Clinicul and Experimental Pharmacology and Physiology (1998) 25, 487-495

BRIEF REVIEW

ROLE OF AFFERENTS IN THE DEVELOPMENT AND CELL SURVIVAL OF THE VERTEBRATE NERVOUS SYSTEM

Rachel M Sherrard" and Adrian J Bower?

*Neuroscience Laboratory, School of Life Sciences, Queensland University of Technology, Brisbane and tDepartment of Anatomical Sciences, The University of Queensland, St Lucia, Queensland, Australia

SUMMARY

1. During normal development of the vertebrate central nervous system, a considerable number of neurons die. The factors controlling which neurons die and which survive are not fully understood.

2. Target populations are known to maintain their inner- vating neurons. However, the role of afferents in maintaining their targets is still under review.

3. In the developing nervous system, deafferentation of a neuron population is difficult to achieve because plasticity (structural re-organization) can cause re-innervation of the area. Re-innervation alters, rather than removes, the afferent supply.

4. Afferent input is important for neuronal survival during development because deafferentation increases neuronal death by 2030% and increasing input diminishes neuronal death.

5. Deafferented neurons die at the normal time for cell death for any given population. This occurs after the arrival of affer- ent axons but before the completion of connectivity and the onset of function.

6. Neuronal survival is maintained by any input, such as re- innervation by inappropriate fibres, but for optimal survival, morphological maturation and the acquisition of normal physi- ology, the correct input is required.

7. Afferents maintain their target neurons via a combination of electrical activity and delivery of trophic agents, which ad- just intracellular calcium, thereby facilitating protein synthesis, mitochondria1 function and suppressing apoptosis.

8. Evidence from animal and in vitro experiments indicates that afferents play an extremely important role in the survival of developing neurons in the immature vertebrate nervous system.

Key words: cell survival, central nervous system, deaf- ferentation, programmed cell death.

INTRODUCTION

All neuroscientists 'know' that afferent input is essential to the survival of neurons. Yet, when the literature is examined, there is

Correspondence: Dr RM Sherrard, School of Life Science, QUT, GPO Box 2434, Brisbane, QLD 4001, Australia. Email: a.sherrard@qut.edu.au>

Received 6 January 1998; revision 17 March 1998; accepted 26 March 1998.

little direct evidence for the truth of that statement. In order to elu- cidate the role of afferents in cell survival during development, ex- perimental evidence from a wide range of systems, involving all developmental stages from embryo to adult, and using a large number of techniques must be analysed. Historically, knowledge that afferent input is essential for neuronal survival in adult animals was used as a tract-tracing tool. Severing afferent fibres leads to antero- grade transneuronal changes in the target neurons.' The degree of change depends on the purity of the afferent input; systems with most afferents from a single source (e.g. the visual system) show the most marked changes. Rarely is cell death seen in adult systems; normally, the transneuronal changes relate to the size and morphology of the target neurons.' However, the result of removing the target is differ- ent. In this case, there is retrograde degeneration of neurons pro- jecting to the target. The implication of this is that because removal of afferent input does not cause neuronal death, but removal of target neurons does, afferent input is not as important for neuronal sur- vival as the factors derived from the target neurons. As will be shown in the present review, this is not the case in the developing nervous system. Target-derived growth factors are still important, but affer- ent input may be equally so because, in the developing animal, re- moval of afferent input does lead to anterograde transneuronal death.3

During development of the nervous system there are several complex steps, ranging from initial formation of the neural plate and neurogenesis through to the development of critically defined con- nections and, ultimately, the death of unnecessary, unwanted or in- appropriate neurons? These unwanted neurons die during a period that is specific for each population and this occurs when a neuron innervates its target! It is this observation that underlies the neuro- trophic hypothesis, that a target produces a signal for its innervating population that limits the population's cell death.5 However, the fac- tors involved in directing which neurons will die and which survive are not fully understood and these have been the subject of ongoing research over many years. While the mechanisms whereby target tissue maintains innervating neurons is well understood,6 it is only in the peripheral nervous system that the interplay between afferents and their targets on neuronal survival has been demonstrated. Deafferentation of autonomic ganglia increased death in the target (ganglion) population. Also, loss of a target results in increased developmental cell death in the innervating popula t i~n .~-~

