Retrograde factors in peripheral nerves.

Pharmac. Ther.Vol. 56, pp. 265-285, 1992 Printed in Great Britain. All rights reserved

0163-7258/92$15.00 © 1993PergamonPress Ltd

Specialist Subject Editor: C. BELL

RETROGRADE FACTORS IN PERIPHERAL NERVES IAN A. HENDRY

Neurobiology Research Group, Division of Neuroscience, The John Curtin School of Medical Research, The Australian National University, G.P.O. Box 334, Canberra, A.C.T. 2601, Australia Abstract--The relationship between the neuron and its target is explored and the possible mechanisms for achieving correct connections are analysed. The most plausible mechanism is the presence of a retrograde intra-axonal message from the target to the neuronal cell body. The molecular form of the message and the mechanisms to achieve this signal transduction are discussed and it is proposed that there are two types of neurotrophic factors. One has a short-acting second messenger, itself incapable of surviving for the time required for transport to the cell body and thus requiring the transport of the message-generating complex to the cell body. The other has a long-lasting second messenger complex which is well able to survive the transport to the cell body so that there is no need for the transport of the neurotrophic factor itself. Thus all neurotrophic factors do not themselves require retrograde axonal transport and such non-transportable factors may generate intricate messages due to associations of signal transduction molecules via binding sites such as phosphorylated tyrosin~.s and the src homology domain 2.

CONTENTS i. Introduction 2. Developmental Cell Death 2.1. Mechanisms of survival and cell death 3. Mechanisms to Achieve Correct Connections 3.1. Pathway to target tissue 3.2. Target selection 4. Phenotypic determination by target tissue 4.1. Target-controlled regulation of central connections 4.2. Mechanism of phenotypic change 5. Mechanisms to Convey Information from Target to Neuron 5.1. Circulatory factors 5.2. Retrograde transport of factor 5.2.1. Transport of active neurotrophic factor 5.2.2. Transport of active receptor 5.2.3. Transport of second messenger 5.2.4. Transport of factor-receptor complex 5.2.5. Control of Tissue Retrophin Levels 5.3. No Retrograde Transport of Factor 5.3.1. Does a neurotrophic factor require to be retrogradely transported? 5.3.2. Transport of permanently modified receptor 5.3.3. Transport of second messenger 5.3.4. Cessation of retrograde transport of death molecule 5.4. Physical mechanisms 5.4.1. Electrical 5.4.2. Cessation of neurite outgrowth 6. Conclusions References

266 266 267 267 268 269 269 270 271 272 272 273 273 273 274 274 275 275 275 276 276 278 278 278 278 279 279

Abbreviations--aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; CAT, choline acetyltransferase (EC 2.3.1.6); CNTF, ciliary neurotrophic factor; FGF, Fibroblast growth factor; IGF, insulin-like growth factor; NGF, nerve growth factor; SCG, superior cervical ganglion. 265

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1. A. HENDRY 1. I N T R O D U C T I O N

The problem of understanding the mechanism by which a neuron achieves the correct innervation of its target tissue has remained unsolved by neurobiologists. During development a neuron must grow an axon from its cell body, choose the correct pathway and eventually synapse with its correct target. There have been many theories proposed to explain the extremely accurate wiring in the nervous system, and all involve some interaction between the target cell and its innervating neuron. The most extreme of these was the chemoaffinity hypothesis of Sperry (1951) that suggested a unique chemical coding between each innervating neuron and its target cell. It is unlikely, however, that there would be sufficient genetic information in a vertebrate to achieve such a coding within the central nervous system. It is more likely that there is a hierarchy of strategies used by the neuron to achieve these connections with different neuronal populations using various combinations of common themes. Guidance factors along the potential pathway and recognition factors in the target tissue will be critical for correct development. Some of these elements may be lost in adults accounting for their impaired ability to regenerate. For example, pathway guidance for the developing axons may be along routes that are present only for short times during the formation of the embryo or consist of transiently expressed components of the extracellular matrix and cell surface adhesion molecules on the cells in the axon's path. One-on-one communication between the target cell and the potential innervating neuron commences when the growth cone reaches the region of the target tissue and the first filopodial tip palpates the surface of the target cell. A complex exchange of information must then occur, ranging from affinities due to matching of cell surface adhesion molecules and thus selection of best matching pairs, through to two-way activation of receptors leading to alteration of nuclear expression of proteins. During regeneration, the growth of sprouts and elongation of the axon recapitulates the axon growth during embryonic development, although there are several significant differences between regeneration and development. The neuron can maintain an axon not connected to its target for a considerable time during regeneration and the guidance cues available in the embryo to the sprouting axon may not always still be present in the adult. Final maturity of the regenerating nerve and its presynaptic contacts are totally dependent on functional contact with the target tissue. Failure to innervate the target tissue results in persistence of chromatolysis and, eventually, death of the neuron (Matthews and Nelson, 1975; Purves, 1975; Hendry, 1975b). The main thrust of this review will be to examine in detail the potential ways in which a target cell can signal to the nucleus of its innervating neuron relevant information leading to the survival of the most appropriate cell to contact it and the resultant production of the exquisitely complex wiring of the adult vertebrate nervous system.

2. D E V E L O P M E N T A L CELL D E A T H No analysis of the formation of neuronal connections can be made without an understanding of developmental cell death. In most nerve centres there is a wave of cell death that occurs at the precise time that the target tissue is being innervated. At this stage a large proportion, usually more than 50%, of the total neuronal population dies (Hamburger and Levi-Montalcini, 1949). In addition, if ti'.e target tissue is removed prior to innervation nearly all the neurons that are destined to supply it will die at the time when innervation would have taken place (Hamburger, 1934). An artificial increase in the size of the target field (Hamburger, 1939), or a reduction in the numbers of neurons innervating the same size of target, leads to an increase in the extent of neuronal survival (Pilar et al., 1980) showing that it is the ratio between the number of innervating neurons and the size of the target that is important for the final number of surviving neurons. Thus the target tissue must provide some message to the innervating neuron which is essential for its survival. The earliest experiments suggested that the number of surviving neurons innervating a target tissue was regulated by the size of that target tissue (Prestige, 1974). It was not known whether this was due to an increase in neuronal proliferation due to a mitogenic factor, or due to a decrease in cell death caused by a survival factor. While the final numbers of neurons in any nerve centre seem to depend on the size of the available target, regional variations can occur due to differential neuroblast proliferation, for example in the chick embryo spinal cord (Oppenheim et al., 1989).

