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Target dependence of Nissl body ultrastructure in cat thoracic motoneurones
Neuroscience Letters, 61 (1985) 201-205 Elsevier Scientific Publishers Ireland Ltd.
201
NSL 03599 TARGET DEPENDENCE OF NISSL BODY ULTRASTRUCTURE THORACIC MOTONEURONES
IN CAT
I.P. JOHNSON, A.H. PULLEN and T.A. SEARS* Sobell Department of Neurophysiology, Institute of Neurology, Queen Square, London WC1N 3BG ( U.K.)
(Received July 20th, 1985; Revised version received August 1st, 1985; Accepted August 2nd, 1985)
Key words: axotomy - target - neuroma - Nissl - motoneuron - horseradish peroxidase - cat
The effects on Nissl body (NB) ultrastructure of muscle reinnervation or neuroma formation were determined in cytochemically identified cat thoracic motoneurones subjected to axotomy by either nerve crush or nerve section with proximal ligation. Normal NB ultrastructure comprised highly ordered lamellae of rough endoplasmic reticulum (RER) with associated linear arrays of unbound polyribosomes. This ultrastructural orderliness was lost following axotomy, with or without light microscopic chromatolysis. While NBs were seen in the light microscope at late stages following both nerve crush or ligation, normal NB ultrastructure was only observed following nerve crush, An inductive effect of the periphery on NB ultrastructure is proposed and the implications of NB ultrastructure discussed in relation to protein synthesis.
M a t u r e m o t o n e u r o n e s d e m o n s t r a t e a m a j o r d e p e n d e n c e on the p e r i p h e r y for n o r m a l m a i n t e n a n c e as revealed t h r o u g h their r e t r o g r a d e responses to a x o t o m y , interr u p t i o n o f a x o n a l t r a n s p o r t or b l o c k a d e o f n e u r o m u s c u l a r t r a n s m i s s i o n [5, 8, 15]. T o investigate further this t a r g e t - d e p e n d e n c e , we have used the p a r a d i g m o f 'reversible' a x o t o m y (nerve crush) o r ' c h r o n i c ' a x o t o m y (transection with p r o x i m a l ligation) o f intercostal nerves in a d u l t cats to s t u d y changes in Nissl b o d y (NB) ultrastructure as a m e a s u r e o f altered p r o t e i n synthesis. This a p p r o a c h follows o u r experience with the t o p o g r a p h i c a l l y distinct N B located p o s t s y n a p t i c a l l y to the Ctype s y n a p s e [10]. W i t h the c h r o n i c p a r t i a l central d e a f f e r e n t a t i o n o f intercostal m o t o n e u r o n e s that occurs following spinal hemisection, there is h y p e r t r o p h y o f this synapse a c c o m p a n i e d b y an increase in size a n d a c h a n g e in the r i b o s o m a l o r g a n i z a tion o f its NB. Since the synthesis o f p a r t i c u l a r classes o f p r o t e i n is associated with p a r t i c u l a r forms o f r i b o s o m a l o r g a n i z a t i o n [7], f u n c t i o n a l correlates can be inferred [11], a n d in the p r e s e n t study this a p p r o a c h has been carried to the analysis o f N B s in n o r m a l a n d a x o t o m i z e d m o t o n e u r o n e s . I n t e r c o s t a l nerves were e x p o s e d in two n o n - a d j a c e n t segments 20 m m f r o m the m i d - d o r s a l line a n d either (a) c r u s h e d with fine forceps, o r (b) tightly ligated, sect i o n e d a n d a 5-ram p o r t i o n o f nerve r e m o v e d distally. A x o t o m i z e d m o t o n e u r o n e s were e x a m i n e d 1, 4, 8, 33, 64 a n d 208 d a y s later. T o p r o v i d e an i n d e p e n d e n t m a r k e r *Author for correspondence. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
202 of axotomized motoneurones, motoneurones were retrogradely labelled by horseradish peroxidase (HRP). For the one-day survival period, H R P (40~o in saline) was applied directly to the site of the original axotomy; for longer survivals, animals were anaesthetized one day prior to perfusion, the nerves were reexposed and newly lesioned 2 m m proximal to the original lesion for application of the HRP. Animals were perfuse-fixed with 2~o glutaraldehyde-l~o paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and 70-#m slices of spinal cord processed to demonstrate H R P activity [1]. Osmicated sections were dehydrated and fiat-embedded in a thin layer of Araldite between two PTFE-coated microscope slides [12]. HRP-labelled cell bodies were identified by light microscopy (LM) in trans-illuminated 70-#m plastic sections; the same motoneurones were reidentified by LM in 0.5-#m toluidine bluestained sections and subsequently analysed by electron microscopy (EM). Using classical criteria, normal NBs were identified by LM in 0.5-pm toluidine blue-stained sections as irregular patches of cytoplasmic basophilia (Fig. l a). Adjacent ultra-thin sections revealed that the ultrastructure of the same NBs comprised highly ordered stacks of rough endoplasmic reticulum (RER) and linear arrays of polyribosomes between individual lamellae (Fig. 2a). These polyribosomes are described as 'lamellae-associated polyribosomes' to distinguish them from ribosomes elsewhere in the cytoplasm and single ribosomes bound to the endoplasmic reticulum, a nomenclature we introduced previously for the C-type synapse NBs [1 1].
