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The objective of this article is to review the clinical presentation and neurobiology of degeneration of the enteric nervous system with emphasis on human data where available. Constipation, incontinence and evacuation disorders are frequently encountered in the ageing population. Healthy lower gastrointestinal function is essential for successful ageing as it is critical to maintaining independence and autonomy to pursue further activity. One clinical expression of enteric neurodegeneration is constipation. However, the aetiology may be multifactorial as disturbances of epithelial, muscle or neural function may all result from neurodegeneration. There is evidence of loss of excitatory (e.g. cholinergic) enteric neurons and interstitial cells of Cajal, whereas inhibitory (including nitrergic) neurons appear unaffected. Understanding neurodegeneration in the enteric nervous system is key to developing treatments to reverse it. Neurotrophins have been shown to accelerate colonic transit and relieve constipation in the medium term; they are also implicated in maintenance programmes in adult enteric neurons through a role in antioxidant defence. However, their effects in ageing colon require further study. There is evidence that 5-HT(2) and 5-HT(4) mechanisms are involved in development, maintenance and survival of enteric neurons. Further research is needed to understand and potentially reverse enteric neurodegeneration.

The precursor form of the nerve growth factor (proNGF), forms a heterotrimeric complex with the receptors p75 and sortilin; this complex has been implicated in neuron cell death. However, it is not known whether proNGF and the receptors p75 and sortilin contribute to age- and disease-related neurodegeneration. Here we show that proNGF induces cell death in subpopulations of basal forebrain and peripheral sympathetic neurons of old, but not of young, adult rodents. In contrast, proNGF appears to induce neurite outgrowth rather than cell death of young adult sympathetic neurons. We have examined the neurotoxic role of proNGF in old age, and find that proNGF protein is elevated during ageing in the projection areas of some populations of vulnerable central and peripheral neurons; caloric restriction, which has known neuroprotective effects, partially prevents these increases. Sortilin was found to play a significant part in the observed patterns of age-related proNGF-mediated neurotoxicity. In particular, survival of aged neurons was rescued by neurotensin, an alternative sortilin ligand that blocks the sortilin-mediated effects of proNGF. Furthermore, sortilin immunoreactivity increases markedly in ageing rodent basal forebrain and sympathetic neurons; in contrast, p75 levels are either unchanged or reduced. From these data we propose that selective age-related neuronal atrophy and neurodegeneration may be mediated by increased sortilin expression in neurons, together with elevated levels of proNGF expression in some targets.

Several studies have sought to demonstrate that neurodegeneration during disease and in old age is associated with reduced neurotrophic support. Little positive evidence has been forthcoming, either in relation to the availability of neurotrophins or to expression and function of the relevant receptors. Recently, a novel way in which neurotrophins could contribute to neurodegeneration has been suggested. In contrast to the well-known neurotrophic functions of the mature beta-form of nerve growth factor (mNGF), its precursor proNGF has recently been shown to be abundant in the adult brain and in the brains of patients with Alzheimer's disease. proNGF is synthesized as 25 and 32 kDa isoforms, which are glycosylated to form a principal 40 kDa species. Studies of the cortical targets of NGF-responsive basal forebrain neurons show that the 40 kDa form of proNGF is secreted in response to nerve stimulation, along with the proteases needed to generate the 13 kDa mNGF, or to degrade it. We have recently found that levels of 40 kDa proNGF are elevated in the aging brain and also in targets of peripheral NGF-responsive neurons. proNGF has been shown to be neurotoxic when bound in a heterotrimer with the p75 receptor and the receptor sortilin (identical to the neurotensin receptor NTS3). Interestingly, we find that sortilin levels increase in aged central and peripheral neurons, perhaps making these neurons more vulnerable to age-related increases in proNGF. Whether elevated levels of proNGF in targets or of sortilin in neurons contribute to known patterns of age- and disease-related neurodegeneration has not been previously investigated. Using in vitro models, our preliminary data now indicate that proNGF is indeed neurotoxic for aged, but not young, NGF-responsive basal forebrain and sympathetic neurons and that blockade of sortilin rescues proNGF-induced cell death. We therefore propose that increased proNGF in targets combined with increased sortilin expression in projecting neurons contributes to age-related neuronal atrophy and degeneration.

We have studied the mechanisms underlying nonpathological age-related neuronal cell death. Fifty per cent of neurons in the rat enteric nervous system are lost between 12 and 18 months of age in ad libitum (AL) fed rats. Caloric restriction (CR) protects almost entirely against this neuron loss. Using the ROS-sensitive dyes, dihydrorhodamine (DHR) and 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) in vitro, we show that the onset of cell death is linked with elevated intraneuronal levels of reactive oxygen species (ROS). Treatment with the neurotrophic factors NT3 and GDNF enhances neuronal antioxidant defence in CR rats at 12-15 months and 24 months but not in adult or aged AL-fed animals. To examine the link between elevated ROS and neuronal cell death, we assessed apoptotic cell death following in vitro treatment with the redox-cycling drug, menadione. Menadione fails to increase apoptosis in 6-month neurons. However, in 12-15mAL fed rats, when age-related cell death begins, menadione induces a 7- to 15-fold increase in the proportion of apoptotic neurons. CR protects age-matched neurons against ROS-induced apoptosis. Treatment with neurotrophic factors, in particular GDNF, rescues neurons from menadione-induced cell death, but only in 12-15mCR animals. We hypothesize that CR enhances antioxidant defence through neurotrophic factor signalling, thereby reducing age-related increases in neuronal ROS levels and in ROS-induced cell death.

