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Timothy Cowen, Department of Anatomy and Developmental Biology, University College London, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK. Tel. +44 (0)207 8302181; e-mail: email@example.com
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.
Interest in the contribution neurotrophin signalling makes to plasticity in the adult and aging mammalian nervous system is not new. However, despite almost two decades of investigation, it remains unclear precisely how this system contributes to adult neuronal plasticity, and whether impairments in the system contribute to age-related neurodegeneration.
A challenging characteristic of aging is its selective effect on particular regions of the nervous system or subgroups of neurons (Morrison & Hof, 1997). This phenomenon of selective vulnerability (Cowen, 2002) is seen in a range of central and peripheral neurons, including small subsets of neurons in the entorhinal cortex (Morrison & Hof, 1997) and gut neurons of the enteric nervous system (Cowen et al., 2000). Impairments range from loss of synapses and the dying back of dendrites and axons to outright cell death. Much remains to be understood about these processes. For example, the relationship between structural and functional impairments in the aging nervous system is not always clear. Furthermore, little is known about why these particular groups of neurons succumb in various specific ways to the stresses of aging. In the present study, we focus on small groups of sympathetic neurons, each of a few hundred neighbouring cells, in the superior cervical ganglion (SCG). SCG neurons which project to the cerebral blood vessels supplying the brain (CV) are vulnerable to loss of axons and dendrites during aging (Thrasivoulou & Cowen, 1995), while those projecting to the iris of the eye are relatively protected and continue to grow in old age (Gavazzi et al., 1996).
The fact that selective vulnerability is associated with projections to particular target tissues suggests strongly that some aspect of nerve–target interaction is involved. This in turn makes neurotrophin signalling (Huang & Reichard, 2001; Ernfors, 2001) a prime candidate for investigation. During development, target tissues secrete neurotrophic factors which are bound and retrogradely transported by the ingrowing axons of innervating neurons (Davies, 1996). Neurons compete for a limited supply of neurotrophic factors and those which are unsuccessful die. By this means, targets regulate the population of innervating neurons. Because neurotrophic factors also affect neuronal growth, targets influence the size (extent of axonal and dendritic arborizations and cell soma) of innervating neurons and other aspects of phenotype, as well as their number. This is likely to provide a means by which neurons and targets adjust physiologically to each other. Nerve growth factor (NGF) was the first neurotrophic factor to be discovered, principally through its role in the survival and outgrowth of sympathetic neurons (Levi-Montalcini, 1987), and is now established as the founder member of the neurotrophin family of related proteins. Sympathetic neurons develop survival-dependence on NGF around embryonic stage E14 in mice (Wyatt & Davies, 1995).
The neurotrophin NT-3 also plays a part in the development of sympathetic neurons (Francis et al., 1999). Like NGF, NT-3 is present in target tissues and is retrogradely transported in rat sympathetic neurons (Zhou et al., 1997). Developing sympathetic neurons are relatively unaffected by the absence of NT-3 in null mutant mice (Wyatt et al., 1997). However, around and soon after birth, SCG neuron numbers decline significantly in these animals. Dependence of sympathetic neurons on NT3 was confirmed by loss of sympathetic neurons following treatment of postnatal rats with anti-NT3 antibodies (Zhou & Rush, 1995).
Our initial studies evaluated a simple ‘neurotrophic hypothesis of aging’– availability of the neurotrophic factors NGF and NT-3 in different target tissues would determine selective vulnerability of sympathetic neurons (Gavazzi & Cowen, 1996). However, investigations in the pineal gland (Kuchel et al., 1999), where axons are lost to a similar extent to those supplying CV, failed to demonstrate any age-related reduction in level of NGF. Neurotrophin levels were also unchanged in the aging rat iris (Cowen et al., 1996). It also became clear from in vitro studies that postnatal NGF-dependent sensory and sympathetic neurons lose their short-term (1–2 weeks in culture) survival dependence on neurotrophins during the first weeks of postnatal life (Easton et al., 1997; Orike et al., 2001a,b). These data argue against a direct relation between neurotrophic factor availability and survival of adult and aged neurons.
