rapidly disappeared when NGF treatment ceased but were maintained when the NGF treatment was prolonged. This is consistent with the maintenance role played by NGF in vitro (Banthrope et al., 1974). Both Levi-Montalcini and Zaimis with their coworkers had concluded from the qualitative studies that NGF treatment induced both hyperplasia and hypertrophy of sympathetic neurons. This impression was confirmed by Black, Hendry and Iversen (1972) who showed that treatment of 4 day mice with NGF for 10 days approximately doubled the number of neurons in the superior cervical ganglia. The cell counts on which this conclusion was based were corrected for the split cell error by the method of Abercrombie (1946). More recently, Hendry (1976) has described a more accurate method of assessing cell numbers, applicable where a given treatment results in a significant change in cell size. Using this method, Hendry has shown that in neonatal rats, NGF causes a much smaller increase in cell number (ca. 25% in 5 days) even when the treatment is started immediately postpartum. The increase in ganglion volume in rats was shown (Hendry and Campbell, 1976) to be due largely to proliferation of non-neuronal cells. This finding contrasts with the report (Aloe, et al., 1975), that NGF treatment for 4 days from birth doubles the number of neurons in the rat superior cervical ganglion. The estimates of cell number in this work are, however, only semi-quantitative. No standard corrections were applied, nor were the data analysed statistically. It does seem that the responses of rats and of mice to NGF are quantitatively rather different. Experiments in vitro (Charlwood et al., 1972; Chamley et al., 1973) have shown that NGF may influence the orientation of sympathetic fibres to their end organ and, furthermore, it has been suggested (Hendry and Iversen, 1973) that the end organ may maintain sympathetic neurons by production of NGF which is transported up the axon to the cell body. Retrograde axonal transport of NGF has since been confirmed (Hendry et al., 1974; Stoeckel et al., 1974; Paravicini et al., 1975). If such transport were physiologically significant, it might be expected that interruption of the supply of NGF from the end organ by postganglionic axotomy would result in neuron death and that such death might be prevented by treatment of axotomized animals with NGF. We wish to report the morphological results obtained from total postganglionic axotomy of the superior cervical ganglia of two week mice and the effect of treatment of the axotomized animals with NGF. A preliminary account of these results has been given (Banks and Walter, 1975). Meanwhile, the results of a similar study on rats (Hendry and Campbell, 1976), and a related study on guinea pigs have also been published (Purves and Nj~, 1976). Materials and methods Nerve growth factor NGF was purified from adult male mouse salivary glands according to the method described by Varon et al., (1967). A dose level of 20 mg/Kg/day was used for all experiments involving NGF.
E f f e c t s o f a x o t o m y a n d N G F o n m o u s e s u p e r i o r cervical ganglia
289
Axotomy 14 day Porton albino mice were anaesthetized with halothane and the right superior cervical ganglion was exposed. Both major postganglionic nerves were sectioned close to the ganglion using a pair of sharp scissors. The ganglion itself was manipulated as little as possible. The left superior cervical ganglion served as an unoperated control. Sham operations were also performed in which the ganglion was exposed and manipulated minimally as before but the postganglionic nerves were left untouched. After the operations the mice were divided into two groups. The mice of one group were each given an injection of 20 mg/Kg of NGF while the control animals of the second group received saline injections. These injections were repeated daily until the day before the mice were killed.
Histology The ganglia were dissected out immediately after the mice were killed and weighed individually on a Kahn balance. They were then fixed in a mixture of 80% ethanol, glacial acetic Acid and 40% formaldehyde (18:1:1 by volume), dehydrated in 90% and absolute alcohol, cleared in benzene and embedded in paraffin wax (m.pt. 56 ~ C). 5/lm sections were cut and stained using Cresyl Fast Violet. To estimate the cell density, the sections were projected on to paper at a final magnification of 2400, using a drawing-mirror attached to a microscope. Neurons were recognized as large cells with a darkly-stained cytoplasm and a lightly-stained round or oval nucleus. The number of neuron nuclei reflected on to a given area of paper (624 cm 2) from a randomly chosen area of each section was determined together with the nuclear diameters. This procedure was carried out for every section from each ganglion. All scores were carried out blind. The apparent density of neurons (NA) in the projected areas was corrected for the inclusion of nuclei of average diameter D in more than one section of thickness T by the method of Abercrombie (1946). The number of units that should be counted, NV, is given by:
T was in all cases 5 ym.
