The nerve cell body response to axotomy

EXPERIMENTAL

48,

NEUROLOGY

The

Nerve

NO.

3,

Cell BERNICE

Department

PART

2, 32-51 (1975)

Body Response GRAFSTEIN

of Physiology, Cornell New York, New

to Axotomy 1

University

Medical

College,

York 10021

INTRODUCTION The nerve cell body is essential for the growth and maintenance of the axon. When the axon is cut, the nerve cell body usually undergoes profound alterations in structure, metabolism, and physiological activity. These changes may be viewed as being specifically appropriate for the repair of the damage, so that the cell body response to axotomy appears to represent a change to a functional state especially conducive to regeneration of the axon. In confronting the question of whether, and how effectively, a severed axon can regenerate, it is therefore germane to consider the nature of the cell body responseand the manner in which this response may contribute to axonal outgrowth. Although most of the available information has been obtained from neurons with axons that emerge from the central nervous system (CNS), the general principles are likely to be equally valid for intrinsic neurons of the CNS. 1 In the preparation of this report, I was greatly aided by the comments and suggestions of a number of investigators to whom I submitted a preliminary version of the manuscript. They are: Dr. Kevin D. Barron, Albany Medical College of Union University, Albany; Dr. David Bodian, Johns Hopkins University School of Medicine, Baltimore; Dr. Brian G. Cragg, Monash University, Clayton, Victoria, Australia; Dr. Michael Goldberger, Medical College of Pennsylvania, Philadelphia; Dr. Matti Hark&en, University of Helsinki, Helsinki; Dr. Frederick C. Kauffman, University of Maryland, Baltimore ; Dr. A. Robert Lieberman, University College Cornell University Medical College, London, London; Dr. Irvine G. McQuarrie, New York; Dr. Marion Murray, Medical College of Pennsylvania, Philadelphia ; Dr. Robert A. Ross, Cornell University Medical College, New York; Dr. Ansgar Torvik, Ullevil Hospital, Oslo; Dr. William E. Watson, University Medical School, Edinburgh; Dr. Erich E. Windhager, Cornell University Medical College, New York. I am grateful for their contributions, and have diligently tried to incorporate as many of them as possible into my presentation, in order that it might reflect more adequately the present state of the field. Nevertheless, this report remains a personal evaluation, and I must myself take responsibility for any errors, omissions, or injudicious points of view that it may contain. Supported by NIH research grant number NS-09015 from NINDS. 32 Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved

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This report has three main sections: the first summarizes the characteristic changes that occur in the cell body in response to axotomy ; the second raises a number of questions about the nature and significance of the response, which focus attention on the current preoccupations of investigators in this area ; the third indicates some prospects for further exploration of the problems. CHARACTERISTICS

OF

THE

REACTION

TO

AXOTOMY

Axotomy usually involves removal of a significant portion of the nerve cell volume, but most of the synthetic machinery of the cell, because it is localized in the soma, is left intact, and the defect produced in the surface membrane at the site of amputation is small in relation to the total cell surface that remains. Nevertheless, the changes that ensue from the injury involve the whole cell, including the cell body, dendrites, and any remaining collateral branches of the severed axon. The typical morphological changes in the cell body, first recognized by Nissl (60)) include swelling of the cell, migration of the nucleus to an eccentric position in the cell, and the apparent disappearance of basophilic material (“Nissl substance”) from the cytoplasm. The prominence of the latter phenomenon led to the general application of the term “chromatolysis” for the response to axotomy, but it has become increasingly clear that the morphological manifestations of this response are different in different cells, and that chromatolysis itself is not invariably seen. Hence the terms “axon reaction,” “retrograde reaction,” or, as in this report, “cell body response,” have come to be considered more appropriate to designate the whole range of alterations that may occur. The characteristics of these alterations have been considered in great detail in recent reviews by Cragg (13), and by Lieberman (48, 49), and therefore in the present report it has frequently been convenient to make reference to specific pages in their three papers to substantiate points for which an extensive literature exists, adding thereto some references to papers that have appeared more recently. At least some of the variability in the response to axotomy results from the fact that more than one kind of process is occurring: on the one hand, the cell may show the catabolic activities associated with structural remodeling or even complete dissolution; on the other hand, it may be mobilizing the anabolic processes which will make survival or even recovery possible. The balance among these forces would presumably determine the particular array of changes elicited by axotomy in a given cell. Cell MelPzbraPze Properties. Although a large proportion of cell membrane may be removed by axon amputation, the defect produced in the cell membrane has a relatively small area. Within a few hours this defect