However, the requirements for neuronal survival in the central nervous system (CNS) are not well understood. In particular, the evidence indicating that afferents play a crucial role in neuronal

488 RM Sherrard and AJ Bower

List of abbreviations

BDNF Brain-derived neurotrophic factor CNS Central nervous system CNTF Ciliary neurotrophic factor FGF-2 Fibroblast growth factor-2 IGF-I Insulin-like growth factor- 1 LIF Leukaemia inhibitory factor

NGF Nerve growth factor NOT NT3 Neurotrophin-3 NT4/5 Neurotrophin-4/5 PDGF Platelet-derived growth factor

Nucleus of the optic tract

survival during development of the CNS is less clear. Experimental evidence shows that electrical activity in afferent paths increases the survival of rat tectal neurons as more of these neurons survive when retinal ganglion cells are spontaneously active.* Furthermore, an in vifro study on retinal ganglion cell survival has shown that these neurons are maintained by several growth factors, some of which are target derived from the tectum. But these growth factors are much more effective when they are combined with circumstances in- dicative of electrical activity (e.g. depolarization by high K', gluta- mate or a rise in CAMP by forskolin).' These authors conclude that this may show that target-derived agents need afferent activity to work properly. Thus, CNS neurons have a different requirement from those of the peripheral nervous system (i.e. greater dependence on afferent activity, in particular that which raises CAMP).

However, this in vitro study' leaves many questions unanswered. It is not known whether afferents have a general non-specific role in neuronal survival (i.e. any afferent will do) or whether an input has to be specific for each neuronal population. One of the diffi- culties facing investigators of the CNS is that the immature nervous system is capable of much plasticity and re-organization of axons4 Therefore, any given population of neurons rarely remains dener- vated, but receives an alternative input, which makes interpretation of the results difficult.

Studies trying to identify whether afferents do play a specific and crucial role in neuronal survival have been divided into three groups: (i) those looking to see the effect of deafferentation on neuronal sur- vival; (iif those assessing whether increasing afferent input can in- crease neuronal survival; and (iii) those that identify whether cell survival can be enhanced by factors associated with afferent input. Because the importance of target-derived support has been widely examined and reviewedk7 it will not be covered in the present re- view. Therefore, the purpose of the present review is to identify what is known about the role afferents may have in the survival and/or death of their target neurons in the developing nervous system. Furthermore, we will propose mechanisms explaining how this may occur. Last, how this information leads to a greater understanding of the role of afferents in the development of the normal, non- interfered with, brain will be summarized.

DEAFFERENTATION-INDUCED NEURONAL DEATH

One of the major strands for investigating the role of afferent input on neuronal survival during development has been to assess the degree of neuronal death and/or survival in target neurons following their deafferentation.

Most early evidence relating afferent input to neuronal survival has come from changes identified in the adult mammalian visual

system following lesions during development. After enucleation (eye removal) in the neonatal period, fewer neurons remain in the deaf- ferented target area for the retinal ganglion cells (optic tectum) of adult rats." This indicates a transneuronal effect of afferent input on target cell survival. Furthermore, the degree of neuronal deatWsurviva1 seems to depend on the amount of afferent loss. In the brainstem parabigeminal nucleus, there is neuronal death after lesions of its afferent source, the optic tectum," such that the number of remaining neurons in the parabigeminal nucleus correlated highly with the size of the innervating population left in the tectum.

However, the above results from adult animals, do not indicate what changes may be taking place within the developing denervated area to cause fewer surviving neurons in the mature brain. Therefore, studies have been performed during development to assess the time course of events.

When do neurons die following deafferentation?