Relative cell n u m b e r per ganglion To obtain a measure of the total number of cells present in each ganglion it was assumed that the specific gravity of the ganglia was unaltered by either growth or postganglionic denervation. Under these conditions the ganglion weights are proportional to the ganglion volumes, and therefore the products of the ganglion weights and the mean cell densities will be proportional to the total number of cells present in the different ganglia. These products yield a series of values that give a measure of any change that occurs in the average number of cells per ganglion as a result of postganglionic denervation or the action of NGF. Results T h e values o b t a i n e d f o r t h e m e a n g a n g l i o n w e i g h t s , t h e a p p a r e n t n e u r o n d e n s i t i e s ( a r b i t r a r y u n i t s ) a n d m e a n n u c l e a r d i a m e t e r s are given w i t h t h e s t a n d a r d e r r o r s o f Table 1. Mice killed at 14 days postpartum
Total no. of ganglia 9
Mean ganglion weight (rag) 156 + 9
No. of fields counted 310
Apparent neuron density NA
Mean nuclear diameter D (#m)
True neuron density Nv
Ganglion weight x neuron density
2 3 . 0 -+ 0.4
7.6 + 0.2
9.1
1.41
8 2 6 8 3 7
Control Sham Operated
Control Sham Operated
199 • 14 217 • 41 152 -+ 22
157 • 10 190 • 20 137 + 18
(mg)
Mean ganglion weight
419 184 322
106 169
176
No. o f fields counted
20.6 • 0.26 14.2 + 0.46 4.8 + 0.19
20.3 + 0.4 16.2 • 0.6 7.1 + 0.4
Apparent neuron density, NA
8.2• 9.1• 8.4•
7.1• 8.9• 6.8•
D (/am)
Mean nuclear diameter
8 2 6 8 3 5
Control Sham Operated
Control Sham Operated
* Identical values for the two ganglia.
35 days
Killed at
21 days
Killed at
Treatment
Total no. o f ganglia
530 • 25 370 + 28 310-+ 43
330-+ 24 390* 310 -+ 38
(mg)
Mean ganglion weight
502 210 343
225 114 277
No. o f fields counted
15.6 + 0.16 15.7 -+ 0.26 12.3 • 0.29
17.3 -+ 0.3 12.4 -+ 0.4 14.0 + 0.4
Apparent neuron density, NA
10.4• 9.4• 9.1•
9.4• 9.0• 8.1•
D (gin)
nuclear diameter
Mean
Table 3, Response to a x o t o m y and NGF. All animals received NGF (20 mg/Kg) postoperatively.
35 days
Killed at
21 days
Killed at
Treatment
Total no. o f ganglia
Table 2. Response to axotomy. All animals received saline postoperatively.
neuron
5.05 5.45 4.36
5.99 4.42 5.36
density, NV
neuron
True
7.8 5.0 1.8
8.0 5.8 3.0
2.67 NC 2.01 NS 1.35 NE
1.97 NC 1.72 NS 1.66 NE
density
neuron
Ganglion weight x
1.56 CC 1.09 CS 0.27 CE
1.25 CC 1.11 CS 0.41 CE
density
density,
Nv
G a nglio n weight x
True neuron
291
Effects of a x o t o m y and NGF on mouse superior cervical ganglia 3.0
.>.