Fig. 1. Toluidine blue-stained sections (0.5-/~msections) of cat thoracic motoneuronal cell bodies, a: normal motoneurone with prominent NBs (arrow). b: motoneurone 4 days following nerve crush, NBs (arrow) can still be seen. c: NBs (arrow) within a motoneurone 64 days following nerve transection, d: NBs (arrow) within a motoneurone 64 days following nerve crush. Bar = 25/*m.
203
Fig. 2. Electron micrographs of the NBs arrowed in Fig. 1. a: normal NB exhibiting highly organized RER lamellae and numerous interposed 'lamellae-associated' polyribosomes(arrows). b: abnormal NB 4 days followingnervecrush comprisingan aggregateof polyribosomesinterspersedwith short RER fragments, c: abnormal NB 64 days followingnerve transection, d: normal NB 64 days followingnerve crush. Bar= 1/lm.
Early stages following axotomy (1-33 days). Normal NBs were seen by both LM and EM one day following either chronic or reversible axotomy. Apparently 'normal' NBs were still present by LM 4 days following reversible axotomy (Fig. lb), but by EM the same NBs had lost their normal orderly structure. They completely lacked the stacks of parallel lamellae of RER and were composed instead simply of aggregates of polyribosomes within which only short fragments of RER were present (Fig. 2b). By 4 days, following chronic axotomy, LM showed early chromatolysis in which the motoneurons exhibited diffuse, cytoplasmic basophilia due to Nissi fragmentation and dispersion. By EM the Nissl fragments were comprised simply of small aggregates of polyribosomes and no lamellae were present. Thus, the difusse basophilia in the light microscope corresponded to a reduction in size of the polyribosomal aggregates close to the limits of resolution by LM, but not the NB disorganization per se. By LM, chromatolysis was fully developed between 8 and 33 days following reversible or chronic axotomy, the ultrastructure of the two types o f axotomized motoneurones being indistinguishable. Late stages following axotomy (64-208 days). By LM, many motoneurones were still chromatolytic at 64-208 days, while others, irrespective of whether axotomy was chronic or reversible, had apparently reformed normal NBs (cf. ref. 13). However, by EM, a striking difference in NB ultrastructure was apparent. In chronically axotomized motoneurones, NBs were composed of dense aggregates of randomly sited polyribosomes, interspersed with only short fragments of RER (Figs. lc and 2c). In contrast, with reversible axotomy, NBs now displayed their normal, ordered appear-
204
ance due to the regeneration of long lengths of RER arranged in stacks with lamellaassociated polyribosomes (Figs. ld and 2d). From measurements on photomontages of individual perikarya, we estimate that for chronically axotomized motoneurones, the total length of R E R within the ribosomal aggregates is only 30--40~ of that organized as parallel lamellae in motoneurones subject to reversible axotomy. Examination of the distal nerve stumps at 64 and 208 days confirmed that axonal regeneration had, or had not, occurred following reversible or chronic axotomy, respectively. To demonstrate further this dependence of the normal Nissl ultrastructure on the periphery, axons terminating in a neuroma of the external intercostal nerve were resectioned at 49 days and allowed to regenerate into the distal stump of a newly sectioned internal intercostal nerve of the same segment. When examined 64 days later, normal NBs had reformed within some of the HRP-identified external intercostal motoneurones, but none were present in control, chronically axotomized motoneurones in the adjacent-but-one segment. By utilizing an independent marker of