In attempting to understand the potential role for afferents in normal neuronal developmental survival or death, the timing of cell death after denervation has to be assessed. Following deafferentation, in- creased neuronal death takes place at the time that normal develop- mental death occurs.'* Therefore, if the afferents are removed before the normal time for developmental cell death, there is a delay in the onset of transneuronal death. This delay may vary between hours in the optic tectum13 to days in the spinal cord,14 auditory n ~ c l e i ' ~ . ' ~ and motor ~ o r t e x . ' ~ The length of the delay appears to depend on the relative timing of the denervation and onset of physiological cell death within each system. Thus, whatever the perturbation to the developmental process caused by the experiment, the implication is that afferent input appears to be important for neuronal survival during specific stages of normal development; that is, the timing of cell death does not change, only the number of neurons that degenerate changes.

How many neurons die?

Developmental cell death has been proposed to be the mechanism that matches the size of target and innervating neuronal populations in different parts of the nervous system.6 Therefore, it is likely that afferents are involved in this process and modify cell survival/death in order to match the size of two neuronal populations. Unfortunately, the current literature contains contradicting effects of deafferentation, some s t ~ d i e s ' ~ ~ ' ~ ~ ' ~ ~ ' ' showing population match- ing and othersi7 that do not show this effect.

Support for the possible role for afferents in matching target and innervating populations comes from consideration of the numerical effects that deafferentation has on various populations. Following deafferentation, the amount of transneuronal death so induced

Role of afferents in neuron survival 489

appears to be quite consistent and seems to relate to the degree of afferent loss. After significant deafferentation, the increase in cell death is in the order of 20-30% and is consistent across widely diverse systems. In the chick spinal cord, transection increases motoneuron cell deathI4 to the same degree that cochlea removal induces death in the nucleus magnocellularis of the chick brainstem’* and ablation of retinal ganglion cell input induces in the rat optic tectum.I3 While in each case a major afferent source has been re- moved, each of the above target areas retains other afferents that presumably are able to maintain the residual population. Remaining afferents to the above systems are interneurons and primary affer- ents to spinal cord motoneurons, descending axons from the di- encephalon to the nucleus magnoceliularis7 and input from the visual cortexzo and parabigeminal nuclei” to the optic tectum. Furthermore, if only a small proportion of afferents disappears, there may not even be any increased death.”

However, there are other studies in which the degree of cell death does not clearly match the proportion of afferents lost. In the zebra finch (Poephilu guttata), the robust nucleus of the archistriatum (RA) is the motor control region for song and it has only a single affer- ent source. When this source is destroyed, cell death in the RA only increases by 50%, with many neurons surviving the denervati~n.’~ There could be two reasons for the apparent difference between this and the studies cited earlier. Either the central location of the RA allows its empty post-synaptic sites to be taken over by inputs nor- mally terminating in adjacent areas; therefore, re-innervating the region, or the neurons themselves have an ability to adapt to the altered conditions.

When are afferents required for neuronal survival? In summary, removal of afferents increases the number of cells that die, but the timing of afferent removal does not affect the timing of cell death. The question now is whether the arrival of afferents heralds the process of cell death during normal development.

Early studies indicated that neurons appear to become dependent on their afferents for survival at the time when such afferents nor- mally innervate that population. This was first demonstrated in the chick cochlear nucleus7 in which neuronal death following early deafferentation does not start until the age at which afferents usually invaded the nucleus.” Also in the chick, primary vestibular afferents reach the vestibular nuclear neurons on embryonic day 3 (E3), but these afferents do not synapse on their targets until E7. If these affer- ents do not arrive due to otocyst ablation, the neurons of the vestibu- lar nuclei continue to develop normally until the time for natural developmental cell death starting on E7.22 These studies were felt to confirm that the onset of afferent dependence and normal develop- mental cell death occurred at the time of afferent arrival. However, recent evidence does not confirm this, as afferents may be present much earlier than previously supposed. Ultrastructural analysis of the vestibular nuclei revealed a few synaptic inputs from centrally located neurons from E5,” which indicates that afferents act on their targets very early in development, preceding the time for develop- mental cell death. These studies illustrate the difficulty of investi- gating the role of afferents in CNS development because it is almost impossible to ensure all afferents are removed. Nevertheless, these studies do suggest two effects of afferent input on target neurons. First, the survival of very immature neurons is either relatively in- dependent of afferent support, because a small input seems adequate

to maintain a whole neuronal population, or survival is always affer- ent dependent, although the types and quantity of inputs that are required may change with maturation. Second, neurons reach a developmental stage when they require their appropriate input and, if it does not arrive, their survival will be compromised and cell death will ensue. Therefore, while the time of onset for afferent depen- dence is not clearly defined, the cited studies provide evidence to show the importance of input for neuronal survival during devel- opment.