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becomes “plugged up” with accumulated constituents of the axoplasm (50, 87), and the plasma membrane presumably reforms. As an immediate effect of the mechanical trauma, however, there is a depolarization of the membrane at the site of the injury. This localized depolarization, which lasts for several hours (15), would engender current flow from intact regions of the cell, resulting in a widespread lowering of membrane potential. During the period of altered membrane potential, there may be abnormal movements of ions across the membrane, and possibly significant water movement as well. It is not clear whether such water movement is the cause for the initial swelling that may be seen in some axotomized neurons (48, p. 54), but the fact that this swelling occurs may be indicative of a significant alteration of membrane permeability, whether initiated by membrane depolarization or by some other factor. Alteration of the neuronal membrane after axon injury also manifests itself in certain modifications of the relationship between the injured cell and the elements that impinge upon it, i.e. the perineuronal glial cells and presynaptic nerve terminals. Thus the attachment between glial cells and neurons is weakened (37, 78) ; and in at least some neurons, glial cells become interposed between the cell body and the terminals of some of the presynaptic fibers ending on it (7, 35, 69). It is not clear whether this kind of localized deafferentation, which can develop within a few days after axotomy, is caused by an active disruption of the synaptic cleft by glial cell processes or by the disengagement of either the presynaptic or postsynaptic elements. The deafferentation may be accompanied by a reduced sensitivity of the axotomized cell to the synaptic transmitter (52). In some cases there are also enzyme changes in the presynaptic terminals, e.g. depletion of acetylcholinesterase in cholinergic terminals (32)) that are likely to affect synaptic transmission. All these changes in the synaptic apparatus may contribute to the alteration in the pattern of synaptic activation that can be observed in axotomized cells. This alteration consists of a decrease in the activity of some synapses, particularly those on the cell body (45, 52, 54, 63), and an enhancement of the activity of other synapses, notably those on dendrites, which acquire a capability for the initiation of spike potentials (44). Cell Size. An increase in cell size has frequently been observed after axotomy (48, pp. 53-55), but the distinction between an increase due to swelling, i.e. water uptake, and an increase due to augmented dry mass, has not always been well drawn. Swelling, which results in dilution of the cellular contents, is usually associated with the early stages of the response (48, p. 54) and in some cases may precede any obvious alterations in neuronal metabolism (79) ; whereas an increase in dry mass is seen during the later stages of the reaction, when the enlarged cell body presumably serves as a reservoir of materials for axon formation.

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RNA Metabolism. One of the earliest events in the axotomized neuron is a change in RNA metabolism. In most cells there is an increase in RNA synthesis, as indicated by an increased incorporation of RNA precursors (48, pp. 75-76; 57) and sometimes an increase in nucleolar size (48, pp. 59-60) ; there may also be an increase in the activity of enzymes required for synthesis of RNA precursors (48, p. 90; 2s). In many cases the increased synthesis leads to an increased cytoplasmic R;“\‘A content, although the RNA concentration may remain constant, or even decrease, due to cell swelling (48, pp. 73-75) or increased RNA catabolism. In addition to changes in the amount of RNA, there are also changes in the configuration of the RNA. These involve disorganization of the ordered arrays of rough endoplasmic reticulum that constitute the Nissl substance, together with loss of some of the membranous component and an increase in the proportion of free polyribosomes; sometimes there may be further disaggregation of the polyribosomes into single ribonucleoprotein particles (4S, pp. 65-69; 4, 5s). The change in the form of the RNA and the decrease in its concentration are the basis for the histologically detectable features of chromatolysis, but it is evident from the large number of variables involved that significant changes in RNA metabolism might occur without the development of chromatolysis. The initiation of the increased RNA synthesis presumably depends on some process of gene activation. An increased thymidine uptake by some injured neurons was seen in one study, suggesting that DNA synthesis might be increased (77). Also, nuclear binding of actinomycin-D, an inhibitor of RNA synthesis which appears to act by complexing with DNA, can be shown to be increased (55). Moreover, application of actinomycin-D to the cell body within 9-12 hr of axotomy prevents the full development of the cell reaction (7 2, 73), suggesting that the change in genomic activity is a necessary step in the reaction. I’roteilL Metabohn. Increased synthesis of ribosomal RNA would be expected to presage an increase in protein synthesis, and this has been confirmed by numerous observations of increased incorporation of radioactive protein precursors (48, pp. 77-79), although there are a few reports of decreased incorporation (14, 43). The increased synthesis may lead to an increased protein content and concentration (48, pp. 76-77) but need not do so, since there is often an increase in proteolytic activity, as indicated by a proliferation of electron microscopically identifiable lysosomes, and an increase in histochemically detectable acid phosphatase activity (48, pp. 91-95; 53). A most significant fact which has recently emerged is that while the overall level of protein synthesis may increase, there is actually a decrease in the neuronal content of certain specific proteins, many of them associated with synaptic transmission. In noradrenergic neurons, for example,