axotomized motoneurones, we have identified two distinct forms of NB ultrastructure. A 'normal' one comprising stacks of RER lamellae with linear arrays of lamella-associated polyribosomes between individual RER cisternae. The other ('abnormal') is disorganized, comprising aggregates of free polyribosomes interspersed with short R E R fragments (abnormal Nissl). The former characterizes both normal motoneurones and motoneurones from 64 days post-axotomy when axonal regeneration is successful (reversible axotomy). The latter characterizes axotomized motoneurones ending in a neuroma (chronic axotomy). These differences in Nissl ultrastructure are unlikely to depend on the synaptic loss associated with axotomy (cf. ref. 2), since normal Nissl ultrastructure, including that of the C-type synapse, persists in the face of extensive synaptic loss due to central deafferentation [10], comparable in extent to the synaptic depletion associated with axotomy of intercostal motoneurones [4]. Nor is axonal elongation a sufficient condition, since this was actively proceeding at 33 days when Nissl regeneration was absent. These observations also discount Schwann cells or the peripheral nerve sheath as sources of a signal for NB regeneration, notwithstanding the role such signals play in axonal elongation [6, 9]. Thus 'normal' Nissl ultrastructure appears to depend on either the presynaptic motor nerve terminal or muscle. In the former case, an orthogradely transported inactive protein might be converted post-translationally (e.g. by tRNA-mediated aminoacylation [3, 16]) to serve, after retrograde transport, as a signal governing gene expression, either causing their induction de novo, or acting as a repressor of the growth-associated proteins (GAPs) that characterize the regenerating state [17]. Alternatively, a muscle-derived signal, acting independently or enabling the presynaptic mechanism discussed above, would represent a further postsynaptic example of a target-dependent neurotrophic signal [14]. Whatever its source, the signal(s) is likely to operate through transcriptional controls that codetermine on the one hand the synthesis of additional endoplasmic reticulum bearing membrane receptors (ribophorins, signal recognition proteins, etc.) that allow the co-translational insertion and vectorial discharge of membrane and secretory proteins; and on the other, the formation of cytoskeletal elements that create the highly ordered structure
205 o f t h e n o r m a l N B . T h i s m a t u r a t i o n m i g h t well d e p e n d o n the a p p e a r a n c e o f a crossl i n k i n g n e u r o f i l a m e n t p r o t e i n m a i n t a i n i n g t h e o r d e r l y c o n f i g u r a t i o n o f the R E R a n a l o g o u s to the M - p o l y p e p t i d e , w h o s e a p p e a r a n c e d u r i n g d e v e l o p m e n t m a r k s t h e t r a n s i t i o n o f the a x o n a l c y t o s k e l e t o n f r o m a state o f g r o w t h to a state o f stability [17]. S u p p o r t e d b y g r a n t s to T . A . S . f r o m T h e N a t i o n a l F u n d f o r R e s e a r c h I n t o C r i p pling Diseases and The International Spinal Research Trust. 1 Adams, J.C., Technical considerations on the use of horseradish peroxidase as a neuronal marker, Neuroscience, 2 (1977) 141-145. 2 Blinzinger, K. and Kreutzberg, G., Displacement of synaptic terminals from regenerating motoneurones by microglial cells, Z. Zellforsch. Mikrosk. Anat., 85 (1968) 145-157. 3 Ingoglia, N.A., Zanakis, M.F. and Chakraborty, G., Transfer-RNA-mediated posttranslational aminoacylation of proteins in axons. In J.S. Elam and P. Cancalon (Eds.), Axonal Transport in Neuronal Growth and Regeneration (Advances in Neurochemistry, Vol. 6), Plenum Press, London, 1984, pp. 119-136. 