Are afferents always required for neuronal survival? Having developed afferent dependence, neurons appear to lose it at the onset of function, a time when all other connections have been established. This has been demonstrated in the gerbil cochlear nucleus.I6 Therefore, the window of dependency on afferent input appears quite short, between the onset of innervation by the major afferent and the completion of connectivity (both afferent and efferent). It could, therefore, be argued that the onset of function correlates with the end of cell death.

However, deafferentation of mature neurons still adversely affects them. Loss of a major afferent input leads to atrophy of dendrites with associated alterations in soma size,’.’ but it is clear that the dependence on input for survival is much less than during maturation.

ROLE OF AFFERENTS IN PREVENTING CELL DEATHKONVERSE OF DEAFFERENTATION

The increase in neuronal death following deafferentation does not implicitly indicate that afferent input is important in normal neur- onal survival. In order to assess this, the converse should be true: increasing the afferent input ought to increase cell survival. While this is technically difficult to do in the vertebrate CNS and implies complex strategies, like transplantation of extrasensory organs such as eyes, there are some studies that seem to indicate that this con- verse argument is true.

Taking advantage of the plasticity that follows surgical interven- tion in the immature nervous system, an increase in the afferent input to an area within the CNS can be induced. In the rat visual system, retinal ganglion cells project to the lateral geniculate body of the thalamus, the nucleus of the optic tract (NOT) and t e c t ~ m . ~ ~ Lesions to the visual cortex cause retrograde degeneration of the lateral genic- ulate body, thus removing one of the targets of the retinal ganglion cells. This induces increased projection to the NOT and optic tec-

which results in a 39% increase in neuronal survival in the NOT.24 Similar evidence arises from retinal ganglion cells by the nature of their partly crossed and partly uncrossed projection to the lateral geniculate body of the thalamus. Transection of one optic tract induces degeneration of retinal ganglion cells in the lateral half of the retina that project to the ipsilateral thalamus, leaving those in the medial half of the retina, which project contralaterally, to survive, There is an increased survival among the neurons of the medial half of the retina.25 Because the target of these retinal ganglion cells has not been affected, the increased neuronal survival is presumed to be associated with an increase in afferent availability.2s

Therefore, these two experiments from the rodent visual system seem to indicate that afferents do play a significant role in normal neuronal survival. However, in view of the differences in neuronal

490 RM Sherrard and AJ Bower

response to deafferentation seen in different systems, it is quite pos- sible that the results of these overload experiments are specific to retinal ganglion cells and may not be found in other areas of the vertebrate nervous system.

LACK OF DEAFFERENTATION-INDUCED CELL DEATH: DOES THIS MEAN AFFERENTS

ARE NOT REQUIRED?

One of the difficulties facing neurobiologists trying to assess the role of afferents in neuronal survival during development is the contra- dictory nature of many of the results. In addition to the evidence for increased neuronal death following deafferentation in develop- ment (vide supru), other studies do not appear to support the impor- tance for afferents in neuronal survival.

Again, much of the evidence arises from studies of the visual system. Following early enucleation, the optic tectum loses neurons with dorsally directed dendritesz6 Also, in dark-reared cats, Y-cells disappear from the lateral geniculate body2' and the visual cortex loses neurons that respond to visual stimuli.28 However, these physio- logical studies are not supported by the quantitative data. Although the optic tectum loses neurons with dorsally directed dendrites, it contains a greater percentage of neurons with different dendritic morphologies and physiological properties, responding to somato- sensory rather than visual input.26Also, there is no evidence for neur- onal loss in the rodent lateral geniculate body after retinal ganglion cell ina~t iva t ion ,~~ a result that probably reflects the extra somato- sensory afferents that re-innervate this nucleus:" suggesting a change of function for these neurons. It is unclear from these studies whether there is overall loss of neurons or whether they have just changed their morphology and function.

