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Although the presence of neurotrophin p75 receptor on sympathetic nerves is a well-recognised feature, there is still a scarcity of details of the distribution of the receptor on cerebrovascular nerves. This study examined the distribution of p75 receptor on perivascular sympathetic nerves of the middle cerebral artery and the basilar artery of healthy young rats using immunohistochemical methods at the laser confocal microscope and transmission electron microscope levels. Immunofluorescence methods of detection of tyrosine hydroxylase (TH) in sympathetic nerves, p75 receptor associated with the nerves, and also S-100 protein in Schwann cells were applied in conjunction with confocal microscopy, while the pre-embedding single and double immunolabelling methods (ExtrAvidin and immuno-gold-silver) were applied for the electron microscopic examination. Immunofluorescence studies revealed "punctuate" distribution of the p75 receptor on sympathetic nerves including accompanying Schwann cells. Image analysis of the nerves showed that the level of co-localization of p75 receptor and TH was low. Immunolabelling applied at the electron microscope level also showed scarce co-localization of TH (which was intra-axonal) and p75. Immunoreactivity for p75 receptor was present on the cell membrane of perivascular axons and to a greater extent on the processes of accompanying Schwann cells. Some Schwann cell processes were adjacent to each other displaying strong immunoreactivity for p75 receptor; immunoreactivity was located on the extracellular sites of the adjacent cell membranes suggesting that the receptor was involved in cross talk between these. It is likely that variability of locations of p75 receptor detected in the study reflects diverse interactions of p75 receptor with axons and Schwann cells. It might also imply a diverse role for the receptor and/or the plasticity of sympathetic cerebrovascular nerves to neurotrophin signalling.

Uterine sympathetic innervation undergoes profound remodelling in response to physiological and experimental changes in the circulating levels of sex hormones. It is not known, however, whether this plasticity results from changes in the innervating neurons, the neuritogenic properties of the target tissue or both. Using densitometric immunohistochemistry, we analysed the effects of prepubertal chronic oestrogen treatment (three subcutaneous injections of 20 microg of beta-oestradiol 17-cypionate on days 25, 27 and 29 after birth), natural peripubertal transition and late pregnancy (19-20 days post coitum) on the levels of TrkA and p75 nerve growth factor receptors in uterine-projecting sympathetic neurons of the thoraco-lumbar paravertebral sympathetic chain (T7-L2) identified using the retrograde tracer Fluorogold. For comparative purposes, levels of TrkA and p75 were assessed in the superior cervical ganglion (SCG) following prepubertal chronic oestrogen treatment. These studies showed that the vast majority of uterine-projecting neurons expressed both TrkA and p75. Both prepubertal chronic oestrogen treatment and the peripubertal transition increased the ratio p75 to TrkA in uterine-projecting neurons, whereas pregnancy elicited the opposite effect. Prepubertal chronic oestrogen treatment had no effects on levels of TrkA or p75 in sympathetic neurons of the SCG. Taken together, our data suggest that neurotrophin receptor-mediated events may contribute to regulate sex hormone-induced plasticity in uterine sympathetic nerves, and are in line with the idea that, in vivo, plasticity in uterine nerves involves changes in both the target and the innervating neurons.

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.

Chronic administration of oestrogen to rats during the infantile/prepubertal period provokes, at 28 days of age, complete loss of noradrenaline-labelled intrauterine sympathetic nerves. It is not known whether oestrogen inhibits the growth or causes the degeneration of developing uterine sympathetic nerves, or whether the uterus recovers its innervation following cessation of infantile/prepubertal oestrogen treatment. In the present study, we analysed the time-course of the effects of oestrogen on the development of uterine sympathetic nerves in the rat, using histochemical methods. In addition, the pattern of sympathetic reinnervation of the uterus of intact and ovariectomised females was assessed 3 and 6 months after cessation of chronic oestrogen treatment. The ability of sympathetic nerves to reinnervate the oestrogenized uterine tissue was assessed in intraocular transplants of uterine myometrium into ovariectomised host rats. Early exposure to oestrogen did not inhibit the approach of sympathetic nerves to the uterus, but prevented the normal growth and maturation of intrauterine sympathetic fibres and abolished the innervation that reached the organ before initiation of treatment. Three or six months following cessation of oestrogen treatment, most of the sympathetic nerves were restricted to the mesometrium and mesometrial entrance, whereas intrauterine innervation remained persistently depressed as a consequence of a sustained oestrous-like state provoked by ovarian dysfunction (polycystic ovary). An organotypic regrowth of uterine sympathetic nerves was observed in ovariectomised infantile/prepubertal oestrogen-treated animals. After 5 weeks in oculo, the innervation of oestrogenized myometrial transplants was reduced by 50%, and substantial changes in the pattern of reinnervation were observed. In control transplants, 86% of the nerves were terminal varicose myometrial and perivascular nerve fibres, whereas 14% were preterminal nerve bundles. In oestrogenized myometrial transplants, 83% of the noradrenaline-labelled intercepting nerves were enlarged preterminal bundles and only 17% were terminal fibres. These results indicate that the oestrogenized myometrium is unattractive for sympathetic nerves and inhibits organotypic sympathetic reinnervation.