This study aimed to examine the expression and function of P2 receptors of the rat tail and mesenteric arteries during maturation and ageing (4, 6 and 12 weeks, 8 and 24 months). Functional studies and receptor expression by immunohistochemistry revealed a heterogeneous phenotype of P2 receptor subtypes depending on artery age. The purinergic component of nerve-mediated responses in the tail artery was greater in younger animals; similarly responses to ATP and alpha,beta-meATP and the expression of P2X1 receptors decreased with age. Contractile responses to 2-MeSADP decreased with age, and were absent at 8 and 24 months; P2Y1 receptor expression followed this pattern. UTP-induced contractions and P2Y2 receptor expression also decreased with age. The mesenteric artery contracted to UTP, responses at 4 and 6 weeks were larger than at other ages although P2Y2 receptor expression did not significantly differ with age. 2-MeSADP induced relaxation of the mesenteric artery, responses being greatest at 6 weeks and decreased thereafter, which was mimicked by the P2Y1 receptor immunostaining. We speculate that the dramatic changes in expression of P2 receptors in the rat tail artery, compared to the mesenteric artery, during development and ageing are related to the role of the tail artery in temperature regulation.

We have examined the hypothesis that differences in nerve growth factor (NGF) uptake and transport determine vulnerability to age-related neurodegeneration. Neurons projecting to cerebral blood vessels (CV) in aged rats are more vulnerable to age-related degeneration than those projecting to the iris. Uptake of NGF was therefore examined in sympathetic neurons projecting from the superior cervical ganglion (SCG) to CV and iris in young and old rats by treating the peripheral processes of these neurons with different doses of I125-NGF. Total uptake of I125-NGF was reduced in old CV-projecting, but not iris-projecting, neurons. Numbers of radiolabelled neurons projecting to each target were counted in sectioned ganglia. The data showed age-related reductions in numbers of labelled neurons projecting to CV, but no change in numbers of neurons projecting to the iris. Calculation of uptake of I125-NGF per neuron unexpectedly showed no major age-related differences in either of the two neuron populations. However, uptake per neuron was considerably lower for young and old CV-projecting, compared to iris-projecting, SCG neurons. We hypothesized that variations in NGF uptake might affect neuronal survival in old age. Counts of SCG neurons using a physical disector following retrograde tracing with Fluorogold confirmed the selective vulnerability of CV-projecting neurons by showing a significant 37% loss of these neurons in the period between 15 and 24 months. In contrast, there was no significant loss of iris-projecting neurons. We conclude that vulnerability to, or protection from, age-related neurodegeneration and neuronal cell death are associated with life-long low, or high, levels of NGF uptake, respectively.

Many individuals experience gastrointestinal (GI) dysfunction more frequently as they age, and the segment of the human population that is growing the most rapidly is the 'oldest old', who are >/= 80 years old. There has recently been renewed interest in the age-related changes intrinsic to the gut, and these investigations may help physicians understand the 'normal' aged GI tract, as distinct from disordered bowel function that is the result of comorbid conditions and/or GI side effects of medications used to treat those conditions. In this concise review we summarize recent data that suggest age-related neurodegenerative changes in the enteric nervous system (ENS) are key to functional changes observed with advanced age. Morphological studies are reviewed that demonstrate clearly the loss of enteric neurones in both submucosal and myenteric plexuses in humans and in rodents. Recent studies that indicate selective preservation of nitrergic, but not cholinergic, neurones are reviewed, as are preliminary findings that intrinsic sensory neurones may be among the most 'age-labile' subpopulations of the ENS. Caloric restriction remains the only intervention known that prevents neurodegeneration of ageing in the ENS, and mechanisms involved in this phenomenon are discussed. The field of ageing research in enteric neurobiology is ripe for rapid progression from phenomenology of age-related losses of neurones and associated functional changes to discovery of therapeutic approaches that may help ameliorate deterioration of bowel function and thereby contribute significantly to improved quality of life in advanced age.

Selective vulnerability of particular groups of neurons is a characteristic of the aging nervous system. We have studied the role of neurotrophin (NT) signalling in this phenomenon using rat sympathetic (SCG) neurons projecting to cerebral blood vessels (CV) and iris which are, respectively, vulnerable to and protected from atrophic changes during old age. RT-PCR was used to examine NT expression in iris and CV in 3- and 24-month-old rats. NGF and NT3 expression in iris was substantially higher compared to CV; neither target showed any alterations with age. RT-PCR for the principal NT receptors, trkA and p75, in SCG showed increased message during early postnatal life. However, during mature adulthood and old age, trkA expression remained stable while p75 declined significantly over the same period. In situ hybridization was used to examine receptor expression in subpopulations of SCG neurons identified using retrograde tracing. Eighteen to 20 h following local treatment of iris and CV with NGF, NT3 or vehicle, expression of NT receptor protein and mRNA was higher in iris- compared with CV-projecting neurons from both young and old rats. NGF and NT3 treatment had no effect on NT receptor expression in CV-projecting neurons at either age. However, similar treatment up-regulated p75 and trkA expression in iris-projecting neurons from 3-month-old, but not 24-month-old, rats. We conclude that lifelong exposure to low levels of NTs combined with impaired plasticity of NT receptor expression are predictors of neuronal vulnerability to age-related atrophy.

Data are presented in support of the idea of antagonistic pleiotropy that features which are adaptive during early life may become maladaptive during the ageing process, when selective pressure is reduced. A model of selective vulnerability to age-related neurodegeneration involving neighbouring subpopulations of vulnerable and protected sympathetic neurons is presented. The two groups of neurons are morphologically and physiologically distinct, indicating advantageous adaptation to particular functions. Neurotrophin signalling is investigated in these different groups of neurons, revealing significant differences between them: neurotrophic factor expression in their target tissues is markedly different, same as their neurotrophin uptake characteristics. Preliminary evidence is presented that the mechanism linking neurotrophin signalling and age-related neurodegeneration may involve the capacity of neurons to buffer free radical generation, hence reducing the effects of attrition by free radical damage.