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these proteins include dopamine-p-hydroxylase (38, 65)) tyrosine hydroxylase ( 11) and monoamine oxidase (26, 11 )-and noradrenaline itself is similarly affected (9, 11, 26, 34). In cholinergic neurons, cholinesterase (48, pp. 70-71) and choline acetyltransferase (29) are reduced. The relatively slow onset and prolonged duration of these alterations are difficult to explain in terms of a simple loss of material from the cell, e.g. by leakage from the cut axon tip, particularly in the face of an elevated level of overall protein synthesis. Thus it is evident that the response to axotomy involves a change in the relative proportions of different types of materials synthesized by the neuron. In general terms, there is a shift away from the production of materials required for transmitter function and toward the production of structural components required for restitution of the axon. In at least some respects, the pattern of protein synthesis in the axotomized cell comes to resemble that in the immature neuron (25). In view of the complexity of the changes in protein synthesis, it is perhaps not surprising that investigations of changes in various cell organelles, including the golgi system, neurofilaments, microtubules, and mitochondria, have yielded extremely variable results (48, pp. 81-89). The smooth endoplasmic reticulum, however, generally appears to proliferate (48, pp. 69-70; 4). Lipid Synthesis. A significant increase in lipid synthesis may begin very soon after axotomy (27, 55). This presumably corresponds to an increased synthesis of membranous constituents of the neuron rather than myelin, since new myelin formation does not usually begin until later, when outgrowth is well under way. Energy Metabolism Studies of the activity of various respiratory enzymes following axotomy have yielded variable results (48, pp. 89-90). However, it is remarkable that in some cases marked changes in the cell body can occur in the absence of gross alterations in energy metabolism (27). Movement of Materials Along the Axon. Since most of the synthesizing capacity of the neuron is confined to the cell body, the maintenance and growth of the axon depends on materials that are continually being supplied by axonal transport (reviewed in 21, 23). An axotomized cell might be expected to show changes in axonal transport, since there would be a drastic reduction in the volume of axoplasm which would have to be maintained by the supply line. Moreover, since axonally transported material accumulates at the cut end of the axon, damming of the flow would be expected. Nevertheless, axotomy does not appear to have any immediate effect on either the amount of material that continues to move along the length of axon remaining attached to the cell body, or the rate at which this material moves (22, 24).