While we do not abandon the question of neurotrophic factor availability, other evidence suggests that receptor expression or its regulation might contribute to selective neuronal vulnerability during aging (Cowen & Gavazzi, 1998). Neurotrophin responsiveness in adult sympathetic neurons involves the trkA receptor tyrosine-kinase and p75 – a member of the tumour necrosis factor receptor family. That NGF and NT3 both bind and activate trkA is based on in vitro studies using SCG neurons from trkC-deficient mice (Davies et al., 1995) and a mutant NT-3 that signals through trkA rather than via trkC (Belliveau et al., 1997). p75 plays an important but controversial role in the neurotrophin response of sympathetic neurons (Miller & Kaplan, 2001) where it can either induce apoptosis or protect neurons against apoptosis following neurotrophic factor deprivation. Evidence suggests that, when co-expressed as they are in the majority of sympathetic neurons, p75 and trkA oligomerize to generate high-affinity binding sites for NGF (Hempstead et al., 1991; Esposito et al., 2001). Since sympathetic and sensory neurons from p75−/– mice exhibit reduced survival-responsiveness to NGF (Lee et al., 1994), these sites are likely to enhance neuronal survival. Similarly, sympathetic and sensory neurons exhibit reduced responsiveness to a mutated NGF protein with reduced binding to p75 but wildtype binding to trkA (Horton et al., 1997). Activated p75 and trkA mediate positive growth responses to NGF in adult sympathetic neurons (Orike et al., 2001b) and are required for NGF uptake and retrograde transport in adult sympathetic neurons (Gatzinsky et al., 2001), despite the possibility that p75, when activated alone, exerts negative effects on growth (Kohn et al., 1999). In sympathetic neurons, NT3 appears also to act through trkA and through p75 (Brennan et al., 1999) rather than through its cognate receptor, trkC (Davies et al., 1995; Dechant et al., 1997). In addition to its actions via trkA, NT3 may influence cholinergic phenotypes via its cognate receptor, trkC, in a subset of sympathetic neurons, at least during development (Brodski et al., 2000).
Studies of sympathetic neurons have shown that trkA is developmentally regulated in chick (vonHolst et al., 1997) and mouse (Wyatt & Davies, 1995). In the mouse, expression levels increase substantially between E14 and E18 but are not affected by treatment with NGF (Wyatt & Davies, 1995). In contrast, expression of the p75 receptor increases during late gestation and early postnatal life and is up-regulated by NGF treatment. Prolonged treatment of the eye in young adult rats with high doses of NGF (Miller et al., 1994) further showed that trkA expression was not affected by NGF, whereas p75 was up-regulated substantially. These data suggest that p75, at least when co-expressed with trkA, may exert a positive influence on the responsiveness of sympathetic neurons to developmental alterations in trophic factor availability such as those induced by target growth.
A range of evidence points to reduced neurotrophin uptake, binding and/or retrograde transport in age- and disease-related impairments in the nervous system. Several groups of neurons are affected, including neurons of the basal forebrain (Cooper et al., 1994; 2001) and motor (Bartlett et al., 1998; Frolkis & Tanin, 1999) and sensory neurons (Delcroix et al., 1997) of the spinal cord. In sympathetic neurons, we have shown reduced levels of NGF protein in the aged SCG, coupled with unchanged peripheral levels of NGF (Cowen et al., 1996) which are indicative of reduced uptake and retrograde transport. As predicted, ganglionic levels of NGF were reduced following axotomy in young animals but were unaffected in old ones, supporting the same explanation. Furthermore, reduced accumulation of NGF has recently been observed in NGF-treated CV-projecting sympathetic neurons of aging rats (Kudwa et al., 2002). These cases point either to impaired receptor binding, internalization or retrograde axonal transport as affectors of aging sympathetic neurons. Thus, reduced growth-responsiveness was observed in iris-projecting sympathetic neurons in aging rats, coupled with reduced p75 immunoreactivity on peripheral axon arborizations (Gavazzi et al., 2001). p75 levels were restored following NGF treatment suggesting again that, in the aged nervous system, peripheral p75 expression may continue to regulate neuronal responsiveness to locally available neurotrophic factors.
Our aims in the present study were (1) to extend our previous observations on the expression of neurotrophic factors in target tissues innervated by ‘vulnerable’ and ‘protected’ neurons, paying attention to the possibility that pre- and post-transcriptional changes might affect the availability of these factors; (2) to examine receptor expression and regulation of expression by neurotrophic factors, in vulnerable and protected subpopulations of neurons. Our results indicate that alterations in receptor expression, and in the capacity of particular subpopulations of neurons to regulate receptor expression during aging, correlate strongly with capacity to resist age-related neurodegeneration. These features of vulnerable and protected neurons are linked, respectively, with low and high patterns of expression of neurotrophic factors in the particular target tissues which are retained throughout adult life.