.~ONC
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_ --.0
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i
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4 Age (weeks)
S
Fig. 1. Effects of a x o t o m y and N G F o n n e u r o n n u m b e r in m o u s e superior cervical ganglion. N, - - - - N G F t r e a t e d animals, C, - - saline i n j e c t e d controls. C, c o n t r o l ganglia, S, s h a m o p e r a t e d ganglia, E, a x o t o m i z e d ganglia. A x o t o m y was carried o u t at t w o weeks.
the mean in the three tables. In Table 1, the results refer to animals killed at 14 days, that is at the same age at which a x o t o m y was carried out on littermates. In Table 2, results are for animals that had been subjected to unilateral axotomy or to sham operations at 14 days and then allowed to survive to 21 days or to 35 days, during which time they received daily injections of saline. In Table 3, the results are T a b l e 4. S u m m a r y o f t h e effects of a x o t o m y a n d t r e a t m e n t w i t h N G F o n relative n u m b e r s of n e u r o n s in m i c e s u p e r i o r cervical ganglia
Ganglion specification
21 days
35 days
Effect of NGF treatment
NC - CC NS - CS NE -- CE
0.69 0.61 1.24
1.11 0.91 1.09
Effect of sham operation
CC - CS NC - NS
0.17 0.31
0.46 0.66
E f f e c t o f a x o t o m y plus manipulation
CC -- CE
0.86
1.30
E f f e c t of a x o t o m y , m a n i p u l a t i o n and NGF treatment
NC - NE
0.31
1.32
E f f e c t of a x o t o m y a l o n e
CS -- CE
0.69
0.84
E f f e c t of a x o t o m y a n d N G F treatment
NS - NE
0.06
0.66
E x p e c t e d value*
NE -- CE
1.34
1.85
*Value e x p e c t e d if N G F p r e v e n t s t h e cell d e a t h d u e t o a x o t o m y (CS - CE) a n d also increases t h e n u m b e r of n e u r o n s ( M e a n of NC - CC, NC - CS a n d NE -- CE)
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BANKS and WALTER
given for animals treated in the same way except that the post-operative injections were of NGF in place of saline. The results are summarized in Fig. 1. The terminology is as follows. The two post-operative treatments are designated C for saline (control) and N for treatment with NGF. The ganglia are designated E for axotomized, S for sham and C for the control (contralateral) ganglion. Each ganglion is therefore specified by two letters, one for the treatment of the animal and the other for the treatment of the ganglion. The problem under investigation is that of cell death. The results, as summarized in Table 4, are given as arbitrary measures of the change in neuron number that results from given treatments and operations at the two post-operative stages of seven and 21 days. It is not possible to give the statistical significance of these figures because groups of ganglia from a given treatment were pooled prior to sectioning. Discussion
Cell death due to axotomy Postganglionic axotomy of sympathetic ganglia has long been known to result in morphological changes in the cell body and axon degeneration distal to the lesion (see Lieberman, 1971). Matthews and Nelson (1975) have reported a 30% decrease in the numbers of neuronal nuclei in sections of rat superior cervical ganglia at some 40 days after axotomy while Purves (1975) has shown that in adult guinea pigs, postganglionic axotomy of the same ganglion results in the death of roughly half the neurons in 30 days. Hendry and Campbell (1976) have recently shown that the numbers of neurons in rat superior cervical ganglia decrease by 30% between 6 and 28 days postpartum in the absence of axotomy and by as much as 90% in animals in which the postganglionic fibres are crushed at 6 days. In the present work on mice, no cell death was found to occur in unoperated animals between 14 and 35 days but section of the major outflows close to the superior cervical ganglia of 14 day mice resulted in a 50% reduction in the number of neurons at 7 days post operation and a 60% drop over three weeks post operation. Comparison of the results of different workers is complicated because the severity of the cell reaction depends on the age of the animal (Lieberman, 1971) the type of injury, i.e. crush, section or ligation (Watson, 1965; Cragg, 1970; Matthews and Raisman, 1972) the proximity of the lesion to the cell bodies (Watson, 1968; Cragg, 1970) and the presence or absence of intact axon collaterals (Fry and Cowan, 1972). The cell death reported here is that due to axotomy alone. A considerable amount of cell death was found to result from sham operation (c.a. 30% in 3 weeks postoperation) and this has been subtracted from the total cell death resulting from axotomy. Treatment of animals with NGF did not effectively reduce the cell death resulting from sham operation (Table 4). The effect of NGF in tbe absence of axotorny A hyperplastic response to NGF was first reported by Levi-Montalcini and Booker (1960) working with mice and confirmed by Zaimis (1972) in rats and kittens. Both
Effects of axotomy and NGF on mouse superior cervical ganglia
293
studies were qualitative. In rats, two to three-fold increases in neuron number have been reported to result from the treatment of neonatal animals with NGF (Thoenen et al., 1971, Aloe et al., 1975) but this degree of hyperplasia has not been found by Hendry (1976) when cell counts have been carefully corrected for the split cell error (Banks et al., 1977). In neonatal rats, NGF has been found by Hendry (1976) to give only a 25% increase in neuron number whereas in neonatal mice, the hyperplastic response is considerably greater (Black et al., 1972; Banks et al., 1975). In the latter reference, no corrections for the split cell error were made but application of a simple correction procedure (Abercrombie, 1946) does not alter the conclusions regarding the reported hyperplasia under the influence of NGF. The choice of the type of correction procedure to be used is determined by the effect of a given treatment on the size of the cell or nucleus being counted. In the case of rats, NGF produces a considerable increase in nuclear and cell size (Thoenen e t al., 1971; Aloe et al., 1975; Hendry, 1976), necessitating the more complex correction procedure of Hendry (1976). In mice, on the other hand, the increase in nuclear size is minimal (see Table 5) and therefore the simple correction procedure of Abercrombie (1946) is adequate.