The light microscopical and ultrastructural morphology of the innervation of the major cerebral arteries and pial vessels is described, including the origins of the different groups of nerve fibres and their characteristic neurotransmitter phenotype. Species and region specific variations are described and novel data regarding the parasympathetic innervation of cerebral vessels are presented. The dynamic nature, or plasticity, of cerebrovascular innervation is emphasized in describing changes affecting particular subpopulations of neurons during normal ageing and in Alzheimer's disease. The molecular controls on plasticity are discussed with particular reference to target-associated factors such as the neurotrophins and their neuronal receptors, as well as extracellular matrix related factors such as laminin. Hypotheses are presented regarding the principal extrinsic and intrinsic influences on plasticity of the cerebrovascular innervation., (Copyright 2001 Wiley-Liss, Inc.)

Altered neuronal responses to trophic factors may play a role in neuronal maintenance in adulthood and may also be involved in neuronal atrophy in old age. We have investigated this issue in the rat iris, studying responsiveness of sympathetic and sensory iridial nerves to a range of NGF concentrations in mature and aged rats. We show here that growth responses of sensory nerves to NGF, as measured by quantitative immunohistochemistry and image analysis, were unchanged in old rats. In contrast, there was a small but significant reduction in responsiveness of aged sympathetic neurons. The shapes of the dose-response curves for sensory and sympathetic neurons were different, with a larger response over a narrower range of concentrations in sensory neurons. Lower levels of p75 immunoreactivity were observed in iridial nerves from old compared to young rats. NGF treatment had no effect on receptor staining in young rats but restored 'young' levels of p75 staining in old rats. Our results do not support the hypothesis of a primary role for NGF in maintenance or atrophy of nerves in ageing.

Background: Alone among autonomic neurones, enteric neurones are known to be vulnerable to age related cell death; over 50% may be lost in aging rodents. A previous study demonstrated unexpectedly that neurones of the myenteric plexus from rats fed a restricted diet appeared not to suffer from extensive cell death in contrast with previous studies of ad libitum fed animals., Aims: To compare myenteric neurone numbers in the ileum of young and aging male Sprague-Dawley rats fed either ad libitum or a restricted diet., Methods: Neurones were counted in whole mount preparations of rat ileum stained immunohistochemically for the pan-neuronal marker PGP9.5, for choline acetyltransferase, or for nitric oxide synthase, or with NADH or NADPH histochemistry., Results: Neurone numbers in the rat myenteric plexus were substantially affected by the dietary regimen: ad libitum feeding (50-60 g per day of standard rat chow) resulted in the death of about 50% of myenteric neurones in 24 month Sprague-Dawley rats, while numbers were unchanged when the daily dietary intake was halved between the ages of six and 24 months. Animals fed a double restricted diet (15 g per day) showed no cell loss at 30 months, as well as the predicted increase in longevity. Neurone loss was largely complete by 16 months in ad libitum fed animals. Numbers of cholinergic (possibly motor) neurones, as demonstrated by choline acetyltransferase immunohistochemistry, were substantially reduced in ad libitum fed aging rats but not in animals fed a restricted diet. Loss of cholinergic neurones after ad libitum feeding was confirmed by reduced numbers of neurones of a size range matching that of cholinergic neurones., Conclusions: Ad libitum feeding of adult rats has adverse effects on the survival of myenteric neurones, neurone loss commencing before 16 months of age. Cholinergic neurones appear to be particularly vulnerable to the effects of diet. Restricting dietary intake from six months of age prevents neurone loss almost entirely up to 30 months of age in these rats.

The factors that determine the ability of some, but not all neurons, to sustain their axonal projections during aging remain largely unknown. Because sympathetic neurons remain responsive to nerve growth factor (NGF) in old age, it has been proposed that the selective decrease observed in the sympathetic innervation to some targets in aged rats may be the result of a deficit in target-derived NGF. In this study we utilized two different techniques to demonstrate decreased target innervation by sympathetic fibers in the aged rat pineal gland, which is an appropriate and relevant model for examining mechanisms of neuron-target interactions in aging. Tyrosine hydroxylase immunoreactive profiles were quantified in pineal glands of young and aged male Sprague-Dawley rats. The density of tyrosine hydroxylase-immunoreactive fibers was 30% lower in aged pineals, although the remaining fibers contained 20% more tyrosine hydroxylase-immunoreactivity. Othograde tracing of the pineal sympathetic innervation using biotinylated dextran revealed that average axon length, varicosity numbers, branch point numbers, and numbers of terminations were all decreased by approximately 50% in aged tissues, indicating possible functional deficits. These findings suggest that whole branches, along with their associated varicosities were lost in old age. A sensitive quantitative ribonuclease protection assay and a two-site ELISA assay were used to examine whether reduced NGF availability might correlate with sympathetic nerve atrophy. No significant differences were detected in either NGF mRNA or NGF protein levels when comparing young and aged pineal glands, suggesting that atrophy in aged sympathetic neurons is not causally related to reduced availability of NGF at the target. Our results indicate that mechanisms other than NGF expression need to be explored in order to explain the age-related axonal regression observed in this target.

Previous studies on ageing animal and human subjects have demonstrated a significant overall decline in neuronal numbers in the myenteric plexus of the enteric nervous system (ENS). Our study aimed to confirm this observation by counting myenteric neurons stained with the panneuronal markers PGP 9.5 and NADH-diaphorase. We also wished to examine the possibility that particular subpopulations of neurons are vulnerable. Therefore, we have immunostained and counted a number of nerve cell groups within the myenteric plexus of old and young Sprague Dawley rats using markers which reflect some of the neuronal phenotypes present, including ChAT and VIP. The number of neurons demonstrating NADH-diaphorase activity was significantly reduced (P < 0.05) by approximately 15 % in old rats. However, the number of neurons stained for PGP 9.5 immunohistochemistry was not reduced and demonstrated larger numbers of neurons than the NADH-diaphorase method. None of the other neuronal markers studied showed any significant reductions with age. In contrast to previous work, this study has gathered little evidence for extensive cell loss in the myenteric plexus of the aged rat, either in overall populations, or in any of the principal functional groups of neurons.