Neurotrophin expression in target tissues
Quantitative RT-PCR demonstrated that levels of NGF in the iris were approximately 10-fold higher than those in CV from 12-week-old and 24-month-old rats (Fig. 1A; old compared to young, P < 0.001 for both). NGF mRNA exhibited no significant age-related changes in iris or CV. ELISA assay further showed six-fold higher levels of NGF protein in iris compared to CV (young iris vs. young CV, P < 0.005; old iris vs. old CV, P < 0.02); no significant age-related change was seen in NGF protein levels in either tissue (Fig. 1B). Levels of NT3 mRNA were about three-fold higher in iris compared to CV (young iris vs. young CV, P < 0.02; old iris vs. old CV, P < 0.001) (Fig. 1C). As with NGF, no significant age-related differences were shown in either tissue.
Neurotrophin receptor expression in SCG
Levels of trkA, trkC and p75 receptor expression were examined in the SCG using quantitative RT-PCR (Fig. 2). trkA and p75 expression were markedly (about 100-fold) higher than expression of trkC (Fig. 2). trkA expression remained unchanged throughout postnatal life including in old age. trkC expression fell by about 50% between 1 and 12 weeks of postnatal life (P < 0.001) but showed no further significant change at 24 months. p75 expression rose significantly (P < 0.02) between 1 and 12 weeks of postnatal life and then, between 12 weeks and 24 months, fell back (P < 0.01) to a level similar to that seen 1 week after birth (Fig. 2).
Neurotrophin receptor expression in subpopulations of SCG neurons
Co-localization studies using immunocytochemistry for p75 and trkA and confocal microscopy showed that the majority of SCG neurons exhibited staining for both receptors (Fig. 3A,B). Unexpectedly, a few small trkA-positive neurons (Fig. 3B) were negative for p75 (Fig. 3C). Also, larger neurons appeared to be more intensely stained for p75 than small ones (Fig. 3A). Co-localization of immunolabelling for receptor staining and Fluoro-Gold retrograde label revealed that FG-positive neurons were generally p75- as well as trkA-positive in both iris and CV-projecting neurons (data not shown).
Effect of age on cell size and plasticity of neurotrophin receptor expression
In the vehicle-treated groups, the size of iris-projecting neurons was significantly larger (young and old: P < 0.01) than that of CV-projecting neurons (Fig. 4; Andrews et al., 1996). There were no significant effects of age on the size of vehicle-treated neurons projecting to either target. Treatment of 12-week CV-projecting neurons with NGF and NT3 resulted in significant (20%; P < 0.05) increases in cell size with each factor (Fig. 4). In 12-week iris-projecting neurons, NGF and NT3 again produced a small but significant increase in size (P < 0.05 for both factors). However, at 24 months, neither subpopulation of neurons exhibited a growth response to NGF or NT3.
p75 protein levels measured by immunocytochemistry (GV/cell) showed slightly higher levels (P < 0.05) in iris-compared to CV-projecting neurons in the absence of neurotrophin treatment (Fig. 5A). No change was seen with age in p75 protein levels in iris-projecting neurons under these conditions (Fig. 5A) while, in contrast, CV-projecting neurons exhibited a significant (P < 0.05) reduction of p75 protein at 24 months compared to 12 weeks of age, resulting in three-fold higher levels of p75 in 24-month iris compared to 24-month CV neurons (P < 0.01). Treatment with NGF increased p75 protein in 12-week iris projecting neurons (P < 0.05), but importantly had no effect in 12-week CV-projecting neurons (Fig. 5A). NGF treatment had no effect on p75 protein levels in either group of 24-month neurons and NT3 treatment had no effect in any of the experimental groups. Variance is high due to the relatively small numbers of traced neurons seen (less than 40 per condition, amassed from four different experiments). Hence the relatively low levels of statistical significance.