Table 5. Effect of NGF treatment on neuronal nuclear size. Rats*
Micet Average nuclear diameter (/~m)
Average nuclear diameter (/~m) Treatment
Treatment Control Treated
10 mg/Kg. NGF 8 days from 6 days 15 days
10 10
14.5 15.5
Control Treated
20 mg/Kg NGF from 14 days
7 days 21 days
7.6 8.6
8.8 9.6
*Data from Hendry and Campbell, 1976 tData from present work
In mice, the effect of NGF on neuron number is roughly the same in the control and sham operated ganglia both at one week and at three weeks postoperation, amounting to increases of 50% and 70% respectively. It therefore appears that 14 day mice are more responsive to NGF, with respect to neuron number, than are 6 day rats (Hendry and Campbell, 1976). The higher sensitivity of mice than rats may well be due to the presence in the former of a larger number of primitive or pluripotential cells as first suggested by Zaimis (1972). The increase in neuron number between 14 and 35 days under the influence of NGF is not due to mitosis since mitotic figures are not seen beyond 9 days p o s t p a r t u m (Levi-Montalcini and Angeletti, 1968).
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The effect of NGF following axotomy The cell death that follows axotomy of six day rats has been reported (Hendry and Campbell, 1976) to be reversed during a fifteen day treatment with NGF, although it appears from the data given that the number of neurons in the axotomized ganglia of NGF treated animals was consistently lower than in the control ganglia of similarly treated animals. The result was consistent with earlier findings relating to the amounts of the enzymes tyrosine hydroxylase and dopamine decarboxylase in the axotomized ganglia of NGF treated and control animals, (Hendry, 1975). The activities of both of these key enzymes in the synthesis of adrenaline were somewhat decreased by axotomy though the drop was not as dramatic as in the case of the number of neurons. NGF treatment increased the activities of the two enzymes but, in axotomized ganglia, the enzyme levels were never as high as in the control ganglia from NGF treated animals. Both the biochemical and morphological findings have been taken as support for the earlier suggestion (Hendry and Iverson, 1973) that contact with the end organ is necessary for the survival of neurons in sympathetic ganglia. In the present work on 14 day mice, NGF has been shown to prevent the cell death that occurs in the first week after axotomy but at 3 weeks postoperation, the cell death was the same in the axotomized ganglia of mice treated daily with NGF as in the axotomized ganglia of animals receiving only saline. The number of neurons in the axotomized ganglia from NGF treated animals at 3 weeks exceeds that in the axotomized ganglia from saline treated animals but only to the same extent as the difference in cell number induced by NGF in unoperated or sham operated ganglia. If NGF had prevented the cell death due to axotomy as well as causing the normal increase in neuron number, the difference between the neuron numbers in the axotomized ganglia from NGF treated and control animals would have been 70% higher than the difference observed at 35 days. The contrast between the results of treatment of axotomized animals for 7 days and for 21 days is of interest. It suggests that, as in vitro, NGF does play a maintenance role, assisting in the survival of sympathetic neurons or rather in slowing the rate of neuronal death (Levi-Montalcini and Angeletti, 1968; Banthorpe et al., 1974; Banks et aI., 1975). However, we can find no evidence that prolonged treatment with exogenous NGF can, in the end, prevent the effects of axotomy on the adrenergic neurons in mice. This is not to say that retrograde axonal transport of NGF from the end organ is not an essential feature of the long term maintenance of sympathetic neurons. It does rather suggest that this route of supply of NGF is vital and cannot be replaced by exogenous NGF reaching the neurons by other means. Although both blood-borne NGF and NGF transported axonally have been shown to affect sympathetic ganglion cells, this does not necessarily mean that the mechanisms of action are the same in both cases. Indeed, it is difficult to envisage a common mechanism in view of the lack of evidence for the appearance of axonally transported NGF on the external surface of the cell (Paravicini et al., 1975) while exogenous NGF appears to act specifically on the cell surface. Bannerjee et al., (1973) showed that NGF bound specifically to receptors on the surface of