It is the capacity for plasticity of axon terminals to re-innervate a denervated population of neurons that makes the assessment of the significance of afferents for neuronal survival difficult. The re- innervation of denervated areas by 'inappropriate' afferents from ad- jacent regions has been widely demonstrated in the vertebrate nervous system, including the thalamus and visual cortex after monocular depri~ation,~" the trigeminal nuc leu~,~ ' thalamus and somatosensory cortex after vibrissal a b l a t i ~ n ~ ' . ~ ~ and cerebellar Purkinje cells after climbing fibre ablation.34 Therefore, it may be extrapolated from these results that afferents are crucial for survival and that neurons are maintained by another input that has the capacity to re-innervate them. However, such results could also in- dicate that a neuron's survival depends on its ability to adapt to other functions26 rather than on its input.

What these studies do indicate is that many neurons do not require a specific input for their survival. It would appear that most neurons could be maintained by any afferent. However, a neuron's mor- phology does seem to reflect its input and this needs to be specific for the development of normal neuronal morphology.

MECHANISMS FOR AFFERENT-INDUCED NEURONAL SURVIVAL

While the above sections indicate that afferents are important for neuronal survival and the development of normal morphology, the mechanisms have not been elucidated. Studies on the role of affer- ents fall into three categories: (i) neuronal survival in vitro; (ii)

normal nervous system development; and (iii) the effects of deaffer- entation during development. However, to gain insight into the pos- sible mechanisms by which afferents act on their targets, the results from these three experimental categories have to be amalgamated according to their effect.

Electrical activity in post-synaptic target neurons Initial investigations on neurons in vitro suggested that neuronal sur- vival is greatly increased by decreasing the membrane potential with high [K'] in the culture medium,3s an effect that is mediated by raising intracellular calcium.36 This was interpreted as indicating that afferent input was important for survival by inducing depolarization in the post-synaptic target neuron. This hypothesis is supported by in vivo experiments. Reduction in neuronal electrical activity is fol- lowed by increased death.37 This occurs whether the decrease in activity is by inhibition of the afferent action potentials using tetrodotoxin13 or blockade of excitatory target cell receptors.38339 However, the relationship between depolarizing activity and cell sur- vival is confused by studies on the inhibitory transmitter, y-amino butyric acid (GABA). Some studies indicate that GABA, or its receptor agonists, increases cell death:" but others show the oppo- site effect.41 While these conflicting results are difficult to recon- cile, it is known that activation of GABA type A receptors can depolarize immature neurons and induce calcium influx:' thus explaining the paradox of an inhibitory afferent assisting neuronal survival.

Although the experiments described above suggest that electrical activity is a mechanism by which afferents maintain their targets, biological activity in vivo is more complex, involving synaptic trans- mitter release and receptor activation. Together, these induce a cas- cade of intracellular events, which may themselves (as in the case of GABAA receptors above) maintain the post-synaptic neuron, rather than the change in membrane potential per se (vide infra).

Oxidative capacity However, afferent input does more than just alter the electrical activity in its targets. There is evidence showing that afferents form part of the mechanisms that maintain their target neurons' normal oxidative capacity. When afferents are removed from the chick cochlear nucleus, there is an initial increase in oxidative enzyme activity in the deafferented neurons.43 This suggests that afferents assist in regulating mitochondrial function in their targets and this may be through regulation of intracellular calcium, a mechanism that has been recognized in peripheral nervous system neuron^.^' Afferent activity raises intracellular calcium and, therefore, also pos- sibly intramitochondrial calcium. Furthermore, mitochondrial en- zymes are adversely affected by raised intramitochondrial calcium.45 Therefore, the increased mitochondrial enzyme activity seen after deafferentation correlates with the decreased electrical activity and calcium influx.