The nature of neural plasticity and the factors that influence it vary throughout life. Adult neurons undergo extensive and continual adaptation in response to demands that are quite different from those of early development. We review the main influences on the survival, growth and neurotransmitter expression in adult and ageing sympathetic neurons, comparing these influences to those at work in early development. This "developmental" approach is proposed because, despite the contrasting needs of different phases of development, each phase has a profound influence on the mechanisms of plasticity available to its successors. Interactions between neurons and their targets, whether effector cells or other neurons, are vital to all of these aspects of neural plasticity. Sympathetic neurons require access to target-derived diffusible neurotrophic factors such as NGF, NT3 and GDNF, as well as to bound elements of the extracellular matrix such as laminin. These factors probably influence plasticity throughout life. In adult life, and even in old age, sympathetic neurons are relatively resistant to cell death. However, they continue to require target-derived diffusible and bound factors for their maintenance, growth and neurotransmitter expression. Failure to maintain appropriate neuronal function in old age, for example in the breakdown of homeostasis, may result partly from a disturbance of the dynamic, trophic relationship between neurons and their targets. However, there is no clear evidence that this is due to a failure of targets to synthesize neurotrophic factors. On the neural side of the equation, altered responsiveness of sympathetic neurons to neurotrophic factors suggests that expression of the trk and p75 neurotrophin receptors contributes to neuronal survival, maintenance and growth in adulthood and old age. Altered receptor expression may therefore underlie the selective vulnerability of some sympathetic neurons in old age. The role of neural connectivity and activity in the regulation of synthesis of target-derived factors, as well as in neurotransmitter dynamics, is reviewed.

Whilst the potent effects of NGF and laminin on developing neurons are well documented, relatively little is known about the effects of, or altered availability of or altered responsiveness to, these substances on the growth of adult neurons. We have therefore examined this question using explant cultures of sympathetic neurons from the superior cervical ganglion (SCG) of mature and aged rats. Explants were grown on substrata containing different doses of laminin, either with or without added NGF in culture medium containing FCS. Individually, laminin and NGF had relatively small effects on neurite outgrowth and length, which tended to be reduced in old neurons. In contrast, laminin in the presence of exogenous NGF exerted a powerful effect on nerve growth which was substantially greater than the sum of the effects of the individual factors. This synergy was evident in all experimental groups and was greatest in old explants at high doses of laminin, where growth was comparable to that of mature neurons. The dose-response curve of old neurons to laminin in the presence of added NGF indicated reduced responsiveness. These results suggest that variations in the availability of laminin and/or exogenous NGF, together with altered patterns of neuronal responsiveness, may contribute to impaired neuronal plasticity in old age.

Axonal atrophy may reflect earlier and more reversible events in neurodegeneration and ageing than somatic atrophy. Innervation density by sympathetic fibres from the rat superior cervical ganglion (SCG) to the middle cerebral artery (MCA) decreases dramatically in old age, while that to the iris is largely unchanged. In situ hybridization was used in conjunction with retrograde tracers to examine the role of the neuronal cytoskeleton in this selective axonal vulnerability. Using a riboprobe complementary to neurofilament light chain (NF-L) mRNA, there was a 22-25% decrease in the mean grain density in aged neurones when all neurones were examined. A small subset of these neurones was shown to project to the MCA and another to the iris. In young SCG, both subpopulations expressed intermediate grain densities for NF-L mRNA. In MCA-projecting neurones, there was a 40% decline in grain density with ageing (p < 0.05), with no change in iris-projecting neurones. Our results demonstrate that major decreases in NF-L expression may represent cellular markers of selective axonal hypotrophy by aged neurones.

Contact with sweat gland acini causes sympathetic neurons to switch from a catecholaminergic to a cholinergic phenotype during development and following experimental manipulations. Substantial reductions of cholinergic innervation have been shown in the sweat glands of ageing rats and humans. Using in oculo transplantation, we have now studied whether sweat gland target tissues retain the capacity to regulate changes in the phenotype of sympathetic neurons observed in maturity and old age, including a switch from catecholaminergic to cholinergic characters. Markers have been used which indicate changes in nerve fibre morphology (the pan-neuronal marker, PGP9.5) as well as neurotransmitter expression (acetylcholinesterase (AChE), vasocative intestinal polypeptide (VIP) and tyrosine hydroxylase (TH). Sweat glands from young and old donor rats became reinnervated by an organotypic pattern of cholinergic host nerves. Surgical sympathectomy demonstrated that these cholinergic nerve fibres originate from sympathetic neurons of the host superior cervical ganglion (SCG). Retrograde tracing combined with staining for VIP (a marker associated with cholinergic phenotype in neurons supplying sweat glands) showed that SCG neurons projecting to irises with sweat gland implants may be induced to express VIP. We hypothesise that these neurons have been switched from their normal catecholaminergic phenotype to a cholinergic one by contact with the sweat gland implants. Transplants from old donors attracted a density of reinnervation by young host nerves which was appropriate to the age of the donor, thus old sweat glands received a significantly reduced density of innervation compared to young glands. Despite the reduced density of innervation, there was no obvious difference in the ability of young and old implants to induce the switch to a cholinergic phenotype, suggesting that different mechanisms regulate nerve growth and neurotransmitter phenotype.