Retrograde factors in peripheral nerves

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The earliest results leading to the discovery of nerve growth factor (NGF) suggested that this was a diffusible agent which caused an increase in the numbers of neurons in a nerve centre by an effect on proliferation (Levi-Montalcini, 1966). It was subsequently shown that N G F had its main action in causing neuronal survival rather than proliferation (Hendry, 1977b) and it was able to act not only via the general circulation but also by transport within the axons from the target tissue to the neuronal perikarya (Hendry, 1977a). It is the fact that this retrograde transport is confined within individual axons that provides the specificity of the communication between a target and its neuron. 2.1. MECHANISMSOF SURVIVAL AND CELL DEATH Protein and RNA synthesis are required in the embryo for both naturally occurring and lesion-induced cell death (Oppenheimet al., 1990) and, in addition, the death of cultured neurons after the removal of N G F is prevented by inhibitors of RNA and protein synthesis (Martin et al., 1988). Thus neuronal cell death is an active process requiring biosynthetic events. It seems likely that neurotrophic factors have a dual function, both stimulating genes that promote survival and differentiation and suppressing genes that would kill the cell (Oppenheim et al., 1990). The finding that interferon retards cell death in cultures of sympathetic neurons after N G F withdrawal is intriguing and suggestive of a role for 2',5'-oligoadenosine synthetase in the process of neuronal rescue (Chang et al., 1990). As the product of this enzyme can activate an RNAse, interferon may interrupt the 'death program' by causing the degradation of mRNA critical for this program. There are many factors involved in the regulation of cell death which may act synergistically and one attractive model for developmental control is the interaction between presynaptic connections and postsynaptic factors. For example, in the developing sympathetic nervous system both presynaptic and target tissue influences control the final numbers of neurons in the superior cervical ganglion (SCG) (Black et aL, 1972; Hendry, 1973, 1975a). The number of embryonic chick spinal motoneurons that survive during the period of naturally occurring cell death is influenced by factors from both the target tissue (Hamburger, 1934, 1975; Oppenheim, 1981) and an intact descending afferent system (Okado and Oppenheim, 1984). Two distinct factors have been isolated from muscle and spinal cord which clearly promote motoneuron survival and there is synergy between these factors (Dohrmann et al., 1987). Naturally occurring cell death is enhanced in sympathetic and parasympathetic ganglia after blockade of ganglionic neurotransmission with pempidine (Hendry, 1973; Maderdrut et al., 1988). On the other hand, blockade of activity of the target with curare leads to an increase in survival of motoneurons (Oppenheim, 1981, 1984) and, conversely, increase in activity by electrical stimulation of the limb muscles in the chick embryo increases cell death (Oppenheim and Nfifiez, 1982). Similarly, neuronal survival is enhanced by blockade of postsynaptic transmission. Some of these apparently activity-related phenomena may be due to changes in the target size, for example, sympathetic preganglionic neuron cell death is reduced by treatment with N G F (Oppenheim et al., 1982a) and hemicholinium (Oppenheimet al., 1982b). This may be due to the enlargement of the sympathetic ganglia and the supply of the preganglionic requirement for neurotrophic factors by the hypertrophied target. Taken together, these results suggest that increased activity in presynaptic neurons regulates cell survival, enhancing the formation of correct connections. Increased activity, indeper_Jent of neuronal firing, in the postsynaptic target can promote cell death as the target is already innervated by more appropriate neurons.

3. MECHANISMS TO ACHIEVE CORRECT CONNECTIONS Developmental cell death occurs at the precise time of innervation and this has led most investigators to speculate that it must be involved in the selection of the correct neurons. This has led to the development of the neurotrophic theory of cell death (Hendry, 1976) which is outlined in Fig. 1. This theory suggests that the target releases limiting amounts of a neurotrophic factor such that the nerves innervating the tissues compete for the factor. Those that compete successfully will survive and those that do not make appropriate or sufficient contacts will die. In addition,

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I.

Nerve

A.

HENDRY

Cell Bodies

Target Tissue

0

Early axonal outgrowth

+

NGF

Critical

Correct connections leading to retrograde axonal transport of NGF

Period

Post target innervation

Survival of neurones that make correct connections Neuronal death

FIG. I. Schematic diagram outlining the neurotrophic theory of cell death. Neurons extend axons in the presence of a low circulating level of neurotrophic factors. At a critical point in their development, usually corresponding to the period of target tissue innervation, the neuron becomes acutely dependent on the neurotrophic factor for its survival. At this stage those that make appropriate connections in the periphery obtain sufficient factor for their survival. Those not making correct connections do not obtain sufficient factor and die.

that do not make contact with their correct target, or contact an incorrect target providing an inappropriate neurotrophic factor, also die (Fig. 1). However, there are many lines of evidence that suggest the simple concept that neurons making the correct connections live while those that make incorrect connections die is unlikely to be the whole truth. There are several possibilities as to how a nerve makes the correct contact with its target and these are by no means mutually exclusive. neurons

3.1. PATHWAY TO TARGETTISSUE The mechanisms for guidance have been extensively reviewed (Tosney and Landmesser, 1985b; Hill and Vidovic, 1992) and will only be briefly touched on here. The main point is that for many neuronal populations there is a stereotype of patterned outgrowth, such that they all grow along the correct pathways to their target region with little or no deviations along the route (Mark, 1980; Tosney and Landmesser, 1985a). A large number of mechanisms for this has been proposed and all are likely to play a role (Purves, 1988; Landmesser, 1980). These include genetic preprogramming of fibre direction or gradients, topographical organization, of connections, birthdates of neurons and the timing of axon outgrowth. Nerves in a nerve centre all grow to the same target as, for example, is seen for chick and amphibian motoneurons (Mark, 1980). There may be growth of axons along preformed anatomical pathways such as blood vessels. Axons may adhere to specific molecules along their route in the extracellular matrix or on surfaces of cells (such as guidepost cells in invertebrates). Gradients of trophic factors may be present in the pathways leading to chemotactic direction of growth, although stable gradients over long distances are unlikely and such gradients are more likely to be based on the matrix or on cell surfaces. In the chick motoneuron system, it has been shown that all the neurons in a motor nucleus destined to innervate a certain muscle, in fact, grow directly to that muscle and, even at the earliest stages of innervation, do not appear to make any gross errors in the muscle they contact (Tosney and Landmesser, 1985a). The retino-tectal system has been extensively examined and it would appear that even at the earliest stages of the innervation of the tectum there are only minor mistakes in innervation (Marotte, 1990). Thus these neurons, at the grossly anatomical level, get to the correct target. It may be that there is a more subtle difference so that there is, within the muscle, a topography that requires to be established and thus that it is not the innervation of the whole muscle but of individual fibres within it that is important. In the frog muscle such a change in the topographical