Examination of p75 mRNA using in situ hybridization combined with retrograde tracing in general confirmed the results of immunocytochemistry by showing that there were similar levels of baseline (vehicle-treated) expression in CV- and iris-projecting neurons (Fig. 5B). In addition, whilst no age-related changes were seen in baseline p75 expression in iris-projecting neurons, 24-month CV-projecting neurons exhibited a substantial (four-fold; P < 0.05) decrease in expression (Fig. 5B), leaving values significantly lower than those of 24-month iris-projecting neurons (P < 0.01). Unexpectedly, treatment with NGF or NT3 failed to generate any changes in p75 expression in either group of neurons at either of the ages studied. Thus, the increase in protein expression in 12-week iris-projecting neurons in response to NGF (Fig. 5A) was not matched by up-regulation of p75 mRNA.
Immunocytochemistry for trkA revealed a 24% higher level in iris-projecting compared to CV-projecting neurons in the vehicle-treated 12-week samples (P < 0.05; Fig. 6A). A comparable difference was retained in these groups at 24 months (P < 0.05). No significant reductions were seen in trkA immunolabelling in 24-month iris- and CV-projecting neurons in the vehicle-only groups. Treatment of CV-projecting neurons with NGF and NT3 failed to demonstrate any alterations in receptor immunofluorescence at either 12 weeks or 24 months of age (Fig. 6A). Treatment of iris-projecting neurons with NGF and NT3 also had no effect on trkA immunolabelling in 24-month neurons. However, treatment with NGF in 12-week iris-projecting neurons resulted in a 17% increase in GV/cell (P < 0.05), while NT3 had no effect on this group (Fig. 6A).
In situ hybridization for trkA mRNA supported the results of immunocytochemistry by showing higher levels of trkA expression in iris- compared to CV-projecting neurons in 12-week, but not 24-month, neurons from the vehicle-only groups (P < 0.05; Fig. 6B). Both groups of neurons exhibited small age-related reductions in expression, in both cases of about 50% which were not statistically significant. Treatment of CV-projecting neurons with NGF or NT3 had no effect on trkA expression at either 12 weeks or 24 months of age (Fig. 6B). However, treatment of iris-projecting neurons with NGF generated strongly increased (> two-fold; P < 0.01) expression of trkA in 12-week neurons, which was not seen with NT3. The effect of NGF on trkA expression in iris-projecting neurons was lost at 24 months.
In situ hybridization for trkC mRNA (Fig. 7) showed significantly higher levels in 12-week iris- compared to CV-projecting neurons. Neither age nor treatment with neurotrophins altered trkC expression in either group of SCG neurons.
Why are certain groups of neurons vulnerable to age-related degeneration whilst others appear to be protected? Here we investigate this question with neighbouring groups of rat sympathetic neurons that have different responses to age. We find higher levels of trkA expression in iris-projecting compared to CV-projecting neurons in the absence of neurotrophin treatment at both 12 weeks and 24 months of age; p75 and trkC levels were also higher in iris neurons, but to a lesser extent than trkA. As a function of age, substantial reductions were seen in p75 expression (protein and mRNA) in CV- but not in iris-projecting neurons. The small reductions in trkA expression (protein and mRNA) were not statistically significant. Treatment with NGF resulted in increases of p75 (protein only) and trkA (protein and mRNA) expression which were restricted to 12-week, iris-projecting neurons.
In general, neurotrophin protein and mRNA levels reveal a correlation between vulnerability to age-related neuronal atrophy and target levels of neurotrophin expression. Vulnerable SCG neurons (Thrasivoulou & Cowen, 1995) project to target tissues (e.g. CV) that exhibit, probably throughout life, low levels of NGF and NT3 expression, while, in marked contrast, neurons which are protected from age-related neuronal atrophy and degeneration (Gavazzi et al., 1996) project to target tissues (e.g. Iris) with high levels of neurotrophin expression. That these high and low levels of neurotrophins translate to differences in the respective groups of neurons is supported by evidence of substantially lower levels of uptake of I125-NGF in the cell soma of CV compared to iris-projecting neurons (Thrasivoulou, Gatzinsky and Cowen, unpubl. obs.).
Here we now see that neurotrophin levels do not change with age in target tissues of either vulnerable or protected neurons. While previous data are insufficient to rule out variation in neurotrophin levels between 3 and 24 months of age, a 3-month male Sprague–Dawley rat is fully grown and sexually mature and we conclude that neurotrophin levels remain more or less constant in both target tissues throughout adult life, as previously reported (Cowen et al., 1996). Thus, age-independent levels of neurotrophin expression in target tissues looks like the best correlate and predictor of neuronal vulnerability.