4 Johnson, I.P., Morphological Correlates of Altered Protein Synthesis: an Ultrastructural Analysis of Axotomy and Diphtheritic Intoxication, Ph.D. Thesis, University of London, 1983. 5 Lieberman, A.R., The axon reaction: a review of the principle features of perikaryal responses to axon injury, Int. Rev. Neurobiol., 14 (1971) 49-124. 6 Lundborg, G., Longo, F.M. and Varon, S., Nerve regeneration model and trophic factors in vivo, Brain Res., 232 (1982) 157-161. 7 Palade, G., Intracellular aspects of protein synthesis, Science, 189 (1975) 347-358. 8 Pilar, G. and Landmesser, L., Axotomy mimicked by localized colchicine application, Science, 177 (1972) 1116--1118. 9 Politis, M.J., Ederle, K. and Spencer, P.S., Tropism in nerve regeneration in vivo. Attraction of regenerating axons by diffusible factors derived from ceils in distal stumps of transected peripheral nerves, Brain Res., 253 (1982) 1-12. 10 Pullen, A.H. and Sears, T.A., Modification of 'C' synapses following partial deafferentation of thoracic motoneurones, Brain Res., 145 (1978) 141-146. 11 Pullen, A.H. and Sears, T.A., Trophism between C-type axon terminals and thoracic motoneurones in the cat, J. Physiol. (Lond.), 337 (1983) 373-388. 12 Romanovicz, D.K. and Hanker, J.S., Wafer embedding: specimen selection in electron microscopic cytochemistry with osmiophilic polymers, Histochem. J., 9 (1977) 317-327. 13 Soreide, A.J., Variations in the axon reaction after different types of nerve lesion. Light and electron microscopic studies on the facial nucleus of the rat, Acta. Anat., 110 (1981 ) 173-188. 14 Varon, S. and Bunge, R.P., Trophic mechanisms in the peripheral nervous system, Ann. Rev. Neurosci., 1 (1978) 327-361. 15 Watson, W.E., The response of motor neurones to intramuscular injection of botulinum toxin, J. Physiol. (Lond.), 202 (1969) 611-630. 16 Willard, M. and Skene, J.H.P., Molecular events in axonal regeneration. In J.G. Nicholls (Eds.), Repair and Regeneration of the Nervous System, Dahlem Konferenzen, Springer, Berlin, 1982, pp. 7189. 17 Willard M., Skene, J.H.P., Simon, C., Meiri, K., Hirokawa, N. and Glicksman, M., Regulation of axon growth and cytoskeletal development. In J.S. Elam and P. Cancalon (Eds.), Axonal Transport in Neuronal Growth and Regeneration (Advances in Neurochemistry, Vol. 6), Plenum Press, London, 1984, pp. 171-183.
201
NSL 03599 TARGET DEPENDENCE OF NISSL BODY ULTRASTRUCTURE THORACIC MOTONEURONES
IN CAT
I.P. JOHNSON, A.H. PULLEN and T.A. SEARS* Sobell Department of Neurophysiology, Institute of Neurology, Queen Square, London WC1N 3BG ( U.K.)
(Received July 20th, 1985; Revised version received August 1st, 1985; Accepted August 2nd, 1985)
Key words: axotomy - target - neuroma - Nissl - motoneuron - horseradish peroxidase - cat
The effects on Nissl body (NB) ultrastructure of muscle reinnervation or neuroma formation were determined in cytochemically identified cat thoracic motoneurones subjected to axotomy by either nerve crush or nerve section with proximal ligation. Normal NB ultrastructure comprised highly ordered lamellae of rough endoplasmic reticulum (RER) with associated linear arrays of unbound polyribosomes. This ultrastructural orderliness was lost following axotomy, with or without light microscopic chromatolysis. While NBs were seen in the light microscope at late stages following both nerve crush or ligation, normal NB ultrastructure was only observed following nerve crush, An inductive effect of the periphery on NB ultrastructure is proposed and the implications of NB ultrastructure discussed in relation to protein synthesis.