We have examined the hypothesis that alterations in target levels of NGF protein lead to selective changes in peripheral nerve fibres in maturity and old age. NGF levels were assayed using 2-site ELISA in the iris of young and old rats. NGF levels in the iris showed no significant change in old age and did not correlate with age-related changes in the density of NGF-sensitive irideal sympathetic and sensory nerve fibres. Following sympathectomy, NGF levels increased substantially in old but not in young irises, possibly because the lower density of aged sensory nerves resulted in reduced capacity for NGF uptake. Content and uptake of NGF in sympathetic (SCG) neurones was shown to be reduced in old age, while NGF content of the sensory (trigeminal) ganglion increased in old age. Thus NGF levels in neurones, as well as targets, fail to correlate with peripheral nerve fibre growth or atrophy.

In the present study the intrinsic nerve plexuses of the basal cerebral arteries, derived from aged non-Alzheimer's and aged Alzheimer's disease patients were quantified and compared. A previous study described and quantified nerve density on similar arteries from healthy middle-aged patients. Whole-mount preparations of various segments of the basal cerebral arteries were stained for protein gene product 9.5. The deep nerve plexuses, located at the adventitial-medial border, were quantified by image analysis. Transverse cryostat sections were stained for various markers and quantified. Measurements on whole mounts demonstrated that nerve densities were highest in the posterior communicating artery and in the postcommunicating part of the posterior cerebral artery (PCA) for both aged and Alzheimer's groups. Statistical comparison showed a tendency toward decreased nerve density with aging, which was significant for the internal carotid artery, precommunicating part of the PCA, and the anterior choroidal artery in both non-Alzheimer's and Alzheimer's aged groups. In addition, in Alzheimer's patients nerve density was significantly lower in the precommunicating part of the anterior cerebral artery compared with the healthy aged group. Measurements on sections confirmed the tendency to decreased innervation with aging. It is concluded that densities of deep perivascular nerves of human basal cerebral arteries are subject to localized changes caused by aging and Alzheimer's disease.

We have investigated alterations in the nerve supply to the iris of aged rats and the role of endogenous nerve growth factor in these changes. The overall density of nerve fibres, and the density of calcitonin gene-related peptide containing sensory nerves, were decreased by over 20% on the aged iris, as measured by computerized image analysis on immunostained preparations, while the density of sympathetic innervation was maintained. Whilst the majority of nerves supplying the iris (sympathetic, sensory and parasympathetic) are known to respond to exogenous nerve growth factor during development and in adulthood, the role of endogenous, target-derived nerve growth factor in nerve maintenance in maturity and old age awaits confirmation. Our results showed that localized treatment with anti-nerve growth factor of iridial nerve terminals did not affect sympathetic or sensory neurons in young rats, but caused a dramatic reduction of sympathetic nerve density on irides of old rats. The effect of anti-nerve growth factor treatment on the sensory innervation of old irides was less obvious. We conclude that aged sympathetic nerves are more susceptible to nerve growth factor deprivation than young ones, or than young or aged sensory neurons, perhaps as a result of reduced responsiveness to nerve growth factor with age. Since sympathetic innervation is maintained, whilst sensory innervation is decreased in the aged iris, age-related changes in innervation are unlikely to be due to altered availability of endogenous nerve growth factor.

Axonal atrophy may reflect earlier and more reversible events in neurodegeneration and ageing than somatic atrophy. Innervation density by sympathetic fibres from the rat superior cervical ganglion (SCG) to the middle cerebral artery (MCA) decreases dramatically in old age, while that to the iris is largely unchanged. In situ hybridization was used in conjunction with retrograde tracers to examine the role of the neuronal cytoskeleton in this selective axonal vulnerability. Using a riboprobe complementary to neurofilament light chain (NF-L) mRNA, there was a 22-25% decrease in the mean grain density in aged neurones when all neurones were examined. A small subset of these neurones was shown to project to the MCA and another to the iris. In young SCG, both subpopulations expressed intermediate grain densities for NF-L mRNA. In MCA-projecting neurones, there was a 40% decline in grain density with ageing (p < 0.05), with no change in iris-projecting neurones. Our results demonstrate that major decreases in NF-L expression may represent cellular markers of selective axonal hypotrophy by aged neurones.

Our purpose in this work was to investigate the role of target tissues in the regulation of dendritic morphology from sympathetic neurons during development and aging. Neurons were retrogradely labeled from three targets, the iris, the submandibular gland (SMG), and the middle cerebral artery (MCA). They were then fixed and intracellularly injected to demonstrate their dendritic arborizations. Dendritic geometry varied quantitatively in sympathetic neurons innervating different target tissues at all stages of development. Neurons innervating the iris had the largest cell bodies and most extensive dendritic arborizations, whereas the vasomotor neurons were the smallest. The number of primary dendrites, however, did not vary significantly between the different neuronal populations. The growth of dendritic arborizations during postnatal development and their atrophy in old age were not concordant in the different neuron populations we studied. Neurons innervating the MCA and the iris ceased dendritic growth early in postnatal development, whereas the dendritic complexity of neurons supplying the SMG increased well into adulthood. By contrast, dendritic atrophy was seen in aged MCA- and SMG-projecting neurons but not in those innervating the iris, suggesting, with other evidence, correlated and distinct patterns of growth and atrophy in axons and dendrites of mature sympathetic neurons projecting to different targets. Swollen dendrites and protuberances on cell soma were a prominent feature of aged neurons. In addition to the target-specific variation in neuronal morphology, we observed diversity in neurotransmitter phenotype. For example, neuropeptide Y was expressed in iridial but not SMG-projecting neurons. These results show a range of age- and target-specific differences in the dendritic morphology and neuropeptide content of sympathetic neurons that may be a result of differing trophic interactions with their target tissues.