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innervation of the muscle has been shown (Noakes et al., 1983, 1988). This finding seems to be the exception rather than the rule, however, and the results on which the conclusions are based have been suggested to be ambiguous due to the potential electrical coupling of these immature muscles. 3.2. TARGET SELECTION At the time of initial target tissue innervation the embryo is very small and the filopodia of the growth cones can contact much of the tissue in the target region. Thus it is possible that, over a few hundred microns, surface adhesion molecules could select the correct neuron-target match. Similarly over the same short distances, chemotactic agents could result in the growth cone being directed to the correct target. Target-derived neurotrophic agents may act in two ways: firstly, to stabilize appropriate synaptic contacts and, secondly, to promote the survival of appropriate neurons making correct connections. The above discussion of course leaves open the question as to which is the 'appropriate' neuron to make the 'correct' connection with a target, and it is a matter of semantics as to which neurons are correct. If there are embryonic guidance cues that result in all the neurons from a specific part of the spinal cord reaching a specific muscle, and we define the neurons within this part of the spinal cord as belonging to the motoneuron pool for that muscle, then there can only be 'correct' innervation. If, however, there are within that region of the spinal cord neurons that for various reasons are not suitable to innervate that muscle, then these 'correct' neurons will in fact be wrong. They may be anatomically correct, in that they come from the region of the spinal cord that provides the correct adult connections. However, not all of the embryonic neurons in this region of the cord may be functionally correct and, therefore, may be inappropriate, in spite of coming from the correct area. Furthermore, in order to be functionally correct, a nerve must receive the appropriate presynaptic inputs as well as make contact with the appropriate target. The neurons from the correct part of the cord that make presynaptic connections appropriate for other muscles will need to be eliminated. Thus we must distinguish between anatomically correct and functionally correct, as some neurons that die have made anatomically correct connections (Prestige, 1976). The factors that led to cells from a specific area of the cord being guided to a specific muscle will relate to the guidance cues along the pathway and the response of the growth cones to it. On the other hand, the factors that control the presynaptic innervation of the cell in the cord will relate to the guidance cues for its innervating neuron, its own cell surface markers and neurotrophic signals it provides to its innervating neuron. Cell death will result from incorrect functional connections rather than anatomical ones under these circumstances.

4. P H E N O T Y P I C D E T E R M I N A T I O N BY T A R G E T TISSUE It is not known whether functional specificity is a property of the nerve fibre itself or is due to the end organ with which it makes contact. It is possible that the fibre and target are already specified, or that the target may confer specificity on its innervating neuron or, finally, that the neuron may guide formation of the target, as seen in the development of the taste buds. In the regenerating sympathetic nervous system the role of the presynaptic reinnervation with regard to function is less clear. Cross anastomosis of skin and muscle sympathetic neurons results in both appropriate and inappropriate changes in the reflexes shown by these neurons (J~inig and Koltzenburg, 1991) and in this study it was concluded that the reflex organization of sympathetic neurons can change qualitatively following nerve lesion when sympathetic neurons regenerate and supply inappropriate target tissues. Thus there is a rearrangement of the central connections to generate these reflex changes. The signal for the central rearrangement must be derived from the target tissue to change either the phenotype of the postganglionic neuron or the recognition signals it generates so that it now accepts new and appropriate connections from the preganglionic neurons. Alternatively, the retrograde signal could be transmitted trans-synaptically through the ganglion and alter the phenotype of the preganglionic neuron to alter the connections it receives. Such a mechanism could be mediated by the trans-synaptic transfer of a signal molecule derived

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from the target tissue. The only molecule to show such a trans-synaptic transfer has been tetanus toxin. An equivalent endogenous molecule has yet to be shown. The third mechanism for this alteration of connections could be by regulation of the neurotrophic factor produced by the ganglion neuron which could promote connection of the appropriate preganglionic neurons or result in a phenotypic change in these neurons so they regulate their connections. 4.1. TARGET-CONTROLLED REGULATION OF CENTRAL CONNECTIONS

Sympathetic and sensory neurons from a given nerve centre may grow out more or less randomly and innervate targets of opportunity along their route (Langley, 1895). For most sympathetic innervation this appears to be the case, as there is no topographical representation in the sympathetic ganglion for the target, although the innervation of the mesenteric vessels and enteric neurons seem to form two populations with seperate specificities for target innervation (Hill et al., 1983). It is possible that the target itself is able to modify the phenotype of its innervating neuron, in order for it to select the appropriate presynaptic innervation. Under these circumstances all neurons could be functionally appropriate. Neurons may, however, still be required to contact specific target types (for example, blood vessels or glandular tissues) in order to receive the appropriate neurotrophic factors for their survival, and this more subtle level of specificity may involve cell death. The regulation of the innervation of ganglionic neurons is clearly under neurotrophic control, as can be seen by the abnormal innervation pattern resulting from N G F treatment during the perinatal period. N G F treatment results in the abnormal ingrowth of sensory neurons identified by their calcitonin gene-related peptide content. These abnormal sensory neurons apparently compete with the preganglionic fibres for their neurotrophic factor such that the preganglionic fibres fail to develop as seen by the failure of the preganglionic marker enzyme choline acetyltransferase (EC 2.3.1.6) (CAT) to develop normally (Hendry el al., 1992). Weiss (1942) proposed the hypothesis that sensory neurons become modulated during embryogenesis by the biochemical character of their end-organs and that this acquired characteristic directs the formation of appropriate central synaptic connections. There is a difference between the motor system and the sensory system. The evidence on the motor side favours a parallel differentiation of motoneuron pools in the spinal cord and muscles in the periphery. The correspondence between motoneurons and muscles is then made by prespecified motoneurons growing out to form connections with appropriate muscles (Mark, 1980). Sensory neurons do not have their peripheral connections controlled to such an extent, although their birthdates and segmental localizations do have an influence (Baker et al., 1978). Axolotls develop appropriate reflex wiping movements after cross anastomoses of sensorimotor nerves and reinnervation of the skin (Griersmith and Mark, 1982). Evidence suggests that the underlying central nervous system connectivity is not the result of autonomous differentiation in the dorsal root ganglia paralleling that of the skin, but is imposed by information that is not available to them until they receive morphogenetic signals from the skin (Oriersmith and Mark, 1982). Thus, in the development of the normal reflex it would appear that there is an orthograde pattern of selection of connections such that the peripheral end-organ specifies the sensory neuron which, in turn, seeks out the motoneuron which has already sought out its correct muscle. The role of the target may become more important during regeneration. The effectiveness of functional recovery may be influenced by the target tissue which, in some ways, may alter the innervating neuron to produce a more suitable input. One case of regeneration best explained by this phenomena is in the sympathetic nervous system where, after lesion of the postganglionic nerve trunk, there is a return of full function after reinnervation despite the finding that, anatomically, many reinnervating neurons are incorrect in that they do not return to their original target (Hendry et al., 1986). To demonstrate this the retrogradely transported fluorescent dye, Fast Blue, was injected into the terminal regions of the neurons of the sympathetic ganglion. Fast Blue has the property of remaining within neuronal perikaryon for many months after its initial retrograde axonal transport from the peripheral tissue, even when the axon of the neuron is subsequently transected (Hendry et al., 1986). Thus, neurons can be labelled as to their initial target projection with Fast Blue and the specificity of subsequent regeneration can be followed using a second dye