However, deafferentation of CNS nuclei is not followed by the death of all neurons contained therein, even though they all develop an increase in mitochondrial activity. Dying and surviving neurons can be distinguished in two ways: (i) dying neurons have a small increase in cytochrome oxidase activity, whereas those that survive have a large increase in mitochondrial enzyme activity; and (ii) dying neurons develop morphologically abnormal mitochondria, but sur- viving cells maintain normal mitochondrial ~tructure.'~ Because

Role of afferents in neuron survival 49 1

mitochondrial abnormalities are associated with decreased oxidative function4' and prevention of the increased oxidative enzyme activity results in greater cell death after deafferentation,18 it is clear that in the absence of normal input, neurons must up-regulate their oxidative capacity in order to survive. The reason for the differential response in mitochondria1 activity following deafferentation is unknown, but it indicates an important role for afferents in controlling normal mitochondrial enzyme activity in neurons.

Protein metabolism Another aspect of cellular metabolism within target neurons modi- fied by afferent input is protein synthesis. This was first described by Pilar and Landmessar;' who studied neural development and noted degenerating neurons, which showed cytoplasmic changes of ribosomal disaggregation, consistent with cessation of protein synthesis, prior to death. This was attributed to a neuron's overall connectivity, but was later proposed to be the mechanism for deaffer- entation-induced death.48 In the early stages following deafferenta- tion, amino acid uptake falls, as does subsequent protein synthesis in the target p ~ p u l a t i o n ; ~ ~ therefore, the relationship of protein syn- thesis to deafferentation has been demonstrated.

Confirmation of the link between protein synthesis and afferent input comes from the chick cochlear nucleus in vivo. Stimulation of the eighth cranial nerve induces action potentials and increased protein synthesis in the target neurons of the chick cochlear nucle~s.~ ' Moreover, protein synthesis did not increase after non- physiological, anti-dromic stimulation of these neurons?' These two studies suggest a role for afferent input in stimulating protein syn- thesis in their target neurons. Furthermore, they indicate that this response seems to rely on physiological processes, in particular synaptic activation and transmitter release, and not just electrical activity within the post-synaptic neuron. This suggests that synaptic activity, which combines neurotransmitters or what they are colocal- ized with in synaptic vesicles, and/or receptor activation forms the mechanism for the trophic action of afferents (vide supra).

Delivery of trophic agents by afferent axons In addition to delivering trains of action potentials and inducing elec- trical activity in the post-synaptic cell, there is increasing evidence from adult animals that afferents also transport trophic agents antero- gradely down their axons and deliver them to their target neurons to act as survival or growth-promoting agents. There are five families of growth factors associated with the developing nervous system" and representatives of three of these families are known to undergo anterograde axon t r a n s p ~ r t . ~ ~ ~ "

Much of the evidence tying anterograde transport of growth factors to afferent-induced neuronal survival comes from studies of the visual system. Retinal ganglion cells will take up and transport insulin-like growth factor- 1 (IGF- 1),56 fibroblast growth factor-2 (FGF-2)54.s6 and three neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3)," and release them in their target regions of the lateral genicu- late body and optic tectum. Furthermore, neurotrophin receptors are produced in the visual cortex during dendritic development,5' a time that is associated with afferent arrival and innerva t i~n .~~ Also, neuro- trophins increase neurite outgrowth both in vitro6" and in the visual cortex in v ~ v o , ~ ' possibly by their induction of the expression of neuritin, a gene that increases neurite outgrowth.62 Therefore, antero-

grade transport of growth factors is a likely mechanism by which afferents sustain neuronal survival and morphological development, thus explaining the deafferentation-induced transneuronal degener- ation seen within this system.

Evidence of afferent-delivered trophic agents is also present in other systems and may be universal within the CNS. Anterograde effects of growth factors have been demonstrated for FGF-2 in the nigrostriatal system,S3 BDNF in the hippocampal mossy fibres" and spinal cord in te rne~rons~~ and IGF- 1 in cerebellar climbing fibres.63 The potency/specificity of these trophic agents has been demon- strated within the cerebellum following climbing fibre removal during development. Climbing fibre ablation early in development causes significant loss of some target Purkinje cells64 and dendritic atrophy in those surviving Purkinje cells.65 This occurs despite extensive re-innervation of empty denervated synaptic sites by other remaining (parallel fibre) a f f e r e n t ~ . ~ ~ Because even numerous re- innervating afferents cannot maintain the normal number or mor- phology of Purkinje cells, these results suggest that individual neur- ons require specific afferent-delivered growth factors for their development.