M a t u r e m o t o n e u r o n e s d e m o n s t r a t e a m a j o r d e p e n d e n c e on the p e r i p h e r y for n o r m a l m a i n t e n a n c e as revealed t h r o u g h their r e t r o g r a d e responses to a x o t o m y , interr u p t i o n o f a x o n a l t r a n s p o r t or b l o c k a d e o f n e u r o m u s c u l a r t r a n s m i s s i o n [5, 8, 15]. T o investigate further this t a r g e t - d e p e n d e n c e , we have used the p a r a d i g m o f 'reversible' a x o t o m y (nerve crush) o r ' c h r o n i c ' a x o t o m y (transection with p r o x i m a l ligation) o f intercostal nerves in a d u l t cats to s t u d y changes in Nissl b o d y (NB) ultrastructure as a m e a s u r e o f altered p r o t e i n synthesis. This a p p r o a c h follows o u r experience with the t o p o g r a p h i c a l l y distinct N B located p o s t s y n a p t i c a l l y to the Ctype s y n a p s e [10]. W i t h the c h r o n i c p a r t i a l central d e a f f e r e n t a t i o n o f intercostal m o t o n e u r o n e s that occurs following spinal hemisection, there is h y p e r t r o p h y o f this synapse a c c o m p a n i e d b y an increase in size a n d a c h a n g e in the r i b o s o m a l o r g a n i z a tion o f its NB. Since the synthesis o f p a r t i c u l a r classes o f p r o t e i n is associated with p a r t i c u l a r forms o f r i b o s o m a l o r g a n i z a t i o n [7], f u n c t i o n a l correlates can be inferred [11], a n d in the p r e s e n t study this a p p r o a c h has been carried to the analysis o f N B s in n o r m a l a n d a x o t o m i z e d m o t o n e u r o n e s . I n t e r c o s t a l nerves were e x p o s e d in two n o n - a d j a c e n t segments 20 m m f r o m the m i d - d o r s a l line a n d either (a) c r u s h e d with fine forceps, o r (b) tightly ligated, sect i o n e d a n d a 5-ram p o r t i o n o f nerve r e m o v e d distally. A x o t o m i z e d m o t o n e u r o n e s were e x a m i n e d 1, 4, 8, 33, 64 a n d 208 d a y s later. T o p r o v i d e an i n d e p e n d e n t m a r k e r *Author for correspondence. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
202 of axotomized motoneurones, motoneurones were retrogradely labelled by horseradish peroxidase (HRP). For the one-day survival period, H R P (40~o in saline) was applied directly to the site of the original axotomy; for longer survivals, animals were anaesthetized one day prior to perfusion, the nerves were reexposed and newly lesioned 2 m m proximal to the original lesion for application of the HRP. Animals were perfuse-fixed with 2~o glutaraldehyde-l~o paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and 70-#m slices of spinal cord processed to demonstrate H R P activity [1]. Osmicated sections were dehydrated and fiat-embedded in a thin layer of Araldite between two PTFE-coated microscope slides [12]. HRP-labelled cell bodies were identified by light microscopy (LM) in trans-illuminated 70-#m plastic sections; the same motoneurones were reidentified by LM in 0.5-#m toluidine bluestained sections and subsequently analysed by electron microscopy (EM). Using classical criteria, normal NBs were identified by LM in 0.5-pm toluidine blue-stained sections as irregular patches of cytoplasmic basophilia (Fig. l a). Adjacent ultra-thin sections revealed that the ultrastructure of the same NBs comprised highly ordered stacks of rough endoplasmic reticulum (RER) and linear arrays of polyribosomes between individual lamellae (Fig. 2a). These polyribosomes are described as 'lamellae-associated polyribosomes' to distinguish them from ribosomes elsewhere in the cytoplasm and single ribosomes bound to the endoplasmic reticulum, a nomenclature we introduced previously for the C-type synapse NBs [1 1].
Fig. 1. Toluidine blue-stained sections (0.5-/~msections) of cat thoracic motoneuronal cell bodies, a: normal motoneurone with prominent NBs (arrow). b: motoneurone 4 days following nerve crush, NBs (arrow) can still be seen. c: NBs (arrow) within a motoneurone 64 days following nerve transection, d: NBs (arrow) within a motoneurone 64 days following nerve crush. Bar = 25/*m.
203
Fig. 2. Electron micrographs of the NBs arrowed in Fig. 1. a: normal NB exhibiting highly organized RER lamellae and numerous interposed 'lamellae-associated' polyribosomes(arrows). b: abnormal NB 4 days followingnervecrush comprisingan aggregateof polyribosomesinterspersedwith short RER fragments, c: abnormal NB 64 days followingnerve transection, d: normal NB 64 days followingnerve crush. Bar= 1/lm.