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transported from the same or a different target. When such experiments were carried out on the regenerating SCG it became clear that a recovery of function can occur in a reinnervated target organ in the presence of a large component of fibres that were not originally to be found in that target. This can best be seen in an experiment where Fast Blue was injected into the anterior chamber of the eye and a week later the internal carotid nerve was cut. After regeneration of the sympathetic nervous system normal function returned to the iris and levator occuli. Another dye, Diamidino Yellow, was then injected into the submandibular gland to determine the projections of the regenerated axons. The surprising finding was that there was only a very small percentage of correct reinnervations in spite of a complete functional recovery (Hendry et al., 1986). This specificity of functional recovery must involve the presynaptic connections to the sympathetic neuron. It has been shown that at the segmental level there is correct presynaptic reinnervation (Langley, 1897; Njfi and Purves, 1978). Axon branching allows a small number of neurons to have widespread peripheral effects (Langley, 1900). The repeated branching of sympathetic fibres at the periphery provides for axon reflexes which further widens the sympathetic influences at the periphery. Thus it is possible that few correct neurons may be responsible for the functional reinnervation appearing to be correct with the incorrect units being turned off. Alternatively, the incorrectly projecting neurons are capable of plasticity in their central connections, such that correct presynaptic connections are made. A further possibility is that the sympathetic system is much less, specific than, for example, the motor system and that most nerves mediate similar responses. This latter postulate is unlikely to be true for events such as the pupiliary light reflex and can probably be discarded. 4.2. MECHANISMOF PHENOTYPIC CHANGE

The relationship between the innervating neuron and its target is not clear but the innervation of the sweat gland in the footpad may shed some light on this interaction. In vivo, the sweat gland is initially innervated by adrenergic neurons and then there is a phenotypic change, presumably under the influence of the target, when these cells become cholinergic (Landis and Keefe, 1983; Landis, 1988). Studies of postnatal rat sympathetic neurons developing in vitro have demonstrated that the environment can play a crucial role in the determination of the mature neurotransmitter phenotype. A series of detailed studies have shown that many non-neuronal cells produce a factor that results in a decrease in noradrenergic properties and the neurons acquire cholinergic properties (Patterson and Chun, 1974, 1977a,b; Potter et al., 1986; Kessler et al., 1984; Kessler, 1985a). In addition, environmental cues also influence the expression of neuropeptide levels in the sympathetic neurons, affecting substance P, (Adler and Black, 1985; Kessler et al., 1984; Kessler, 1984b, 1985b; Nawa and Sah, 1990; Bohn et al., 1984), VIP, somatostatin (Nawa and Patterson, 1990) and enkephalin (Bohn et al., 1983; Henschen et al., 1986; Romagnano et al., 1989) levels. The target tissue itself has been implicated as a possible source of a putative environment factor (Schotzinger and Landis, 1988, 1990; Stevens and Landis, 1990). Thus there may be a rearrangement of the central connections to provide a new specificity related to the target tissue innervated by the neuron. A number of proteins influence neurotransmitter traits in sympathetic neurons (Fukada, 1985; Kessler et al., 1986; Saadat et al., 1989). Sympathetic cholinergic differentiating factor and leukemia inhibitory factor are the same 45 kDa glycoprotein molecule (Yamamori et al., 1989) which increases CAT activity and decreases tyrosine hydroxylase activity (Fukada, 1985). A 22 kDa protein isolated from rat sciatic nerve, ciliary neurotrophic factor (Lin et al., 1989; Manthorpe et al., 1986; St6ckii et al., 1989) has a similar effect (Saadat et al., 1989). It appears that the two molecules act via the same receptor complex (Ip et al., 1992). Partially purified extracts of brain also induce cholinergic traits in sympathetic neurons (Kessler et al., 1986) and this is facilitated by plasma membranes and membrane-bound factor (Lee et al., 1990). Depolarization stimulates CAT in mouse spinal cord cultures (Ishida and Deguchi, 1983) but not in rat purified motoneurons (Martinou et al., 1989) suggesting a synergistic component from non-motoneurons. There is no obvious pattern of transmitter induction in sympathetic neurons but the timing of exposure to neurotrophic factors may alter the developmental profile of sympathetic neurons (Schwarting et al., 1990). Depolarization (Black et al., 1971; Black and Geen, 1973; Goodman et al., 1974; Kessler, 1986; Kessler and Black, 1982; Kessler et al., 1981, 1983; Walicke et al., 1977), cAMP analogues

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1. Circulation

,essenger active :tire active Axon

7.

,. Active Factor

2. Retrophin

I

Terminal

second messenger

- B. Factor Transported

~,cM 9. Factor exposed during neurite • FGF Labile second messenger

~

, C. Factor not Transported

|BR Receptor for B tE) Stable second messenger

I (~ Receptor for C •

Binding to extracellular matrix

Extracellular Matrix

FIG. 2. Diagrammatic representation of the ways in which the target can convey information to its innervating neuron. (1) Specific factor via circulation. (2) Transport of active neurotrophic factor. (3) Transport of active receptor. (4) Transport of second messenger. (5) Tansport of factor receptor complex. (6) Transport of permanently modified receptor. (7) Transport of second messenger. (8) Cessation of transport of death molecule. (9) Retrograde electrical activity. (10) Cessation of neurite outgrowth with build-up of molecules. ( G o o d m a n et al., 1974; Keen and Mclean, 1974; Mackay and Iversen, 1972), non-neuronal cells (Kessler, 1984a, 1986; Lefebvre et al., 1991; Patterson and Chun, 1974) all affect transmitter expression in different ways and result in diverse transmitter combinations, suggesting that co-localized transmitters are independently regulated. On the other hand, in vivo, the sympathetic ganglionic neurons do not appear to be affected by depolarization and remain adrenergic in spite of the section of preganglionic nerve trunks (Hill and Hendry, 1979). Substance P levels are increased in sympathetic ganglia by interleukin-I and stimulated splenocytes (Jonakait and Schotland, 1990). It is clear that depending on the interactions between factors, multiple agonists in different concentrations or acting sequentially in different orders can lead to the development of many different phenotypes.