Why should the phenotype of tissue-specific low level of NGF evolve if this trait permits age-dependent neurodegeneration? Most likely, NGF levels during development are adaptive, and any costs that arise from these patterns in terms of senescence are paid only at older ages, when the force of selection has decreased. Specifically, the smooth muscle of CV, in common with other blood vessels, exhibits a ‘multi-unit’ pattern of innervation where the majority of muscle cells are not directly innervated but are activated via gap junctions which connect them to the few innervated cells (Burnstock, 1993). This system provides gradual, graded contraction and relaxation over extended areas of the vascular plexus. In contrast, each iris smooth muscle cell is innervated directly, forming a ‘single-unit’ pattern which mediates the characteristic rapid responses of the iris dilator and constrictor muscles. These contrasting functions are mirrored by different morphologies: CV-projecting neurons are small with relatively short and unbranched axonal and dendritic arborizations, while iris-projecting neurons are large with extensive axons and dendrites (Andrews et al., 1996). Furthermore, it is known that high levels of endogenous NGF (Albers et al., 1994) and prolonged NGF treatment or deprivation (Ruit & Snider, 1991) permanently alter the size and branching patterns of NGF responsive neurons. We therefore hypothesize that low and high levels of target-derived neurotrophic factors in CV and iris, respectively, play a key part in establishing the appropriate structure and function of innervating neurons during development.
Low levels of target-derived neurotrophins may become disadvantageous in old age by rendering neurons vulnerable to age-related stressors. Reactive oxygen species (ROS) are candidate stressors with known cumulative effects throughout life (Beckman & Ames, 1998). We have recently demonstrated that age-related loss of enteric neurons in the rat gut (Cowen et al., 2000) is preceded by elevated levels of intraneuronal ROS and, furthermore, that neurotrophic factors reinforce anti-oxidant defence and protect neurons against ROS-induced apoptotic cell death (Thrasivoulou et al., unpubl. obs.). A similar role for NGF has recently been demonstrated in sympathetic neurons (Dugan et al., 1999; Thrasivoulou & Cowen, unpubl. obs.). Levels of NGF in target tissues may therefore provide a cellular example of antagonistic pleiotropy (Partridge & Barton, 1993) where low levels of target-derived neurotrophic factors on the one hand benefit early development and adaptation of neurons to local physiology while, on the other, rendering these neurons vulnerable to age-related stressors such as ROS.
Since constant differences in neurotrophin levels appear to be associated with vulnerability or protection from age-related neurodegeneration, it becomes important to understand how this might affect expression of the neurotrophin receptors in adult and aging neurons. In particular, is receptor expression in adult and aging neurons regulated independently of target-derived neurotrophic factors, perhaps as a result of intrinsic alterations in neuronal gene expression? At least during embryonic development, trkA expression in sympathetic neurons is regulated independently of neurotrophins while, in contrast, the p75 receptor can be up-regulated by NGF treatment during late stages of embryonic development and through to 6 weeks of age (Miller et al., 1994). Our results correlate well with the idea that p75 expression is regulated by neurotrophins during early life, by showing intense p75 immunoreactivity in the large iris-projecting neurons which are exposed to high levels of target-derived NGF while the smaller CV-projecting neurons, which are exposed to low levels of NGF, exhibit low levels of p75 expression. We therefore suggest that p75 expression may regulate or ‘set’ neuronal responsiveness to varying levels of trophic factors induced by target growth during maturation.
As noted in Cowen et al. (1996) and in our current results, tissue-specific patterns of age-related neurodegeneration do not correlate with declines in target levels of neurotrophic factors. Yet, with age, neurons appear to lose responsiveness to neurotrophic factors. For instance, cholinergic basal forebrain neurons of aged rats have reduced uptake and/or retrograde transport of 125INGF (Cooper et al., 1994) – a pattern we have also observed in rat sympathetic neurons (Gatzinsky & Cowen, unpubl. obs.), including those projecting to CV, where p75 has been shown to be essential for retrograde axonal transport of NGF (Gatzinsky et al., 2001). Recently, reduced accumulation of NGF has been demonstrated in CV-projecting sympathetic neurons (Kudwa et al., 2002). Reduced growth responses to NGF have also been demonstrated in aged irideal sympathetic nerves, coupled with reduced p75 immunoreactivity (Gavazzi et al., 2001). Our present results support this evidence of age-related loss of plasticity by showing a loss of the capacity for growth of the cell soma of both iris- and CV-projecting aged neurons in response to NGF and NT3.