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After 1-2 days, there may be a decrease in the amount of transported transmitter-associated materials (9, 34, 65)) which presumably reflects their decreased content in the neuron. Still later, after axonal outgrowth has been initiated, there may be an increase in the overall amount of protein that is axonally transported (17, 39) or in its rate of transport (18), or both (22, 24). The modification of axonal transport, which does not occur until after the changes of RNA and protein metabolism in the cell body have begun, must therefore be considered to be part of the response leading to an increased supply of materials required for construction of the new axon, and not the signal for initiation of that response. In those nerves in which the transport does not increase during axonal outgrowth, the increased supply of building materials may perhaps be obtained at the expense of the intact portion of the axon, since the diameter of the axons above a lesion may become considerably reduced while outgrowth is in progress (13, p. 12; 39). Ribosomes, which are rarely seen in normal mature axons, may appear in the axons of axotomized neurons (A. R. Lieberman, personal communication), presumably having been conveyed from the cell body by axonal transport (31). This suggests that local protein synthesis in the axon may be more significant during regeneration than under normal circumstances. Axon Collaterals. Changes elicited by axotomy, such as the decrease in axonally transported transmitter-associated materials, may also be manifest in the undamaged collateral branches of the severed axon (65). Moreover, section of one axon branch may result in axonal sprouting at the terminals of the collateral branches (62). Whether the sprouting is initiated by a propagated change in the cell membrane, or whether it is produced as a consequenceof the large metabolic disturbances in the cell body, is still not known. The outcome of these distant effects is that an injury directly involving only a single axon branch may cause alterations in function throughout the field of distribution of the injured neuron. Dendrites. The metabolic changes seen in the cell body continue into the dendrites, which have some of the same protein-synthesizing capacity. The reaction in the dendrites may be less severe than in the cell body, in terms of both loss of presynaptic boutons (7, 69) and depletion of transmitterassociated protein (40). Nevertheless, a significant retraction of the dendritic field has been observed (70). This again emphasizesthe widespread effects that may result from injury of a discrete portion of the axon. Summary of Changes. The response to axotomy is extremely variable, but in general the following events are to be expected: After a transitory depolarization, cell swelling may occur, and there is a loosening of the attachment between the neuron and its neighboring glia. Detachment of

38

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some presynaptic terminals from the cell body and enzymatic changes in the terminals may affect synaptic transmission, so that the pattern of synaptic input to the neuron is modified. RNA synthesis is increased, but RNA concentration may be reduced by cell swelling or increased degradatiton. The change in RNA metabolism, which is accompanied by disorganization of the rough endoplasmic reticulum, may reveal itself in chromatolysis. The overall level of protein synthesis increases, but increased proteolysis also occurs, and there is a decrease in the content of some proteins, notably those related to synaptic transmission. Lipid synthesis increases. These fundamental metabolic alterations need not be associated with gross changes in energy metabolism. The amounts of various materials carried by axonal transport to the axon tip are altered in parallel with the changes of their content in the cell body ; changes in the rate of axonal transport, if they occur at all, appear only after axonal outgrowth has begun. The intact axon collaterals and dendrites of the axotomized cell also participate in the above pattern of response, so that damage of a single axon branch may result in functional alterations throughout the field of distribution of the injured neuron. CURRENT

ISSUES RESPONSE

Do All Cells Show

IN RESEARCH TO AXOTOMY the Response

ON THE

to Axotomy?

The features of the response to axotomy that have been described above can be seen to some degree in most neurons with axons that terminate outside the central nervous system. Even in many cases in which it had originally been claimed that no response could be elicited, subsequent studies were successful in showing that significant changes could occur (49, pp. 79-80). In some instances, the reaction had been overlooked because it did not have the features of typical chromatolysis ; in other cases, one can only assume that the original observations had been made at an inappropriate time after axotomy. For intrinsic neurons of the CNS, the range of response is much more variable: in some neurons there is a greater likelihood that the outcome will be cell death or atrophy ; and, at the other extreme, there are some neurons in which the overt morphological changes are minimal or comsince the response to axotomy involves a whole pletely absent. However, array of changes-some of which tend to oppose one another-it is clear that a significant response could occur in the absence of overt morphological change. For example, the axotomized cells of the locus coeruleus show no morphological change although their synthesis of transmitterassociated materials is greatly reduced (66) and vigorous axonal out-

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growth occurs (6). Therefore it is still not possible to decide whether all CNS neurons can respond to asotonip with at least some inctabolic changes that could support axonal regeneration. Whut Factors

Influence

The Respome?