This proposal is supported by studies on the effect of colchicine, which inhibits axon transport. Colchicine injection into the neonatal eye induces rapid onset of ceIl death in one of its targets, the developing optic t e c t ~ m . ' ~ However, colchicine injection into the adult inferior olive only affects the dendritic morphology of the target neurons.66 These results may reflect the greater impact during development than in the adult of deafferentation and associated trophic deprivation. Moreover, the discordance between experiments as to whether deafferented neurons have n ~ r m a l ~ ' . ~ ' or at- rophiC26,30-34,65 adult morphology (vide supra), may reflect the availability of growth factors provided by remaining afferents or the sensitivity to specific trophic agents, respectively.

Further evidence of the delivery of trophic agents to target areas by its afferents comes less directly from deafferentation experiments. In the spinal cord, motoneurons receive numerous afferents, many of which descend the cord from supraspinal sources. Following spinal cord transection, motoneurons in segments below the lesion die in greater numbers than n0rmaI.6~ However, this deafferentation- induced cell death can be prevented by application of brain tissue extracts and, therefore, presumably factors from afferent areas." Indeed, the exogenous application of purified growth factors present in the brain, including IGF- BDNF and NGF,I2 NT3, FGF-2, cili- ary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), platelet-derived growth factor (PDGF) and the glial protein S IOO,I4 can maintain motoneurons after spinal cord transection. Similar results have been found in the zebra-finch song motor nucleus, where injection of the neurotrophins BDNF, NT3 and neurotrophin-4/5 (NT4/5), which are normally produced in its innervating population, can prevent deafferentation-induced cell death." Thus, these deaf- ferentation experiments strengthen the view that one mechanism whereby afferents maintain their target is by the delivery of growth factors. However, it must be remembered that many growth factors are widely produced within both the CNS and the periphery. Therefore, the assumption that the neuronal rescue by growth factors is indicative of afferent support may be over-optimistic.

It is always difficult to know how much the results following extensive intervention really reflect physiological parameters. However, the effect of exogenous growth factors is supported by studies more physiological in design. Electrical activity in neurons

492 RM Sherrard and AJ Bower

increases the production of neurotrophins within a matter of hours, a phenomenon seen after both normal physiological activity induced by eye opening7' and pathological seizure a~tivity.~' This up-regu- lation is retained for at least 24 h. It may well be that the afferent- induced increase in protein synthesis5" identified above reflects the production of trophic agents. Furthermore, catecholamine trans- mitters act indirectly to increase neuronal survival by stimulating astrocytes to produce trophic agents; 5-hydroxytryptamine causes hippocampal astrocytes to produce S100, which, in turn, is atrophic factor for serotoninergic neurons." Second, noradrenaline stimulates the astrocyte production of NGF in the striatum, cerebellum and cerebral cortex.73 Therefore, these studies indicate that growth factor up-regulation is related to afferent synaptic activity and is specifically

an effect of neurotransmitters, albeit, in part, as an indirect action via glial cells.

Furthermore, the correlation of afferent activity with growth factor production is congruent with the known efficiency of biological systems in using similar mechanisms in different combinations to have multiple effects. It is reasonable to suspect that the mechanisms by which afferents maintain neuronal survival are similar to those by which targets support their afferent populations. After all, it is unlikely that any given neuron will efficiently produce one set of trophic agents for its own afferent population and a different set for the target that it innervates. Target-derived trophic support is known to involve numerous growth-promoting agents and these agents now appear to be important for afferent-induced survival and growth.