Our present results support the view that vulnerability to age-related neurodegeneration is associated with reduced neurotrophin responsiveness involving principally the p75 receptor. Expression of p75, but not of trkA, declines with age in the whole SCG. Critically, when evaluated in subgroups of neurons projecting to iris and CV, the decline in p75 correlated with neuronal vulnerability: thus, p75 protein and mRNA declined significantly only in the vulnerable, CV-projecting neurons, while neither trkA protein nor mRNA were significantly reduced in either group of neurons. p75 expression is therefore the best correlate of plasticity in the aging nervous system.
Importantly, the changes in p75 (and trkA) expression occur independently of changes in extrinsic, target-related levels of neurotrophic factors. Treatment of 12-week and 24-month neurons projecting to CV and iris with neurotrophins further demonstrated that vulnerability to degeneration was associated with a failure of receptor regulation. At 12 weeks, treatment of adult iris-projecting neurons with NGF somewhat up-regulated p75 and trkA protein but increased trkA mRNA more substantially. In contrast, the same regime produced no obvious response in CV-projecting neurons. At 24 months, induction of p75 and trkA by NGF in iris-projecting neurons was lost. Receptor expression in CV-projecting neurons remained uninduceable. The dynamics seen in iris-projecting neurons must presumably arise either from changes in post-transcriptional control of p75 message or from post-translational events in p75 processing (Miller et al., 1994).
We propose that two variables affect the age-dependent neurodegeneration of sympathetic neurons: target-derived factors that affect receptor regulation during postnatal maturation, and intraneuronal events that lead to a down-regulation of receptor gene expression during aging. Thus, compared with protected neurons, vulnerable neurons such as those projecting to CV have, throughout adult life, the following characteristics: (1) they are exposed to low levels of target-derived neurotrophins; (2) they express low levels of neurotrophin receptors; and (3) they are unable to regulate receptor expression in response to extrinsic neurotrophic signals. These features may be adaptive during early adult life as they presumably contribute to the specific physiology of target tissue such as the blood vessels. However, at later ages these traits may promote neuronal vulnerability to age-related stressors, particularly ROS, as suggested by recent evidence that neurotrophic factors and associated signalling pathways provide life-long protection against age-related, ROS-induced neurodegeneration (Cowen et al., 2000) (Thrasivoulou et al., unpubl. obs.). The age-related decline of p75 in CV-projecting neurons may arise from the accumulated effects of such oxidative damage on neuronal gene expression, and this loss may in turn synergize further impairments of neuronal function. Aging sympathetic neurons may therefore represent an example at the cellular level of antagonistic pleiotropy (Partridge & Barton, 1993). We hypothesize that developmental conditioning of neuronal responsiveness to survival and growth signals is crucial to determining vulnerability to subsequent age-related, ROS-induced neurodegeneration, a view that is consistent with evolutionary theories of aging (Lithgow & Kirkwood, 1996).
All experiments were carried out on male Sprague–Dawley rats from a colony maintained at UCL (RFC). All animals were reared with ad libitum (AL) access to BEEKAY Rat and Mouse standard 1 diet (B & K Universal Ltd, Hull, UK), composed of 19% crude protein, 5% oil, 4% fibre and 72% dry matter. Animals were killed by carbon dioxide inhalation at a range of ages from birth to 24 months of age. All procedures were undertaken in compliance with Home Office legislation under the Animals (Scientific Procedures) Act, 1986.