Electrical Activity. In many experiments, axotomy of a particular cell has unavoidably been accompanied by axotomy of some of its presynaptic neurons, and since presynaptic denervation may elicit many of the same effects as postsynaptic axotomy (4), it is conceivable that the presynaptic events may play at least a contributory role in the development of the cell body response in these instances. Even when the presynaptic cells are not directly damaged, some axotomized neurons may suffer from derangement of synaptic transmission (see above). The ensuing reduction in the level of synaptic activity would be expected to cause a reduction in the synthesis of transmitter-associated materials (30), and might therefore be responsible for some of the alterations in the pattern of synthesis associatedwith axotomy. It is probable, however, that not all of the alterations can be explained in this way: in the case of peripheral autonomic ganglia, for example, the changes produced by axotomy (11) are more intense and involve a larger number of enzymes, than those produced by deafferentation (30). Another problem is the possible role of activity generated by the axotomized neuron itself. Although the burst of action potentials directly evoked by the injury is very brief (75), continuous action potential activity may develop in the regenerating axon (36, 74), presumably elicited by random mechanical and chemical stimuli impinging on the emerging axon sprouts. The elevated level of functional activity could lead to increased RNA and protein synthesis (56, 76), and might therefore have a beneficial effect on regeneration by enhancing the synthetic activity of the regenerating cell. Age of the Arziwza.Z. Younger animals show more prominent changes in cell characteristics and a higher incidence of cell death (13, p. 5 ; 49, pp. 73-79). Lieberman has postulated that immature neurons may have a different metabolic program than do adult neurons and that they may lack a number of basic homeostatic mechanisms that appear with maturation. Possibly relevant to this issue is the fact that when adult neurons are axotomized, they shift to a pattern of protein metabolism more nearly resembling that in the young animal (25). Site arzd Nature of the Injury. Axonal lesions close to the cell body, compared to those further away, produce more intense and prolonged chromatolysis and are more likely to result in cell death (49, pp. 83-89). A possible explanation for this effect, suggested by Lieberman. is that with more proximal lesions there is an earlier cutoff of the increase in

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RNA synthesis (7Y), which would result in a smaller increase in cytoplasmic RNA content. A neuron is less likely to be destroyed by the injury when the remaining length of axon is highly branched. This has led to the concept that the intact axonal branches have a “sustaining” effect on the cell body (49, pp. 85-89). It is not clear, however, whether this effect involves a specific influence emanating from the intact collaterals, or whether the presence of a large number of collaterals implies that the injury has produced only a relatively small alteration in the total cell economy. The latter hypothesis may also be relevant in dealing with the case of the dorsal root ganglion cell, which has long been a puzzle, since section of the peripheral axon branch leads to a dramatically more intense cell body response than section of the central branch ( 13, pp. 5-7 ; 48, pp. 97-102)) even though there is no obvious size discrepancy between the two branches (13, p. 6). In recent studies, however, it has been shown that three to five times more axonally transported material normally enters the peripheral branch than the central one (46, 61). It may be concluded, therefore, that the response is more intense under conditions which cause a greater perturbation in the distribution of materials within the cell. The type of injury-e.g. whether the lesion of the axon has been performed by cutting the nerve, by crushing, or by freezing-may also have a significant effect on the nature of the changes in the cell body (34, 67). Since some of the differences related to different types of injury can be detected very soon after the injury, it is possible that the relative ease or rapidity of the initial sprouting of the axons may play an important role in determining the intensity of the response. Reconnection of the Axon. In some cases the cell body recovers before axon regrowth is complete (8)) but more often the duration of the cell body reaction corresponds to the period in which active axonal regeneration is occurring. The changes begin to regress at about the time the axons reach their terminations, but persist to some degree until axonal maturation is complete (10, 59, 79). The final condition of the cell body is influenced by whether or not the regenerating axon makes an effective terminal connection, and nerve cells whose axons fail to do so usually show persistent abnormalities, including chromatolysis, reduced size, retraction of dendrites, and depressed synthesis of RNA and protein (49, pp. 90-97 ; 14). A significant observation made by Watson is that when reconnection of the regenerating axon is prevented, the decline in cell nucleic acid from its elevated level occurs at the same time as it would if reconnection had occurred (79). This suggests that once the cell body reaction is initiated it runs its course, presumably according to the preset genetic program, without feedback from the regenerating axon.

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Do the Glial Cells Play a Role? l’he glial cells adjacent to the cell body of an axotomized ncurou show changes correlated with the neuronal response. The microglial cells near the axotomized neurons divide, and these cells as well as the astroglia show an increase in dry mass and in RNA activity (83). Indeed, the increase in astrocyte dry mass may be detected before any metabolic changes in the nerve cell body (81). Watson has suggested (81, 84) that the glial cell reactions are related to the alteration of the dendritic membrane of the injured neuron and the presynaptic endings on it, rather than to the metabolic activity of the neuron. However, in view of the close metabolic linkage between the glia and the neurons under normal circumstances, it is possible that glial alterations make some contribution to the characteristics of the neuronal response. Which