Early stages following axotomy (1-33 days). Normal NBs were seen by both LM and EM one day following either chronic or reversible axotomy. Apparently 'normal' NBs were still present by LM 4 days following reversible axotomy (Fig. lb), but by EM the same NBs had lost their normal orderly structure. They completely lacked the stacks of parallel lamellae of RER and were composed instead simply of aggregates of polyribosomes within which only short fragments of RER were present (Fig. 2b). By 4 days, following chronic axotomy, LM showed early chromatolysis in which the motoneurons exhibited diffuse, cytoplasmic basophilia due to Nissi fragmentation and dispersion. By EM the Nissl fragments were comprised simply of small aggregates of polyribosomes and no lamellae were present. Thus, the difusse basophilia in the light microscope corresponded to a reduction in size of the polyribosomal aggregates close to the limits of resolution by LM, but not the NB disorganization per se. By LM, chromatolysis was fully developed between 8 and 33 days following reversible or chronic axotomy, the ultrastructure of the two types o f axotomized motoneurones being indistinguishable. Late stages following axotomy (64-208 days). By LM, many motoneurones were still chromatolytic at 64-208 days, while others, irrespective of whether axotomy was chronic or reversible, had apparently reformed normal NBs (cf. ref. 13). However, by EM, a striking difference in NB ultrastructure was apparent. In chronically axotomized motoneurones, NBs were composed of dense aggregates of randomly sited polyribosomes, interspersed with only short fragments of RER (Figs. lc and 2c). In contrast, with reversible axotomy, NBs now displayed their normal, ordered appear-
204
ance due to the regeneration of long lengths of RER arranged in stacks with lamellaassociated polyribosomes (Figs. ld and 2d). From measurements on photomontages of individual perikarya, we estimate that for chronically axotomized motoneurones, the total length of R E R within the ribosomal aggregates is only 30--40~ of that organized as parallel lamellae in motoneurones subject to reversible axotomy. Examination of the distal nerve stumps at 64 and 208 days confirmed that axonal regeneration had, or had not, occurred following reversible or chronic axotomy, respectively. To demonstrate further this dependence of the normal Nissl ultrastructure on the periphery, axons terminating in a neuroma of the external intercostal nerve were resectioned at 49 days and allowed to regenerate into the distal stump of a newly sectioned internal intercostal nerve of the same segment. When examined 64 days later, normal NBs had reformed within some of the HRP-identified external intercostal motoneurones, but none were present in control, chronically axotomized motoneurones in the adjacent-but-one segment. By utilizing an independent marker of axotomized motoneurones, we have identified two distinct forms of NB ultrastructure. A 'normal' one comprising stacks of RER lamellae with linear arrays of lamella-associated polyribosomes between individual RER cisternae. The other ('abnormal') is disorganized, comprising aggregates of free polyribosomes interspersed with short R E R fragments (abnormal Nissl). The former characterizes both normal motoneurones and motoneurones from 64 days post-axotomy when axonal regeneration is successful (reversible axotomy). The latter characterizes axotomized motoneurones ending in a neuroma (chronic axotomy). These differences in Nissl ultrastructure are unlikely to depend on the synaptic loss associated with axotomy (cf. ref. 2), since normal Nissl ultrastructure, including that of the C-type synapse, persists in the face of extensive synaptic loss due to central deafferentation [10], comparable in extent to the synaptic depletion associated with axotomy of intercostal motoneurones [4]. Nor is axonal elongation a sufficient condition, since this was actively proceeding at 33 days when Nissl regeneration was absent. These observations also discount Schwann cells or the peripheral nerve sheath as sources of a signal for NB regeneration, notwithstanding the role such signals play in axonal elongation [6, 9]. Thus 'normal' Nissl ultrastructure appears to depend on either the presynaptic motor nerve terminal or muscle. In the former case, an orthogradely transported inactive protein might be converted post-translationally (e.g. by tRNA-mediated aminoacylation [3, 16]) to serve, after retrograde transport, as