Expression of neurotrophins and neurotrophin receptors in SCG neurons and target tissues
Iris, cerebral arteries from the Circle of Willis (CV) and SCG were dissected quickly from untreated 8–12-week and 24-month rats and stored frozen at −70 °C until ready for use. Neurotrophin, neurotrophin receptor and GAPDH mRNAs were quantified using competitive, quantitative RT-PCR (Wyatt & Davies, 1995). In short, known amounts of a synthetic cRNA species, identical to the mRNA of interest apart from an extra three bases inserted between the PCR primer sites, were added to each reverse transcription reaction. The products of each RT-PCR reaction were separated on an 8% polyacrylamide gel, stained with Syber Gold and visualized under UV light. The intensity of the RT-PCR products of the competitor cRNA and the mRNA of interest were quantified on a gel documentation system and the initial amount of target mRNA calculated from the ratio of the signal between the RT-PCR products of the competitor cRNA and the target mRNA. ELISA assays were made for NGF, using previously described methods, in iris to confirm previous data (Cowen et al., 1996) and in CV.
Neurotrophin receptor expression in subpopulations of SCG neurons
Establishment of treatment regime
Neurotrophin treatment combined with retrograde tracing on iris-projecting SCG neurons was carried out in 2- and 12-week-old rats in order to establish appropriate doses and treatment conditions. Animals were deeply anaesthetized with fluothane (2% in 95% O2: 5% CO2). Sympathetic neurons projecting to the iris were treated using transcleral injections into the anterior eye chamber of NGF (Promega, UK) 10 or 200 ng µL−1 in 1 µL (2-week) or 2 µL (12-week) sterile (PBS), NT3 (concentrations as NGF; Genentech, USA), or vehicle alone. Slow infusion and gradual withdrawal of the needle were used to minimize leakage. Fifteen minutes later, 1–2 µL of a 2% solution of Fluoro-Gold (Fluorochrome Inc., USA) was injected by the same route. Four ganglia were examined for each treatment and age using quantitative immunocytochemistry (see below).
Effect of age on plasticity of neurotrophin receptor expression
Using the regime established above, iris-projecting sympathetic neurons from 12-week and 24-month rats were treated with neurotrophins. Sympathetic neurons projecting to cerebral vessels (CV) in the region of the middle cerebral artery were treated with similar doses of NGF, NT3 or vehicle, followed by Fluoro-Gold. In this case, animals were again deeply anaesthetized and their heads held in a stereotactic frame. Following incision of the skin and reflection of overlying muscle, a small craniotomy was carried out in the temporal bone, lateral to the sagittal suture and overlying a principal branch of the middle cerebral artery, using a 1.5-mm-diameter Dental burr. The dura mater adjacent to the artery was incised and a small piece of surgical sponge (Allevyn cavity dressing, Smith and Nephew, UK) pre-soaked in the treatment medium was inserted into the subdural space. Fifteen minutes later, a second piece of surgical sponge soaked in Fluoro-Gold was placed adjacent to the artery. Antibiotic gauze was laid over the incision, the muscle returned to position and the wound closed. The animals were placed in a recovery suite in warm conditions with regular observation. Four ganglia were examined for each treatment and age.
Eighteen to 20 h after treatment, animals were killed as above. Immediately following arrest of breathing and cessation of heart beat, animals were perfused transcardially with 50 mL (2-week) or 150 mL (12-week and 24-month) of 4% paraformaldehyde following ligation of the descending aorta. SCG were dissected, desheathed and fixed for a further 1–2 h in 4% paraformaldehyde. Ganglia were washed in PBS, treated overnight with PBS containing 25% (w/v) sucrose, frozen in cryoprotectant solution noting antero-posterior orientation and cryosectioned at 10 µm onto superfrost slides (BDH, UK). Slides were prepared with sections from all experimental conditions represented on each slide and used within a maximum of 4 weeks to minimize degradation of signal. Slides were allocated to immunocytochemistry and in situ hybridization such that three non-adjacent sections were analysed for each condition.