Characteristics

of the Response Are Likely Rather Than Recovery?

to Lead to Cell Death

This is a particularly important question in connection with the problem of CNS regeneration, since central neurons show a high probability of degeneration in response to axotomy. Thus far, there are no clear criteria for recognizing cells that are fated to die. For example, an increase in proteolytic activity, as indicated by lysosomal proliferation or increased hydrolytic enzyme activity, is no more likely to occur in neurons that are killed by axotomy than in those that are capable of regeneration (48, pp. 91-95; 71). Generally it has been assumed that more intensely chromatolytic cells are more likely to die (48, p. 97). Implicit in this assumption, however, is the view that chromatolysis is essentially a degradative process, whereas in fact chromatolysis may be associated with a significant increase in RNA synthesis and vigorous axonal outgrowth. An important point to be resolved is how the catabolic events that may lead to dissolution of the neuron are linked with the anabolic events culminating in regrowth of the axon. It is possible to imagine either that the catabolic and anabolic events run in parallel but are subject to separate controls, or that the one is not able to proceed without the other. Hozv Does the Cell Body Response

Cofltribute

to Regeneration?

Some neurons may show a typical cell body reaction even when axon outgrowth is blocked (22), and conversely axon outgrowth may continue for a short time even when protein synthesis is inhibited by drugs (86). In spite of these indications that the cell body response and axonal sprouting may proceed independently of each other, it is unlikely that sustained

42

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asonal regeneration could occur in the absence of the eilhanced metabolic support that is mobilized in the cell body reaction. Even in those cases in which it may be established that there is no morphological alteration of the cell body, some important features of the cell body response, such as a shift in the pattern of protein synthesis, might be occurring, as has been demonstrated, for example, for certain central noradrenergic neurons (65, 66). The same is also likely to be true in those cases in which the cell body response appears to involve a decrease in the overall level of protein synthesis (14, 43). It is also possible that retrograde changes are absent in some neurons because axonal outgrowth is supported not so much by the creation of new sources of materials as by the diversion of normally available materials from existing neuronal structures. One fact which does point to the importance of the cell body response in regeneration is that a faster rate of axonal outgrowth occurs after the second of two successive nerve injuries (51) : presumably the cell body response produced by the first injury is already fully developed when the second injury is made, so that the metabolic support for axonal outgrowth is immediately available. Similarly, the effect of thyroid hormone in accelerating the rate of axonal outgrowth (12, 16) is probably attributable to enhanced synthesis of materials required for outgrowth. How

Is the Response

to Axotomy

Initiated?

In spite of the prominent changes that axotomy elicits in the glial or schwann cells both at the site of the lesion and in the neighborhood of the cell body (13, p. 3 ; 83)) it seems quite certain that the initiation of changes throughout the axotomized neuron involves an intraneuronal mechanism rather than some process in the neuroglia, schwann cells or other extraneuronal constituents, since the reaction is limited to those neurons whose axons have been cut. There have been some reports of changes in neighboring neurons and in the corresponding neurons on the opposite side of the body, but these effects generally seem to be accounted for by work hypertrophy (13, p. 8; 48, pp. 106-107), or by collateral sprouting from the intact axons of nearby cells to occupy the sites previously occupied by the damaged fibers (82). It seems reasonable to assume that the initiation of the changes in the cell body depends on the transmission of a “signal” along the axon from the site of injury, since the time required for appearance of the cell body changes is proportional to the distance between the cell body and the lesion (13, p. 4) ; it has been estimated that such a signal ascends the axon at a rate of several mm/day. A number of hypotheses about the nature of -the putative signal have been carefully considered by Cragg (13, pp. &16), who concluded that