a signal governing gene expression, either causing their induction de novo, or acting as a repressor of the growth-associated proteins (GAPs) that characterize the regenerating state [17]. Alternatively, a muscle-derived signal, acting independently or enabling the presynaptic mechanism discussed above, would represent a further postsynaptic example of a target-dependent neurotrophic signal [14]. Whatever its source, the signal(s) is likely to operate through transcriptional controls that codetermine on the one hand the synthesis of additional endoplasmic reticulum bearing membrane receptors (ribophorins, signal recognition proteins, etc.) that allow the co-translational insertion and vectorial discharge of membrane and secretory proteins; and on the other, the formation of cytoskeletal elements that create the highly ordered structure
205 o f t h e n o r m a l N B . T h i s m a t u r a t i o n m i g h t well d e p e n d o n the a p p e a r a n c e o f a crossl i n k i n g n e u r o f i l a m e n t p r o t e i n m a i n t a i n i n g t h e o r d e r l y c o n f i g u r a t i o n o f the R E R a n a l o g o u s to the M - p o l y p e p t i d e , w h o s e a p p e a r a n c e d u r i n g d e v e l o p m e n t m a r k s t h e t r a n s i t i o n o f the a x o n a l c y t o s k e l e t o n f r o m a state o f g r o w t h to a state o f stability [17]. S u p p o r t e d b y g r a n t s to T . A . S . f r o m T h e N a t i o n a l F u n d f o r R e s e a r c h I n t o C r i p pling Diseases and The International Spinal Research Trust. 1 Adams, J.C., Technical considerations on the use of horseradish peroxidase as a neuronal marker, Neuroscience, 2 (1977) 141-145. 2 Blinzinger, K. and Kreutzberg, G., Displacement of synaptic terminals from regenerating motoneurones by microglial cells, Z. Zellforsch. Mikrosk. Anat., 85 (1968) 145-157. 3 Ingoglia, N.A., Zanakis, M.F. and Chakraborty, G., Transfer-RNA-mediated posttranslational aminoacylation of proteins in axons. In J.S. Elam and P. Cancalon (Eds.), Axonal Transport in Neuronal Growth and Regeneration (Advances in Neurochemistry, Vol. 6), Plenum Press, London, 1984, pp. 119-136. 4 Johnson, I.P., Morphological Correlates of Altered Protein Synthesis: an Ultrastructural Analysis of Axotomy and Diphtheritic Intoxication, Ph.D. Thesis, University of London, 1983. 5 Lieberman, A.R., The axon reaction: a review of the principle features of perikaryal responses to axon injury, Int. Rev. Neurobiol., 14 (1971) 49-124. 6 Lundborg, G., Longo, F.M. and Varon, S., Nerve regeneration model and trophic factors in vivo, Brain Res., 232 (1982) 157-161. 7 Palade, G., Intracellular aspects of protein synthesis, Science, 189 (1975) 347-358. 8 Pilar, G. and Landmesser, L., Axotomy mimicked by localized colchicine application, Science, 177 (1972) 1116--1118. 9 Politis, M.J., Ederle, K. and Spencer, P.S., Tropism in nerve regeneration in vivo. Attraction of regenerating axons by diffusible factors derived from ceils in distal stumps of transected peripheral nerves, Brain Res., 253 (1982) 1-12. 10 Pullen, A.H. and Sears, T.A., Modification of 'C' synapses following partial deafferentation of thoracic motoneurones, Brain Res., 145 (1978) 141-146. 11 Pullen, A.H. and Sears, T.A., Trophism between C-type axon terminals and thoracic motoneurones in the cat, J. Physiol. (Lond.), 337 (1983) 373-388. 12 Romanovicz, D.K. and Hanker, J.S., Wafer embedding: specimen selection in electron microscopic cytochemistry with osmiophilic polymers, Histochem. J., 9 (1977) 317-327. 13 Soreide, A.J., Variations in the axon reaction after different types of nerve lesion. Light and electron microscopic studies on the facial nucleus of the rat, Acta. Anat., 110 (1981 ) 173-188. 14 Varon, S. and Bunge, R.P., Trophic mechanisms in the peripheral nervous system, Ann. Rev. Neurosci., 1 (1978) 327-361. 15 Watson, W.E., The response of motor neurones to intramuscular injection of botulinum toxin, J. Physiol. (Lond.), 202 (1969) 611-630. 16 Willard, M. and Skene, J.H.P., Molecular events in axonal regeneration. In J.G. Nicholls (Eds.), Repair and Regeneration of the Nervous System, Dahlem Konferenzen, Springer, Berlin, 1982, pp. 7189. 17 Willard M., Skene, J.H.P., Simon, C., Meiri, K., Hirokawa, N. and Glicksman, M., Regulation of axon growth and cytoskeletal development. In J.S. Elam and P. Cancalon (Eds.), Axonal Transport in Neuronal Growth and Regeneration (Advances in Neurochemistry, Vol. 6), Plenum Press, London, 1984, pp. 171-183.