5. M E C H A N I S M S TO C O N V E Y I N F O R M A T I O N F R O M T A R G E T TO N E U R O N There are a number of ways in which the target can convey information to its innervating neuron including provision of specific factors via the circulation, by retrograde axonal transport of neurotrophins, neurotrophin receptors or other messenger systems, and by a variety of other modulatory effects on neuronal function. 5.1. CIRCULATORY FACTORS As mentioned earlier, the initial neuroembryological studies suggested that the size of a nerve centre was determined by the size of its target. When N G F was first described in tumour tissue it was clear that it acted systemically to affect not only cells innervating the tumour but also other sympathetic and sensory ganglia. Thus a target derived neurotrophic factor was acting via the circulation. Insulin-like growth factor-I (IGF-I) causes enhanced regeneration of the rat sciatic nerve after a freezing lesion (Kanje et al., 1989; Sj6berg and Kanje, 1989). There may be a general role for neurotrophic molecules to act via the circulation to increase the potential size of the


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neuronal pool (Fig. 2.1) and both NGF and insulin-like growth factor-1 (IGF-1) have been suggested to increase mitogenic activity in neuroblasts (Rush et al., 1992). Such an action, however, does not explain the unilateral effects seen after limb removal or limb hyperplasia. IGF-I, which is important in the regulation of peripheral nerve regeneration (Linnemann and Bock, 1986), has been reported to be retrogradely transported in the sciatic nerve (Hansson et al., 1986, 1987). There has been failure, however, to detect high affinity uptake and retrograde axonal transport of IGFs by motoneuron processes in situ (Caroni and Grandes, 1990). There are likely to be many molecules that act directly on the cell bodies of developing neurons via the circulation. While many of these molecules could be produced in the target tissues, and may promote the survival of groups of neurons, it is unlikely that sufficient specificity could be achieved to enable accurate communication between the target tissue and the individual innervating neurons. 5.2. RETROGRADETRANSPORT OF FACTOR It is difficult to imagine an alternative mechanism for a specific target cell to convey information to a specific neuron cell body, other than by transport of a specific signal confined to the axon. In order for there to be a direct influence of the target only on the neuron innervating it, there must be a specific retrograde message carried via the axon from the target to the nucleus. The simplest mechanism for the target tissue to control the innervating neurons is for the target cell to make a neurotrophic molecule which is available only in limiting amounts. This REtrogradely TRansported neurotrOPHIN (Retrophin) (Hendry and Hill, 1980) is then taken up by the nerve terminals or growth cones and retrogradely transported back to the neuronal perikarya, where it acts to promote the survival of the neuron that has transported it (Fig. 2.2). The first neurotrophic molecule to be described to undergo retrograde axonal transport was NGF (Hendry et al., 1974). While the retrograde intra-axonal transport of NGF is now well established, the physiological significance of the specific retrograde axonal transport of NGF or, indeed, any other neurotrophic molecule is not resolved. The possible reasons for the transport of NGF include many of the mechanisms to convey information from the terminal to the cell body that have already been outlined. 5.2.1. Transport of Active Neurotrophic Factor Transport may be the means of getting NGF to the cell body where it or a breakdown product could exert its trophic effect. The site of action of NGF remains controversial with proponents for direct action on cytoskeletal elements and nuclear chromatin (Calissano and Cozzari, 1974; Andres et al., 1977; Bradshaw, 1983) or via second messenger mechanisms (Thoenen and Barde, 1980; Heumann et al., 1981). Some studies have suggested that free NGF reaches the cell cytoplasm and the nucleus where it interacts directly with the cytoskeletal elements leading to fibre outgrowth (Calissano and Cozzari, 1974; Nasi et al., 1982) or with nuclear binding sites (Andres et al., 1977; Bradshaw, 1983) leading to regulation of expression of specific genetic information, such as neurotransmitter biosynthetic enzymes. Studies of the localization of retrogradely transported NGF have not resolved this conflict, as some studies with iodinated NGF have suggested that NGF may reach the nucleus (Hendry et al., 1974; Iversen et al., 1975) but other studies, using either coupling products of NGF and horse radish peroxidase or autoradiographic localization of I t25 NGF, did not provide any evidence that NGF reaches the cytoplasm or, subsequently, the nucleus (Schwab and Thoenen, 1977; Schwab, 1977). The problem is that no detection system can rule out the presence of a small proportion of NGF in the nucleus. Thus this type of localization experiment cannot demonstrate that there is not a small, undetectable amount of NGF that leaves the membrane-confined compartments into which it goes after its receptor-mediated uptake and that may be responsible for the biological effects. In addition, NGF or its receptor could be degraded in the lysozomal compartment and a peptide could be released into the cytoplasm to act directly on the target elements. 5.2.2. Transport of Active Receptor Transport of NGF may be a means of getting the receptor to the perikaryon to act as the trophic

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message (Fig. 2.3). The receptor for NGF is retrogradely transported by neurons both in the periphery and in the central nervous system, as shown by the intracellular localization of the antibody to the receptor (Johnson et al., 1987). NGF receptors are transiently expressed on motoneurons during development in newborn rats and NGF is retrogradely transported by these neurons (Yan et al., 1988). As these receptors are not capable of mediating any traditional neurotrophic effects on survival, this suggests that the retrograde axonal transport of NGF in neurons is not necessarily synonymous with the ability of NGF to exert trophic activity.

5.2.3. Transport of Second Messenger An appropriate second messenger, which is subsequently transported to the cell body, may be generated at the nerve terminal and the retrograde transport of NGF may merely reflect the presence of receptors capable of internalizing NGF. It is unlikely that the neurotrophic factor itself is the message. It is more likely that the neurotrophic factor generates a second messenger, or a cascade of messengers, that is able to interact with the nucleus to result in neuronal survival. Any such second message that is to reach the cell body by its own retrograde transport would need to be very stable (Fig. 2.4).