Effects of axotomy and NGF on mouse superior cervical ganglia
295
sympathetic neurons while Frazier et al. (1973) have demonstrated that NGF bound to Sepharose beads maintains its biological activity and, in this form, the factor must certainly be extracellular. It is therefore possible that axonally transported NGF and blood-borne NGF have two distinct effects on sympathetic neurons. The blood-borne factor appears, at least in mice, to stimulate the production of neurons from more primitive or pluripotential cells and to maintain these newly matured cells throughout the period of treatment. It also maintains, for limited periods only, those mature neurons which, having been deprived of an intracellular source of NGF, will eventually die. These results are consistent with the report by Johnson and Aloe (1974) that NGF treatment can suppress for a limited period only the death of sympathetic neurons that results from treatment of neonatal rats with guanethidine. On the other hand, the effects of 6-hydroxydopamine on the same nerve cells has been found to be prevented over prolonged periods (21 days) by simultaneous administration of NGF, (Aloe et al., 1975), a result that leads to the conclusion that exogenous NGF gained access to the cell bodies through the plasma membrane. The chemical axotomy produced by 6-hydroxydopamine may be presumed to have blocked axonal transport of NGF from the end organ. The prolonged survival of the neurons in spite of deprivation of this possible source of NGF is in conflict with the results reported here on the effects of surgical axotomy in mice.
Physiological effects of axotomy and NGF Postganglionic axotomy in adult animals (rabbit and cat) has long been known to result in a temporary depression of synaptic transmission through the sympathetic ganglia (Brown and Pascoe, 1954; Acheson and Schwarzacher, 1956). A recent morphological study by Matthews and Nelson (1975) on the superior cervical ganglion of the adult rat has shown that the altered synaptic transmission may be due to detachment of otherwise normal presynaptic nerve endings presumably caused by changes in the postsynaptic membrane induced by axotomy. The study of Purves (1975) on the superior cervical ganglion of the adult guinea pig has confirmed that the depression of synaptic transmission in the first week following axotomy is paralleled by a decrease in the number of morphologically distinctive synapses. However, the transmission recovers in the third or fourth postoperative week whereas the loss of synapses is irreversible, as is the reduction in the number of neurons (50%) that occurs after the period of maximum synaptic depression. More recently, a brief report has appeared (Purves and Nj~, 1976) showing that NGF applied locally to the superior cervical ganglion of the adult guinea pig markedly reduces the synaptic depression otherwise occurring in the first seven days after axotomy. The authors suggest that NGF may normally regulate synaptic function in mature, sympathetic ganglia. Although this is an interesting possibility, it is also possible that, as in the mouse, the exogenous NGF may merely delay the onset of chromatolytic or other unrecognized changes that precede neuronal death.
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Acknowledgements We thank the SRC for a postdoctoral award to one of us (S.J.W.) and gratefully acknowledge the financial assistance of the Whitehall Foundation. We also wish to thank Dr D. C. Edwards and Professor C. A. Vernon for their continued interest in this work and for valuable discussions. References ABERCROMBIE,
M. (1946)
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