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it is most likely that several mechanisms are operating together. Watswl has commented that “axotomy interrupts nearly as many signals as cutting a snake in half” (personal communication, 1974). Among the factors to which Cragg assigned a possible role in the initiation of the response are: depolarization of the membrane, loss of action potentials, depletion of axonally transported constituents (such as transmitter-associated materials or a substance which would act as a repressor of the genes regulating protein synthesis), loss of axoplasm and mitochondria, and loss of a trophic substance coming from the periphery. In Cragg’s view, these factors may be operative to different degrees in different situations, which would account for the variety of responses that may be observed. At the present time, Cragg’s formulation, with its detailed presentation and critical evaluation of supporting evidence for the various possible mechanisms, still stands as the beacon for further work on problems of the initiation of the response to axotomy. However, a number of observations made more recently must be considered to have an important bearing : (i) Ch an g es l’k1 e t h ose produced by axotomy can be seen in cells undergoing collateral sprouting (20, 82). These observations, together with those in which a similar effect has been evoked by application to the axon of botulinun toxin (SO) or other drugs (see below), suggest that neither depolarization of the membrane nor loss of axoplasm is essential for the initiation of the cell reaction. Moreover, since the cell body response may be even more intense under conditions of collateral sprouting than in response to axotomy, it is possible that the depolarization or loss of axoplasm may prevent the cell from developing its full capacity for metabolic support of axonal growth, as expressed in the cell body response. In interpreting such experiments, Watson has proposed that the triggering event for the cell body response, whether elicited by axotomy or other means, may be the expansion of the cell membrane associated with axon sprouting. Two facts which appear to conflict with this view are that some of the drugs that can elicit the response have been found to inhibit rather than promote axonal outgrowth (22, 64, 68)) and that in the normal sequence of events, axonal outgrowth may sometimes follow rather than precede the initiation of the cell body changes (22). However, these points need further investigation. (ii) Some extracellular materials originating at the site of the lesion can reach the cell body within the time required for the initiation of the cell body response. Exogenous proteins, such as albumin or horseradish peroxidase, which are carried by retrograde axonal transport from the axon terminals to the cell body (41, 47), may also enter the axon at the site of an injury and reach the cell body within a few hours (42). This

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raises the possibility that the cell body response might be initiated by the arrival of some foreign materials. (iii) Following axotomy the movement of material from the cell body into the length of axon remaining connected to it shows no change in amount or rate before the cell body response is well under way (22, 24). Immediately after the lesion, both the fast and slow components of anterograde axonal transport proceed normally in the axon stump. Some transported material accumulates at the site of the lesion, and there may be an early transient elevation of cell body levels of some transported constituents, e.g. noradrenaline and dopamine-p-hydroxylase (1, 65), but there is no corresponding elevation all along the length of the axon, as would be expected from damming of the transport system. The excess of transported material originally destined for the missing portion of the axon may be disposed of to some extent by an increased loss from the cell, but it is likely, as Frizell and Sjijstrand have suggested (19), that much of it is carried, by the mechanism of retrograde axonal transport (41, 47), back to the cell body, where it might serve in a feedback regulation of protein synthesis. This hypothesis would also explain the increase in the cell body levels of some transported materials, which would be more difficult to account for on the basis of another postulated mechanism (13)) namely that the lesion acts by preventing the arrival of some trophic substance transported from the periphery. (iv) The cell body reaction produced by some drugs has many, but not all, of the features of the response elicited by axotomy. Microtubuledisrupting drugs, e.g., colchicine or vincristine, which are known to block axonal transport (e.g. 33)) may elicit at least some of the following features of the cell body response to axotomy : chromatolysis, nucleolar enlargement, retraction of presynaptic terminals, and loss of synaptic transmission (33, 63, 88; W. R. White and B. Grafstein, unpublished results). In postganglionic sympathetic neurons, guanethidine also has an effect like axotomy (32). There are some indications, however, that the response to the drugs is not necessarily a perfect reproduction of the response to axotomy-in rat dorsal root ganglia neither neuronal enlargement nor glial cell multiplication is seen (88) ; in goldfish retinal ganglion cells, there is no increase in protein synthesis, and cell enlargement is minimal (W. R. White and B. Grafstein, unpublished results). These observations point to the possibility that the array of cell body changes elicited by axotomy is related to an array of axonal alterations produced by the injury, and that the drugs, which produce only some of the axonal alterations (e.g. block of axonal transport) and not others (e.g. loss of axoplasm or disruption of the cell membrane), thereby elicit only some of the cell body changes.