The causes of age-related degeneration in the peripheral nervous system remain unclear. The search for clues has focused on developmental mechanisms and particularly on the neurotrophic hypothesis and its principal player, nerve growth factor, reduced levels of which are thought to cause degeneration of some autonomic and central neurons in old age. Nerve growth factor may well be important in the mature and ageing nervous system, but recent experiments on sympathetic nerves in ageing rats suggest that lack of NGF is not the only limiting factor in neuronal growth and survival. Other candidates include laminin, which is bound in the extracellular matrix and may act in synergy with NGF to regulate neuronal maintenance and growth in maturity. Reduced, region-specific patterns of availability of one or both of these substances may underlie age-related degeneration in autonomic nerves. Different combinations of these factors may influence particular aspects of neuronal plasticity, such as collateral sprouting and regeneration. In addition to extrinsic factors, it appears increasingly likely that altered neuronal responsiveness to neurotrophic factors in old age contributes to structural and functional deficits in autonomic nerves.

Previous studies in our laboratory using a transplantation model have shown that target tissues of some autonomic neurons, including cerebral blood vessels, exert a controlling influence on nerve fibre loss in old age. The present study was undertaken in order to discover whether the influence of targets extends to controlling age changes in specific populations of nerves. In old rats, we have demonstrated a significant decrease of approximately 50% in the sympathetic innervation of middle cerebral arteries, using tyrosine hydroxylase-like immunoreactivity. Following transplantation, tyrosine hydroxylase-like immunoreactive nerve density on both young and old implanted middle cerebral arteries mirrored the nerve densities seen in normal, non-transplanted vessels. Furthermore, implanted tissue from old donors became reinnervated with a nerve density approximately 50% less than that of young implanted vessels. Treatment of transplants with nerve growth factor, however, was able to reverse these age changes and restore the sympathetic innervation of aged middle cerebral arteries to levels above those seen in young middle cerebral arteries. These results suggest that the pattern and density of sympathetic innervation that the middle cerebral artery receives is determined by the target rather than by the neurons supplying the tissue. The ability of nerve growth factor to induce regrowth in sympathetic neurons innervating ageing target tissues implies that age-related neuronal atrophy may be due to reduced synthesis or availability of target-derived neurotrophic factors.

The aim of this study was to investigate whether NGF could reverse the nerve fiber atrophy exhibited by some neurons in old age. We used quantitative immunohistochemical techniques to investigate how the autonomic nerves that supply different blood vessels are affected by aging. Age changes in the nerve plexus were not widespread but were specific to particular vascular targets. Furthermore, where nerve fiber atrophy did occur, it affected different populations of nerves; specifically, sympathetic nerve fibers were lost from the middle cerebral artery while nonsympathetic nerves were lost from the tail vein. Peripheral target tissues have been shown to have a trophic influence on the pattern and density of their innervation, which declines in old age (Gavazzi et al., 1992), possibly as a result of decreased availability of neurotrophic factors such as NGF. Consequently, in an attempt to reverse nerve fiber atrophy, we used miniosmotic pumps to infuse NGF for 2 weeks over involuting nerve fibers in the middle cerebral artery of freely moving aged rats. Our results show that exogenous NGF can induce organotypic neurite outgrowth from aged neurons undergoing nerve fiber atrophy. Moreover, NGF can induce a change in the pattern of the old nerve plexus to one similar to that seen in young animals. NGF had different effects on the extent and manner of neurite outgrowth in young and aged nerves. NGF induced greater nerve growth in old compared to young nerves, causing old nerves to grow within existing nerve bundles and to sprout new nerve fibers, in contrast to young nerves, which tended to grow only within existing nerve bundles. In conclusion, this study shows the potential of exogenous NGF to reverse age-related changes in neuronal morphology.

We have investigated whether dendritic growth can be induced from sympathetic neurons of aged rats by the application of exogenous nerve growth factor to their target tissues. A previous study showed that significant dendritic atrophy (19%) occurs during aging in the sympathetic neurons innervating the middle cerebral artery and that dendritic atrophy correlated with loss of axon collaterals in the same population of neurons. Using retrograde tracing in conjunction with intracellular injection of fixed tissue and confocal microscopy, we now demonstrate that infusion of nerve growth factor over the peripheral processes of the same neurons from aged rats induces significant dendritic (45%) and cell body (60%) growth. However, not all aspects of the dendritic arborization were affected. Primary dendrites and branch points were not altered by nerve growth factor. In contrast, nerve growth factor induced a significant increase in the number of primary dendrites and branch points (100%) of neurons from young adults. Our results demonstrate that sympathetic neurons undergoing dendritic atrophy during aging can exhibit significant dendritic growth in response to the in vivo infusion of nerve growth factor, although the lack of regenerative response displayed by some parts of the dendritic tree leads us to believe that these neurons also show signs of reduced plasticity.

Intracellular injection of a biotinylated probe in fixed superior cervical ganglia followed by confocal microscopy was used to investigate the effects of age on the dendritic arborisation of sympathetic neurons in rats aged 6 wk (young adult), 7 months (fully grown adult) and 24 months (aged). In accordance with other studies considerable dendritic growth was observed during postnatal development. However, in old age dendritic growth did not continue, and significant atrophy was observed. Quantitation of neuronal morphology showed significant reductions in soma size, total dendritic length, number of branch points and total area of dendritic arborisation in old age. Unexpectedly, significant reductions in the numbers of primary dendrites were observed in maturity and in old age. Concomitant with this atrophy there was an increase in age-related morphological abnormalities. The similarities between the atrophy and dendritic abnormalities shown by our aged neurons and those seen in other studies of young adult sympathetic neurons following axotomy or trophic factor deprivation are discussed.