The innervation of the uterus is remarkable in that it exhibits physiological changes in response to altered levels in the circulating levels of sex hormones. Previous studies by our group showed that chronic administration of estrogen to rats during the infantile/prepubertal period provoked, at 28 days of age, an almost complete loss of norepinephrine-labeled sympathetic nerves, similar to that observed in late pregnancy. It is not known, however, whether early exposure to estrogen affects uterine cholinergic nerves. Similarly, it is not known to what extent development and estrogen-induced responses in the uterine cholinergic innervation are affected by the absence of sympathetic nerves. To address this question, in this study we analyzed the effects of infantile/prepubertal chronic estrogen treatment, chronic chemical sympathectomy with guanethidine, and combined sympathectomy and chronic estrogen treatment on developing cholinergic nerves of the rat uterus. Cholinergic nerves were visualized using a combination of acetylcholinesterase histochemistry and the immunohistochemical demonstration of the vesicular acetylcholine transporter (VAChT). After chronic estrogen treatment, a well-developed plexus of cholinergic nerves was observed in the uterus. Quantitative studies showed that chronic exposure to estrogen induced contrasting responses in uterine cholinergic nerves, increasing the density of large and medium-sized nerve bundles and reducing the intercept density of fine fibers providing myometrial and perivascular innervation. Estrogen-induced changes in the uterine cholinergic innervation did not appear to result from the absence/impairment of sympathetic nerves, because sympathectomy did not mimic the effects produced by estrogen. Estrogen-induced responses in parasympathetic nerves are discussed, considering the direct effects of estrogen on neurons and on changes in neuron-target interactions.

Oestrogen is a key factor in the remodelling of uterine sympathetic nerves during puberty and the oestrous cycle; these nerves are influenced by changes in their target uterine tissue. The magnitude of oestrogen-induced responses might however be influenced by the maturation stage of sympathetic nerve fibres, the age of the neurons and/or the developmental state of the uterus. We have therefore compared the sympathetic innervation of the uterus following chronic oestrogen treatment of infantile/prepubertal and young adult intact and ovariectomised rats. Treatment of infantile/prepubertal rats resulted in the complete loss of intrauterine noradrenaline (NA)-labelled sympathetic nerves and a marked reduction in the total NA content in the uterine horn. Chronic treatment of young adult rats had little effect. To examine whether the age of the neurons or the degree of development of the uterus determined responsiveness of nerves to oestrogen, we assessed the effects of oestrogen on the sympathetic reinnervation of intraocular transplants of young adult uterine myometrium into ovariectomised adult host rats. Early treatment (10 days post-transplantation) resulted in less sympathetic innervation than late treatment (30 days post-transplantation). Measurements of nerve growth factor (NGF) levels in the uterine horn of control rats before and after puberty and following infantile/prepubertal chronic oestrogen treatment and acute oestrogen treatment of young adult rats revealed a coordinated increase between the growth of the uterus and NGF protein levels. Thus, developing and recently regrown sympathetic nerves are more susceptible to oestrogen-induced changes in the uterus than mature nerves, differential susceptibility is not related to the age of the neurons or the developmental state of the uterus and changes in NGF protein do not account for the differential susceptibility of developing and mature uterine sympathetic nerve fibres to oestrogen. Growing sympathetic fibres are more vulnerable to oestrogen than mature fibres and nerve fibres that have been in contact for longer periods with their target become less susceptible to oestrogen.

We have developed and tested the biological activity and specificity of a novel fluorescent dextran-Texas Red-nerve growth factor (DTR-NGF) conjugate. DTR-NGF was found to promote survival and neurite outgrowth in cultured dissociated sympathetic neurons similarly to native NGF. The conjugate was taken up and transported retrogradely by terminal sympathetic nerves innervating the iris to neurons in the ipsilateral superior cervical ganglion (SCG) of young adult rats. Uptake and transport was assessed by counting numbers of labelled neurons and by measuring intensity of neuronal labelling using confocal microscopy and image analysis. DTR-NGF labelling in SCG neurons was shown to be dose-dependent with an EC(50) of 75 ng. Similar concentrations of unconjugated DTR resulted in no neuronal labelling. DTR-NGF uptake was competed off using a 50-fold excess of native NGF, resulting in a 73% reduction in numbers of labelled neurons. Pretreatment of nerve terminals with function-blocking antibodies against the low (p75) and high (TrkA) affinity NGF receptors resulted in a large (85-93%) reduction in numbers of DTR-NGF labelled neurons. Anti-p75 and anti-TrkA antibodies had comparable effects which were concentration-dependent. These findings indicate that both receptors are required for uptake of NGF in adult rat sympathetic neurons. In particular, the results provide strong evidence that the p75 receptor plays a more active role in transducing the NGF signal than has been proposed.