RESPONSE

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AXOTOMY

Do C‘NS‘ Neurons Fail to Regmcvatc l’ccause u,f UIL laudequatc Cell Body Response to Axotomy? Detailed comparisons between the cell body responses of neurons with centrally projecting axons and those with peripherally projecting axons might be expected to reveal some basic differences related to their respective capacities for axonal regeneration. In some studies of this kind (2, 3), acid phosphatase activity (which might be involved in RNA synthesis) was found to be decreased in the central neurons, in contrast to its increase in peripherally projecting neurons. Generally, however, it has not yet been possible to recognize a consistent feature in axotomized central neurons that might be responsible for their failure to regenerate. An interesting idea put forward recently is that this failure may be due to an immunological reaction directed against brain autoantigens, and that antibodies or lymphocyte products axonally transported from the tip of the injured axon to the cell body act there to inhibit protein synthesis (5). One implication of this hypothesis is that the cell body response is indeed a critical variable in determining whether central neurons can be successful in axonal regeneration. INDICATIONS

FOR

FUTURE

RESEARCH

The Cell Biology of An-otoneized Neurom. Over the years, there have been many descriptions of the changes evoked by axotomy. However, it is often difficult to draw sufficient information from these early studies since we are now aware that previous criteria, e.g. the presence of chromatolysis, are inadequate either for recognizing cells that are affected by axotomy, or for diagnosing the kinds of changes that these cells are undergoing. This has left us uncertain about the answers to some basic questions, for example, whether or not every kind of nerve cell is capable of some kind of response which might be the basis for regenerative activity. Thus we must welcome further evidence about the manner in which different kinds of nerve cells respond to axotomy ; however, these studies should focus not on the superficial manifestations of the response, but on the basic mechanisms involved, and particularly on the sequence in which these mechanisms operate. Moreover, it will be necessary to consider the cell body events in much greater detail than heretofore: it will no longer be adequate, for example, to evaluate changes in RNA synthesis or protein synthesis as a whole-these will have to be analyzed in terms of changes in individual cell constituents. As a background for such studies, it is necessary not only to investigate the properties of axotomized cells, but also to develop a better picture of how the normal cell functions with respect to the production and delivery

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of materials that are required for regeneration. In this connection, all studies of neuronal metabolic function have some relevance, but the analysis of mechanisms regulating RNA metabolism and the rates of protein and lipid synthesis and degradation is especially important; the routes of intraaxonsl transport of materials and the mechanisms involved in this transport are topics having a direct bearing ; and the nature of the interactions between glial cells and neurons, as well as the nature of the trophic relationships (both anterograde and retrograde) between one neuron and another, are subjects that are appropriate for further consideration. Correlation of the Cell Body Response with Regeneration. It seems probable that the cell body response provides the metabolic support for regeneration. It is still not clear, however, how the cell body changes relate to the process of axonal regeneration. One may still ask, for example, what features of the response are essential for outgrowth? What events are supportive if not essential ? What events represent exhaustion of the cell’s resources? One approach to these problems, which is to compare cells that are capable of regeneration with those that are not, has already been tried in a small number of studies ; but detailed investigations, for example, of the unfolding sequence of events in each case, have not yet been carried out. Another approach would be to examine more closely those cases in which regeneration can occur in the apparent absence of a cell body reaction. fnitiution and Ekmcevzent of the Response. In view of the probable contribution of the cell body response to axonal regeneration, it may be anticipated that evocation or intensification of this response could encourage regeneration in cells whose regenerative capacity might be limited by a defect of the cell body response program or by some inadequacy of the initiating stimulus. One approach to these objectives might be to investigate the effects produced by procedures other than axotomy that are known to elicit a similar response in the cell body, e.g. the application of microtubule-disrupting drugs ; another would be to determine the concomitant effects on the cell body response and on axonal outgrowth of drugs and hormones (e.g. cyclic AMP and GMP, thyroid hormone, somatotrophin, and insulin) which are known to have a significant effect in stimulating biosynthesis ; other processes which are known to modify protein metabolism, e.g. increased functional activity and the induction of collateral sprouting, are also obvious topics for investigation. Eventually, such studies must have a much wider frame of reference than just the problem of axonal regeneration, since questions about the properties of the cell. body response, and whether all nerve cells can be induced to show such a response, are clearly related to the more general questions of how gene action is regulated, and whether, during differen-

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tiation, certain genes are lost completely from the cell or merely repressed. Current ideas on these issues have been developed almost entirely on the basis of experiments carried out on non-neuronal tissues, but the time has now come to develop such ideas more intensively in connection with studies of the nervous system.

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