5.2.4. Transport of Factor-Receptor Complex NGF binds to specific receptors, is internalized at nerve terminals and transported, together with its receptor, back to the cell body in coated vesicles which eventually fuse with lysosomes where the NGF is degraded (Schwab, 1977). It is unlikely that the internalized NGF itself is the active intracellular message, as intracellular NGF, where the cell surface receptors have been by-passed by direct injection of NGF into the cytoplasm and thus able to directly reach the nuclear chromatin cannot mimic the NGF receptor-mediated response (Rohrer et al., 1982; Heumann et al., 1981, 1984). In addition, affinity purified antibodies to NGF injected directly into the cytoplasm of PC12 cells do not affect the cellular response to NGF (Heumann et al., 1984). Fibre outgrowth also is not influenced by intracellular NGF (Seeley et al., 1983; Heumann et al., 1981). The blockade of the lysozomal degeneration of internalized NGF by the proteolytic enzyme inhibitor, leupeptin, influenced neither SCAT induction nor fibre outgrowth, showing that it is also unlikely for degeneration products of NGF to be responsible for its biological activity. Thus it is clear that the mechanism of action of NGF is via a second messenger system, but it is not clear whether the various effects are via the same second messenger or a variety. The formation of a second messenger could be initiated by the interaction of NGF with its receptors either immediately after NGF binding at the cell membrane or following internalisation and retrograde axonal transport of the receptor-NGF complex in vesicles. As the receptor-NGF complex in the transported vesicle is presumably continuously capable of generating a second messenger, it may be that the transport enables a short-lived or unstable second message to reach the cell body. It seems likely the rapid effects of NGF, such as chemotactic effects on growth cones (Gundersen and Barrett, 1980; Seeley and Greene, 1983) and initiation of neurite sprouting (Calissano and Cozzari, 1974; Nasi et al., 1982), could be mediated by locally acting second messengers activated by the interaction of NGF with its receptor at the surface and involving calcium ions and phosphorylation by receptor-associated tyrosine kinase (Kaplan et al., 1991). The longer term effects, involving the regulation of the synthesis of specific proteins in the cell body at the transcriptional or post-transcriptional level (Rohrer et al., 1978; Hefti et al., 1982), require the transport of the second messenger-generating system to the cell body or else the transport of a much more stable molecule. Peripheral administration of NGF to sympathetic nerve terminals leads to a specific increase in tyrosine hydroxylase in the cell bodies (Paravicini et al., 1975) and, subsequently, an increase in the size of the neurons that retrogradely transported the NGF (Hendry, 1977a). Therefore, the messenger responsible for the long-term effects must either be produced at the terminal and be stable enough to survive its own retrograde transport to the nucleus or be generated by the NGF-receptor complex as it is transported along the axon and into


275

the cell body. Thus a much more labile second messenger could be responsible for the longer term actions of NGF (Crouch and Hendry, 1991). 5.2.5. Control o f Tissue Retrophin Levels If a neurotrophic factor is to have in vivo physiological relevance, there must be a mechanism for controlling its availability to neurons. This may be via competition between neurons for a limited amount of trophic factor released from the target which will be exacerbated if the neurons remove the factor. Retrograde transport may serve as a mechanism to remove NGF from the terminal region and therefore be a means to regulate the levels of NGF in the target. Neurotrophic factors for sympathetic and parasympathetic neurons are elevated in the rat ventricle after chemical denervation (Kanakis et al., 1985) and growth factors for ciliary neurons are elevated in skeletal muscle after denervation (Hill and Bennett, 1983). The effects of 6-hydroxydopamine and colchicine on levels of NGF in sympathetic ganglia and sympathetic target tissue strongly suggest that NGF levels in the target is controlled by retrograde axonal transport by the innervating neurons (Korsching and Thoenen, 1985). The availability of trophic factors may be regulated by other mechanisms, such as binding to the substrate, as has been proposed for fibroblast growth factor (FGF) (Eccleston et al., 1985; Rogelj et al., 1989). If this is the case, then availability is regulated by the composition of the target tissue itself and not by the innervating neurons. The most plausible mechanism of action of NGF is that it is internalised together with its receptor into coated vesicles which are retrogradely transported to the cell body, where the NGF-receptor complex continues to generate the labile second messenger required for its action (Fig. 2.5). 5.3. No RETROGRADE TRANSPORT OF FACTOR In spite of the logic of the retrophin model, retrograde axonal transport has been described for only a very few putative neurotrophic molecules. While this mechanism is the simplest, it is not the only way to get a message from the periphery, and an in-depth analysis of some other mechanisms that may lead to the chromatolytic signal has been made previously (Cragg, 1970). If the neurotrophic molecule itself is not the second messenger, then there has to be translocation of some other message from the terminals in the target tissue to the nucleus. Before this can be considered, some consideration must be given as to what is a neurotrophic factor. 5.3.1. Does a Neurotrophic Factor Require Retrograde Transportation? The NGF model has led to the suggestion that all neurotrophic factors must be retrogradely transported (Thoenen et al., 1985); a point of view that is much more rigid than the evidence warrants. If there are ways for a factor to signal from the target in the absence of the transport of the factor itself, then it should be still considered a neurotrophic factor. In order for a potential survival factor identified in culture to be seen as a viable neurotrophic factor it must act in vivo to promote neuronal survival. There are many neurotrophic factors that have been shown to have effects in vivo, but there is always the possibility that the effects of an agent are pharmacological in that it mimics the action of the endogenous factor, often at very high concentrations, rather than being the physiological factor itself. The physiological factor will need to be present in limiting amounts in order to achieve the desired control of survival. The classical studies on NGF have shown that this molecule can cause the survival of sensory neurons (Levi-Montalcini and Angeletti, 1968) and has provided a paradigm for the in vivo investigation of the other putative survival factors. NGF also causes the survival of sympathetic neurons (Hendry and Campbell, 1976; Levi-Montalcini, 1965) and can rescue them after axotomy (Hendry, 1975a) and 6-hydroxydopamine treatment (Levi-Montalcini et al., 1975). There are several members of the FGF family that may be of neurotrophic relevance (Basilico et al., 1989; Delli-Bovi et al., 1988). Polyclonal antibodies to basic FGF (bFGF) have been shown to react to a higher MW molecule which appears to cause survival of ciliary neurons in culture (Grothe et al., 1990). bFGF is found in adrenal medullary chromaffin granules (Westermann et al.,