Fig. 1 A summary diagram of the currently known mechanisms for afferent regulation of neuronal survival. All these afferent actions combine with the known actions of growth factors produced by the neuron's target that bind to surface receptors and are transported back along the axon to have their intracellular effects. 0 Activity in afferents releases neurotransmitters (NT) at the synapse. These interact with receptors (AA) depolarizing the membrane and opening calcium channels (CaC) to allow calcium influx. Calcium modifies mitochondrial function and gene expression involving protein synthesis and the process of apoptosis. Neurotransmitters also induce adjacent astroglia to secrete peptide growth factors (GF), which, in turn, act on the target neuron. 0 Afferent neurons also deliver GF to act via specific receptors (u ) on the target neuron to induce survival. Growth factors are known to act via tyrosine kinases to alter intracellular calcium concentration ([Ca"]) and gene expression. 0 Electrical activity in afferent axons raises the [Ca"] and modifies gene expression to increase growth factor production. This, in turn, is secreted from the neuron and can act via receptors in an autocrine manner to induce s ~ r v i v a l . ' ~ ~ ' ~ * ~ ~ ~ ~

Role of afferents in neuron survival 493

Suppression of apoptosis A final common pathway by which afferents may maintain neuronal survival is that, whatever the intracellular paths involved, afferents may suppress the process of apoptosis. The overall evidence seems to indicate that the process by which deafferented neurons die is an active process rather than a passive wind-down phenomenon. The major evidence supporting this is that deafferentation induces the expression of immediate early genes c-fos and c-jun in a matter of hours in guinea-pig74 and rat7' vestibular nuclei and in mouse spinal cord dorsal horn.76 This up-regulation of immediate early gene expression takes place both in immature neurons and in adult neurons. Whether the expression of these genes is the start of the 'death process' or whether their expression simply indicates altered neuronal is unclear. However, studies from Weaver mutant mice, in which cerebellar granule cells die by apoptosis early in devel~pment?~ indicate that prolonged expression of c-jos at least appears to precede the onset of a p o p t ~ s i s . ~ ~ This is also confirmed by studies on the rodent optic tectum, in which deafferentation- induced cell death is known to be by apopt~sis . '~ Furthermore, protein synthesis inhibitorsI3 block deafferentation-induced death and protein synthesis is a mechanism required for induction of apop- tosis." However, afferent suppression of apoptosis is unlikely to be the sole mechanism of action for two reasons. First, in some neurons deafferentation decreases protein synthesis49 and blockade of pro- tein synthesis does not always prevent deafferentation-induced cell death," suggesting that some neurons die by other mechanisms. Second, deafferentation-induced immediate early gene expression in adult neurons is not followed by cell death.' Because these opposing results were obtained in different systems, apoptosis being in the rodent visual system13 and necrosis in the chick cochlear n u c l e ~ s , ' ~ afferents may have different effects in different regions of the CNS. This is especially true as there is no direct evidence that afferent input suppresses apoptosis, just a series of steps that can potentially be put together when all the literature is reviewed.

CONCLUSIONS

The survival of any given neuron is likely to depend on a combin- ation of factors derived both from its afferent input as well as from its target (Fig. 1) . Any alteration to either of these will compromise the neuron's survival during the vulnerable ages of development. When the literature is reviewed, the following conclusions may be drawn about the role of afferents in neuronal survival. 1. Afferents are more important for neuronal survival during devel- opment than they are in the adult system. 2. Loss of afferents during development does not alter the timing of neuronal death, only the number of neurons that die. 3. While the contribution of afferents to neuronal development be- gins very early in maturation, afferents are only essential for neur- onal survival for a short period while connectivity is established. 4. For correct morphology and function, a neuron requires its specific input, while for survival any non-specific input appears sufficient. 5. Afferent maintenance of neurons is through a variety of mech- anisms, including the actions of neurotransmitters and the delivery (direct or indirect) of growth factors. While the intracellular mech- anisms of these actions are ill-defined, regulation of calcium, pro- tein synthesis and mitochondrial function may contribute to the

suppression of cell death. Thus, it is clear that, in addition to the well-established actions of target-derived factors, the survival of developing neurons is critically dependent on their afferents.

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