In order to compare age-associated neurodegenerative changes in peripheral nerves of laboratory mammals and humans, we have investigated the density and pattern of different nerve populations innervating sweat glands of ageing rats and compared our results with a previous study of the innervation of human sweat glands. We have also studied age-changes in subepidermal afferent nerves that may be involved in reflex activation of sweat glands. Total nerve density, measured by immunohistochemical staining for the general neuronal marker, protein gene product (PGP9.5) and image analysis, showed a significant decline around secretory coils of sweat glands of old compared to young rats. Marked reductions of acetylcholinesterase (AChE) histochemical staining and of vasoactive intestinal polypeptide (VIP)- and calcitonin gene-related peptide (CGRP)-like immunoreactivity were observed in nerves around sweat glands. In the sub-epidermis, PGP- and CGRP-like immunoreactive nerves were significantly reduced in old rats. The age-related changes in sweat gland innervation of old rats were comparable to those reported in elderly human subjects suggesting that these tissues may provide a suitable model for experimental studies of neuronal ageing.

The aim of this study was to investigate the response of sympathetic neurones to prolonged neural stimulation, using cold exposure as a non-invasive experimental paradigm. We examined the effects of prolonged (8 days and 4 wk) cold exposure on tyrosine hydroxylase (TH) protein and activity and neuropeptide Y (NPY) levels in sympathetic neurones of the superior cervical ganglion (SCG), together with NPY levels in the ear artery from young and aged rabbits. The main findings were as follows. In young rabbits, TH levels and TH activity were differentially regulated in response to prolonged cold exposure. TH levels rose whilst TH activity tended to decline. Decentralization of SCG from young animals before cold exposure abolished the rise in TH levels. TH activity in SCG from young rabbits was reduced by decentralization whilst cold exposure resulted in an increase in TH activity. Thus, TH activity was induced in the SCG in the absence of pre-ganglionic input, demonstrating a non-synaptic regulatory mechanism. In old rabbits, cold-induced changes were either delayed or failed to occur, indicating that the responses of sympathetic neurones to cold stress are impaired in old age.

Peripheral target tissues can determine age-related changes in their density and pattern of innervation. We have shown previously that middle cerebral arteries from young and old rats transplanted in oculo in young hosts become reinnervated with a density and pattern of innervation that is typical of the age of the donor, i.e., the density of reinnervation on old transplants is 50% lower than on young transplants. The alterations in the target tissues responsible for their decreased innervation in old age are still unknown. We have investigated the possibility that increasing the availability of nerve growth factor (NGF) might restore the pattern and density of perivascular nerves on old blood vessels to levels of innervation typical of young tissues. Old middle cerebral transplants were therefore treated with NGF or vehicle by three weekly transscleral injections. NGF treatment markedly increased the reinnervation of old transplants, restoring the density and pattern of innervation to one characteristic of young animals. NGF produced an equivalent increase in nerve growth on young and old transplants, thus confirming that the receptivity of old blood vessels to reinnervation is not impaired. Control experiments were performed by treating transplants with saline, bovine serum albumin, or cytochrome c. Unexpectedly, bovine serum albumin was shown to promote axonal growth, although to a lesser extent and with a different pattern than NGF.

Changes occur with age in several areas of the nervous system, including autonomic ganglia and the target tissues of some sympathetic neurons where signs of neuronal degeneration have been shown in old age. In this study, we have employed an intraocular grafting technique to examine whether age changes occur in sympathetic neurons of the superior cervical (SCG) and stellate ganglia that might explain the reduced innervation of some of their target tissues in old age. Ganglia taken from young and old rats were implanted in oculo for 4 or 6 weeks in young hosts whose irises had been previously sympathetically denervated. The extent, pattern, and time course of reinnervation of the host iris was studied by catecholamine histochemistry followed by image analysis. We did not observe any difference in the pattern and time course of reinnervation between ganglia taken from young and old donors. If anything, the extent of reinnervation was greater in irises implanted with SCG taken from 25-month-old animals. Moreover, we did not observe any difference in ability to reinnervate the host iris between ganglia that normally project to the iris (SCG) and ganglia that normally do not project to the iris (stellate), regardless of the age of the donor animal. Our results suggest that sympathetic neurons from aged rats have unimpaired capability of regeneration and retain plasticity, as shown by their ability to reinnervate an inappropriate target. We hypothesize that the decreased innervation observed in some sympathetic targets in old age might be caused by changes in the target tissues themselves rather than being the result of degenerative changes in the neurons.

There are few generalisations that can be made regarding the changes that occur in autonomic nerves during ageing. Old age has different effects, including loss of neurones, loss of axon branches and alterations in neurotransmitters and other intracellular features. However, these age-related events are associated with particular and often small groups of neurones and are frequently species specific. Changes occur at different periods during development and maturity without any obvious age-stage at which neurodegenerative changes come to predominate. Some of the observations regarding neuronal changes in old age can be interpreted as the result of altered interactions between neurones and their peripheral target tissues. Recent studies in my laboratory support this contention. The neurotrophic theory has been used to explain such interactions during early development and it seems possible that, for example, alterations in the access of neurones to target-derived growth factors may underlie some of the changes that have been observed in old age. Plasticity in the mature autonomic nervous system may also be governed by similar relationships between nerves and their target tissues.

Age-related changes in the nervous system might be intrinsic to the neurons or secondary to changes in the target tissues they supply. We have investigated this question by transplanting old and young vascular muscle into contact with young host nerves in oculo and observing the reinnervating nerve fibres. The density and pattern of reinnervation were those typical of the age of the target tissue, thus old transplants imposed an 'old' pattern of reinnervation on young host nerves. Our findings strongly support the hypothesis that target tissues determine the pattern and density of their innervation in old age.