276

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1990) and gel foams soaked in bFGF can rescue adrenal preganglionic neurons after electrolytic destruction of the adrenal medulla (Blottner et al., 1989b). Acidic FGF (aFGF) enhances nerve regeneration in sciatic nerve (Cordeiro et al., 1989) and retinal ganglion cell processes (Lipton et al., 1988) and stimulates adrenal chromattin cells to proliferate and extend neurites, but fails to cause long-term survival (Claude et al., 1988). In addition, aFGF causes the differentiation of ciliary neurons in chick embryos but fails to reverse naturally occurring cell death (Hill et al., 1991). Ciliary neurotrophic factor (CNTF) can rescue adrenal preganglionic neurons after electrolytic destruction of the adrenal medulla (Blottner et al., 1989a) and facial neurons after axotomy (Sendtner et al., 1990). While the FGFs and CNTF are good candidates as ciliary neurotrophic factors (Watters et al., 1989) specific retrograde axonal transport of FGF (Hendry and Belford, 1991) or CNTF (Smet et al., 1991) by ciliary neurons have not been demonstrated, bFGF was also not retrogradely transported in the adult rat sciatic nerve, or from iris to trigeminal or SCG (Ferguson et al., 1990). Most molecules can be shown to have a low level of nonspecific retrograde transport; for instance horseradish peroxidase, FGF, bovine serum albumin, cytochrome C are all only transported to a minor degree. This is presumably as a result of pinocytosis of extracellular fluid in the region of the nerve terminal (Heuser and Reese, 1973) into coated vesicles (Sacks and Saito, 1969). This uptake is dependent upon activity of the nerve (Teichberg et al., 1975; Holtzman et al., 1971), unlike that of the receptor-mediated uptake of NGF, where alterations in the transport of NGF were not seen after alterations in the firing pattern of sympathetic neurons (Lees et al., 1981; St6ckel et al., 1978). From the preceding discussion it can be concluded that there are many putative neurotrophic molecules that do not themselves have a high capacity retrograde transport and there may be two potential types of neurotrophic molecule with either a short-lived second message (Fig. 3A) or a long lived-message (Fig. 3B). One class, like NGF, has a labile second messenger and thus requires the transport of the neurotrophin-receptor complex in order to be able to generate the second messenger near enough to the neuronal nucleus to be effective. The other class must generate a stable second messenger at the nerve terminal and it is the transport of this that results in the specificity of the target/nerve communication. There are three ways in which a long-lived message could be generated. Firstly, activation of the receptor might render it permanently active and this activated receptor could reach the cell body. Secondly, there could be the transport of a stable, activated second messenger complex. Thirdly, the normal transport of some inhibitory or cell death factor might be prevented. 5.3.2. Transport of Permanently Modified Receptor One mechanism by which the signal might be transduced from the nerve terminal is by the permanent activation of a receptor. In platelets, the thrombin receptor is cleaved and this results in its permanent activation. It is interesting that thrombin itself has been shown to have direct effects on central nervous system neurons (Mazzoni and Kenigsberg, 1991). It is possible that proteolytically active molecules secreted by the target tissue could act to modify receptors on the nerve terminal and these may be internalised either alone, or together with other internalised receptor-factor complexes and be transported back to the cell body. Thus there is a mechanism for several signals to act in concert and provide a complex integration of target derived signals to be received by the nucleus. 5.3.3. Transport of Second Messenger One of the major arguments against aFGF or bFGF being neurotrophic factors is the lack of an appropriate secretory sequence (Esch et al., 1985a,b), making regulated release by the target tissue unlikely. If FGF is not a target-derived neurotrophic factor then one of the other members of the FGF gene-related family may be (Brookes et al., 1989; Zhan et al., 1988; Huebner et al., 1988). In this case, the protein would have to share an antigenic epitope with aFGF and may itself be retrogradely transported. If in fact aFGF is an essential neurotrophic factor for ciliary neurons, then the retrograde message must be conveyed by a mechanism other than the retrograde transport


277

of FGF itself. It is not essential, however, for the neurotrophic factor itself to be retrogradely transported but, clearly, some signal must reach the neuronal cell body. A likely alternative is the retrograde axonal transport of one of the second messengers. The most likely candidates for a stable second message are proteins that have undergone some covalent change. 5.3.3.1. G-protein translocation. It has been shown that in platelets there is a translocation of G-protein from the cell membrane to the cytoskeleton (Crouch et al., 1989). It has also been shown that activation of Balb/c 3T3 fibroblasts results in translocation of the g subunit of Gi from the membrane to the nuclear chromatin (Crouch, 1991). A similar phenomenon could result in the retrograde axonal transport of the neurotrophic message for FGF. There is an accumulation of Gi, (Hendry and Crouch, 1991) and Gz~ (Hendry and Crouch, 1992) on the proximal and distal sides of a ligature placed on the mouse sciatic nerve, demonstrating that there is not only transport of these second messenger molecules to the nerve terminal but also transport back to the cell body from the terminals. In this case it has to be assumed that the nerve terminals are normally being activated by endogenous neurotrophic factors and that retrograde axonal transport of the GTP-binding proteins may be a representation of the stable message generated by this activation. 5.3.3.2. Protein phosphorylation. Many neurotrophic factors act through tyrosine kinase receptors which seem to generate cascades of phosphorylation reactions. Any of these may, generate an appropriate stable second message to transport back to the cell body. Furthermore, phosphorylation reactions can provide a mechanism for the interaction of growth factors at the terminal to generate specific alterations in potential retrograde messengers (Greengard, 1987). These phos-

A

B

Labile Second Messenger [NerveTerminaiJ

Stable Second Messenger [Nerve Terminal]

NGFReceptors

Growth Factor Receptors

o oo o o oo NGF o

'

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Growth Factor

NGFbound to Receptors leads to clustering

Retrograde A.xonal Transport of NGF bound to Receptors in Coated Vesicles

"~~.4--sec~t41~ ~ ~

Intemalizadon of NGFintovesicles

Long livedsecond messenger undergoing Retrograde Axonal Transport

Growth Factor - Receptor Complex may generate stable or labile second messengers

Labile

Labile second messenger continues /A ~ ~ tobe generate,din / '~'~m,,~t--"~

~

X

RetrogradeAxonalTransport of Stable Second Messenger

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GF-Receptorcomplex

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~--O-~, ~~ Nucleus

FIG. 3. Two possibilities for the types of neurotrophic factors. (A) Neurotrophic factor with labile second messenger requires the retrograde axonal transport of a receptor/factor complex to enable the generation of active second messenger at the cell body. (B) Neurotrophic factor which generates a stable second messenger which is active after the long time required for its transport to the cell body does not require the transport of the factor itself. JPT