Immunolabelling of trkA and p75 receptors was carried out using a polyclonal antibody raised in rabbits against trkA (a generous donation from Dr David Dawbarn, University of Bristol, UK) or a monoclonal antibody against p75 (mouse hybridoma cell line Ig192). To assess the extent of colocalization of trkA and p75 receptors in SCG neurons, goat anti-rabbit Alexa 488 and donkey anti-mouse Alexa 568 (Molecular Probes, Oregon, USA) were used to label p75 and trkA antibodies, respectively. Sections were imaged in the confocal microscope. To examine receptor levels in subpopulations of SCG neurons, p75 and trkA antibodies were visualized on separate sections using Alexa 568. To ensure that Fluoro-Gold-traced neurons were reliably identified, sections were subsequently labelled with antibodies against Fluoro-Gold (Chemicon, UK) and visualized using Alexa 488 secondary antibody. Intensity of immunohistochemical labelling over Fluoro-Gold-positive (FG+) neurons was measured using densitometric methods of image analysis (Cowen & Thrasivoulou, 1992). Parallel fields of FG+ neurons and receptor label were imaged using an oil-immersion ×40 Planapo objective on an Olympus Vanox fluorescence photomicroscope. All traced neurons in three non-adjacent sections of SCG were located and imaged. Images were digitized and stored using a low-light high-resolution digital camera (Photonics Science, UK). Imaging conditions were standardized at the beginning of the experiment and then at each session using a uranyl glass standard to ensure that image intensity was set at a constant level that was within the linear response range of the camera. The cell and nuclear outlines of FG+ neurons were traced interactively and used to generate a mask within which the intensity of cytoplasmic receptor labelling was measured using Kontron-Zeiss image analysis software (Imaging Associates, Bicester, UK). Multiplicative background subtraction was carried out using adjacent unstained fields within the sample and semi-automatic discrimination of specific signal was achieved using a purpose-written macro. Following evidence that neurotrophin treatment altered cell size (Fig. 4), fluorescence intensity was expressed as mean grey value (GV) per cell in preference to GV µm−2. Cell size data were automatically generated from the mask image. Mean values were tested using anova followed by post hoc testing of data pairs.
In situ hybridization
Slides were stored in a cryoprotectant solution of 120 mL ethylene glycol in 200 mL of PBS containing 120 g sucrose (Watson et al., 1986). Contamination with extrinsic RNAase was minimized by careful handling of specimens and use of DEPC-treated water throughout. In situ hybridization with radiolabelled oligonucleotide probes was used to demonstrate the cellular localization of mRNA for p75, trkA and trkC receptors in retrogradely traced neurons. Standard procedures were used whereby immunolabelling of Fluoro-Gold tracer was conducted prior to in situ hybridization (Michael et al., 1997). Tetramethyl rhodamine isothiocyanate (TRITC) was utilized as the secondary reagent for visualization of Fluoro-Gold immunoreactivity. Oligonucleotides for trkA and trkC have been characterized previously in dorsal root ganglia (Michael et al., 1999). The p75 oligonucleotide is complementary to nucleotides 671–704 of the rat sequence (Radeke et al., 1987). The specificity of the p75 oligonucleotide was assessed in adult dorsal root ganglion sections and confirmed to label cells with the correct distribution as described in the literature. Addition of a 100-fold excess of unlabelled oligonucleotide to the hybridization reactions effectively competed the specific labelling of the radioactive probes and resulted in only non-specific signal. After hybridization, slides were coated with LM1 emulsion (Amersham, UK), kept in the dark at 4 °C for 2–3 weeks (p75 and trkA) or 4–5 weeks (trkC) and then developed and fixed in Kodak reagents. Slides were coverslipped using PBS/glycerol (1 : 3) containing the anti-fading agent, 2.5% 1,4 diazobicyclo (2,2,2) octane (Sigma, UK).
Two methods for quantification by image analysis of the extent of in situ labelling were tested on preselected groups of neurons exhibiting the full range of labelling intensities from sparse to very dense. For both tests, cell soma and nuclear outlines were traced interactively as before using the FG+ image. The mask generated of the cytoplasmic area was superimposed on the same field, now illuminated using epi-polarizing illumination on an Olympus Vanox photomicroscope to visualize the silver grains. In the first approach, the area covered by silver grains was measured and expressed as area per cell. In the second approach, the summed intensity of labelling (GV) was measured for each cell and expressed as GV per cell. GV per cell gave a steeper response curve and a 40% higher maximum reading compared with area per cell after measuring the same groups of neurons and this method was therefore chosen for subsequent experiments. To assess in situ hybridization labelling in the treated groups of neurons, identified neurons projecting to each target were traced round and analysed as before using a similar image analysis procedure with the intensity of labelling expressed as GV per cell. Background subtraction and semi-automatic discrimination of specific signal were again used. All traced neurons in three non-adjacent sections of SCG from each sample were located and imaged. Mean values were tested using anova followed by post hoc testing of data pairs and t-testing, as appropriate.
Funded by Wellcome Trust project grants 049001 and 065580 to T.C.