Age-related changes in the human peripheral sudomotor neuro-effector system have been investigated in six 80-year-olds and six young adults. Histochemical and immunohistochemical studies on forearm skin biopsies showed diminished vasoactive intestinal polypeptide (VIP) and calcitonin gene related peptide (CGRP)-like immunoreactivity and a virtual absence of acetylcholinesterase in the elderly sudomotor nerve endings compared to the young. Reduced size of nerve bundles and decreased density of sympathetic nerve endings adjacent to the sweat glands of old people were shown by the neuronal marker, protein gene product (PGP 9.5), and by electron microscopy. Image analysis techniques were also used to demonstrate a marked regression in secretory coil size with age. Functional decrements accompanying the neurochemical and morphological changes in the neuro-effector system were measured in ten 80-year-olds by local quantitative nicotine axon reflex responses and compared with 12 young adults. These studies demonstrate marked regressive changes in both the nerve endings and target cells in old age and appear to express a significant loss of vigour in trophic interactions.

The effect of old age on the net accumulation of 5-hydroxytryptamine (serotonin) into cerebrovascular nerves of rats was studied using a new, densitometric application of image analysis of immunohistochemical staining. The results showed evidence of reduced accumulation in old animals in nerves supplying arteries of the circle of Willis. Measurements of the density of nerves stained with an antibody to the neuronal marker, PGP9.5, showed that loss of nerve fibres may occur in some cerebral vessels in old age.

The development of noradrenergic and peptide-containing perivascular nerves in common carotid, mesenteric, renal and femoral arteries of the guinea-pig was studied using the glyoxylic acid fluorescence and indirect immunofluorescence techniques on whole-mount stretch preparations at 6 stages between 6 weeks in utero and two years after birth. The noradrenergic plexus was more dense than the peptide-containing nerve plexuses in all the blood vessels, and, in general, calcitonin gene-related peptide-containing nerves were more numerous than substance P-containing nerves which in turn were more numerous than vasoactive intestinal polypeptide-containing nerves. In mesenteric and carotid arteries, noradrenergic nerve density reached a peak at about 4 weeks after birth that was maintained to old age, whereas the peptide-containing vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP) nerve plexuses reached a peak at birth and declined thereafter to about half maximum density in old age. In contrast, in the renal and femoral arteries, peptide-containing nerves reached a maximum density at 4 weeks after birth, while noradrenergic nerve density reached a peak around birth; both noradrenergic and peptide-containing nerve plexuses declined in density in old age. Of the 4 vessels studied, the mesenteric artery showed the greatest density of innervation for both noradrenergic and peptide-containing nerves at all stages of development, while the femoral artery was the least innervated. The possibility that some perivascular peptide-containing nerves play a trophic role during development is discussed.

Variations in ascorbate and thiol concentration in lens and aqueous humour, with age and vitamin C nutrition, are of potential biological importance. To study these relationships, Dunkin-Hartley guinea-pigs were maintained for periods of up to 1.3 yr on diets containing either high or low (marginal) vitamin C. Ascorbate contents of liver, spleen, adrenals, lens and aqueous humour, and acid-soluble thiol of lens and aqueous humour were measured. High vitamin C intake maintained ascorbate levels in the internal organs between five and 30 times the level attained by the low vitamin C intake and aqueous humour vitamin C was 10-20 times higher at high vitamin C intake. Lens ascorbate, however, was only about twice as high at high vitamin C intake than at low intake, and at both intake levels it declined steadily to about half its initial value, after 1.3 yr. Thus an animal aged 1.3 yr on the low intake had about one-quarter to one-fifth of the lens ascorbate level of a young animal receiving a generous intake. Acid-soluble thiol in the lens, in contrast to ascorbic acid, increased significantly with age but was not significantly affected by dietary vitamin C intake. Acid-soluble thiol in the aqueous humour was only 0.5-2% of the concentration found in lens, and unlike the lens thiol level, it declined with age. No sex differences were observed for ascorbate or thiol levels either in lens or in aqueous humour.

Age-related changes in vascular adrenergic nerves of 5 contrasting arteries in the rabbit were studied from before birth through to old age. Adrenergic nerves were demonstrated on stretch preparations using glyoxylic acid fluorescence histochemistry. Quantitative estimates of the densities of nerve fibres and varicosities were obtained by automated light microscopic image analysis. The early stages of development of vascular innervation were similar in all the vessels studied. They consisted of a period of outgrowth of axons; a period of rapid increase in density and formation of varicosities; and a later period of more gradual nerve growth. The timing of these stages varied greatly between the different vessels. The larger vessels, i.e. the carotid, renal and femoral arteries, had a well-developed innervation at birth, whilst the innervation of the smaller mesenteric and basilar arteries was sparse. Nerve loss occurred between 6 weeks and 6 months in the femoral artery and in old age (3 years or over) in the renal and carotid arteries. The large elastic arteries were, in general, more densely innervated than the smaller muscular arteries throughout life. The innervation of the different vessels became increasingly diverse between birth and adulthood, indicating a relationship between the pattern of vascular innervation and local physiological requirements. Factors which could influence age-related changes in nerve pattern and density are discussed.

Quantitative immunohistochemical techniques were applied to whole mount stretch preparations of the femoral, saphenous and cerebral blood vessels of rabbits from before birth to old age (greater than 3 years) in order to study the effects of ageing processes on 5-hydroxytryptamine(5-HT)-like immunoreactive perivascular nerve plexuses. Immunostained perivascular nerve fibres first appeared at 30 days in utero in all the vessels studied with the exception of the saphenous vein where nerves appeared at one day after birth. Perinatal and early postnatal development showed a general increase in nerve density in all the vessels, which continued up to adulthood (6 months) in all vessels except the femoral artery and then showed a general and often statistically significant decline in old age. Comparison of these results with those of noradrenergic perivascular innervation showed by 5-HT tended to appear later in perinatal development and was more widely reduced in old age than noradrenaline.