56/3--B

278

I.A. HENDRY

phorylation reactions can alter proteins such that they can generate new complexes via binding mechanisms involving phosphorylated tyrosines and the src homology domain 2 regions SH2 and SH3 of intracellular molecules that are thought to be important in signal transduction (Coughlin et al., 1989; Cantley et al., 1991; Klippel et al., 1992). For example, the platelet-derived growth factor receptor is a tyrosine kinase that, on activation, can form complexes with phospholipase-C 7-I, GTPase activating protein and phosphatidylinositol 3-kinase via distinct phosphotyrosinecontaining sequences (Fantl et al., 1992). Thus it is possible that at the nerve terminal, there can be a complex interaction between receptors and the intracellular transduction molecules, such that very specific complexes generating a unique message may be generated and this second messenger unit can be conveyed to the cell body via retrograde axonal transport. 5.3.3.3. Acylation or deacylation o f proteins. Alterations in the hydrophobicity of molecules may come about by addition or cleavage of a lipid component, resulting in a molecule suitable for retrograde transport. No matter what the signal is, it must be transported either in vesicles or as a soluble molecule, either alone or in a complex. The evidence for the retrograde transport of coated vesicles is good but there does not seem to be any evidence for the retrograde transport of isolated proteins. Thus there may be two types of protein alteration, either to add a lipid component and enhance the chances of becoming associated with coated vesicles or to remove an acyl group to promote the protein leaving the plasma membrane. The 87 kDa protein phosphorylated by protein kinase C seems to be the same as a 68 kDa protein myristoylated in response to lipopolysaccaride in macrophages (Aderem et al., 1988). This myristoylation could allow targeting of the protein to the membrane, where it is more readily phosphorylated by protein kinase C and thus would prime the cells for a subsequent activarnon. The 87 kDa protein has been described in brain, where it is phosphorylated in response to phorbol esters and depolarisation (Wang et al., 1988, 1989). This protein appears to be myristoylated in murine frontal cortex cells (Aderem et al., 1988). GAP-43 appears in dorsal root ganglion cells and in the dorsal horn in the spinal cord of neonatal rats following peripheral nerve injury (Woolf et al., 1990). Myristoylated alanine-rich C kinase substrate (MARCKS) (Stumpo et al., 1989) is a 87 kDa protein which is a major specific substrate for protein kinase C in rat brain (Ouimet et al., 1990). MARCKS is translocated after phosphorylation in isolated nerve terminals (Wang et al., 1989), an event associated with its release from membranes into the cytosol (Narayanan and Narayanan, 1981). Thus there are many acylation/deacylation reactions occurring in the nerve terminal which may generate a stable, modified protein and result in its becoming available for retrograde transport. 5.3.4. Cessation o f Retrograde Transport o f Death Molecule There may be molecules taken up at the nerve terminals that could promote the death of the neuron. Interaction with the appropriate target could prevent the uptake of this substance and thus prevent neuronal death. 5.4. PHYSICALMECHANISMS 5.4.1. Electrical

Although this type of mechanism has not yet been explored in the literature, it should still be considered when analysing the many different strategies that could be used to signal to the cell from its target. At the time of synaptogenesis there is an intimate communication between neuron and target, often with the formation of gap junctions. The possibility for electrical coupling exists and the generation of retrograde electrical activity could effect neuronal survival or even neuronal phenotype. 5.4.2. Cessation o f Neurite Outgrowth It is not clear what the signal is that results in the cessation of neurite outgrowth, but it could be the effect of good matching of cell-cell recognition molecules or the result of high concentrations of growth factors. The earliest experiments on NGF demonstrated that sensory axons, growing into


279

a tumour synthesising large amounts of N G F , ended blindly, apparently ceasing elongation in the absence of any target contact (Levi-Montalcini and Hamburger, 1951). When a neuron reaches its appropriate target there is also a cessation of axon elongation and growth. Whatever the mechanism of this cessation of growth, there could be a build-up of proteins normally transported away from the cell body. In this way, factors necessary for neuronal survival m a y accumulate.

6. C O N C L U S I O N S It is likely that more than one mechanism is involved in the formation of correct neuronal connections. There must, however, be some feedback from the target to the innervating neuron to uphold and strengthen correct connections. There is little evidence to support the direct effect of a neurotrophic factor on the neuronal nucleus and there must be the mediation of a second messenger. It is proposed here that there are two classes of neurotrophic factor. The first, represented by N G F , has a labile second messenger which requires the transport of a factor-receptor complex capable of continuous generation of the second messenger to the cell body. The second class does not require retrograde transport of the factor, as it can generate a stable second messenger that survives the long-time for transport up the axon. It is probable that the message that is transported via the axon will be a complex resulting from the activation of multiple receptors at the nerve terminal, rather than a single molecule. The result of the transport of the complex will be to give the cell detailed information as to the state of its terminal region and the nature of the target tissue and target cell that it has contacted.

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JPT 56/3--47

Retrograde axonal transport of rapidly migrating proteins in peripheral nerves.

Retrograde axonal transport of rapidly migrating labelled proteins and glycoproteins in regenerating peripheral nerves.

Microsurgery of peripheral nerves.

Autograft suture in peripheral nerves.

Iatrogenic lesions of peripheral nerves.

Noninvasive imaging of peripheral nerves.

Gunshot Wounds of Peripheral Nerves.

Advanced imaging of peripheral nerves.

Properties of tyrosine hydroxylase in peripheral nerves.

Synaptoid profiles in regenerating crustacean peripheral nerves.

Progenitors in Peripheral Nerves Launch Heterotopic Ossification.

Carboxypeptidase M in brain and peripheral nerves.

Normal and sonographic anatomy of selected peripheral nerves. Part II: Peripheral nerves of the upper limb.

Regeneration of peripheral nerves after injury.

Rerouting peripheral nerves for spinal cord lesions.

Peripheral development of avian trigeminal nerves.

Diffusion tensor imaging of peripheral nerves.

Percutaneous recordings from peripheral nerves (preliminary communication).

Course of the peripheral gustatory nerves.

Label-free photoacoustic microscopy of peripheral nerves.

Human endothelial cells secrete neurotropic factors to direct axonal growth of peripheral nerves.

Repair of peripheral nerves of unequal diameters.

Benign fatty tumors of the peripheral nerves.

5th Days of the Francophone Society of Peripheral Nerves: Foreword].