In the guinea pig, pregnancy is associated with a generalised depletion of noradrenaline in uterine sympathetic nerves and, in the areas of the uterus surrounding the foetus, by a complete degeneration of sympathetic nerve fibres. These pregnancy-induced changes have been interpreted as a selective effect of placental hormones on the system of short sympathetic fibres arising from the paracervical ganglia. An alternative explanation is that pregnancy affects the neurotrophic capacity of the uterus. We measured NGF-protein levels in the guinea pig uterine horn, tubal end and cervix at early pregnancy, late pregnancy and early postpartum, using a two-site enzyme-linked immunosorbent assay. For comparative purposes the distribution and relative density of noradrenaline-containing sympathetic nerve fibres were assessed histochemically, and tissue levels of noradrenaline were measured biochemically, using high-performance liquid chromatography with electrochemical detection. In all the uterine regions analysed, NGF-protein levels showed a decline at term pregnancy, but in no case was this change statistically significant. After delivery, NGF-protein levels showed a marked increase in the cervix as well as in both the fertile and empty horns. These results suggest that alterations in NGF-protein do not account for the impairment of uterine sympathetic innervation during pregnancy, but may contribute to their recovery after delivery.

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 have shown that chronic administration of oestrogen to prepubertal rats reduces the total content of noradrenaline in the uterine horn, abolishes myometrial noradrenergic innervation and reduces noradrenaline-fluorescence intensity of intrauterine perivascular nerve fibres. The mechanisms underlying these changes are not known. In the present study we have analysed the effects of prepubertal chronic oestrogen treatment on the synthesis of noradrenaline in the rat uterine sympathetic nerves using biochemical and immunohistochemical approaches. Tyrosine hydroxylase activity was evaluated biochemically, by measuring the in vivo accumulation of dihydroxyphenylalanine (DOPA) in the presence of a DOPA-decarboxylase inhibitor. In addition, nerve fibres were visualised immunohistochemically using antibodies against tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DbetaH) and the general marker protein gene product 9.5 (PGP 9.5). After chronic oestrogen treatment, the total content of noradrenaline of the uterine horn was reduced, whereas the total content of DOPA was increased. In controls, TH-immunoreactive, DbetaH-immunoreactive and PGP 9.5-immunoreactive nerve fibres were distributed in both the circular and longitudinal myometrial layers and in the blood vessels of the intran-myometrial region. After chronic oestrogen treatment the only fibres recognised by the three antibodies were those associated with the blood vessels, but no myometrial-associated fibres could be recognised. These results suggest that noradrenaline synthesis is selectively reduced in myometrial-associated uterine sympathetic nerves, but is preserved in perivascular sympathetic nerves. The increased DOPA levels measured after chronic exposure to oestrogen was interpreted as the consequence of the substantial increase in size and number of blood vessels observed in the uterus of oestrogen-treated animals. A possible neurodegenerative effect of oestrogen on myometrial sympathetic fibres is discussed.

Sympathetic nerves to the enlarged fetus-containing region of the uterus undergo degenerative changes during late pregnancy and show slow regrowth after parturition. It is not known whether this unusual response of sympathetic nerves to smooth muscle hypertrophy is due to the sensitivity of short adrenergic neurons to hormonal changes, or whether the nerves respond to changes in the neurotrophic capacity of the target. We have investigated this question using in oculo transplantation. Small pieces of myometrium from the uterine horn of virgin guinea pigs, or from the region previously occupied by the placenta and fetus in postpartum guinea pigs, were transplanted into the anterior eye chamber. After 3 wk in oculo, the pattern of reinnervation of the transplants was assessed on whole mount stretch preparations stained for tyrosine hydroxylase. The histology of the transplants was examined in toluidine blue-stained semithin sections. Myometrial transplants from virgin donors and uterine artery transplants from both virgin and postpartum donors became organotypically reinnervated by sympathetic fibres from the host iris. In contrast, sympathetic nerves did not reinnervate myometrial transplants from postpartum donors, although they approached the transplants and became distributed in the surrounding connective tissue. All transplanted tissues showed a normal histological appearance. Both the myometrium and uterine artery from postpartum donors retained a hypertrophic appearance after 3 wk in oculo. We interpret these results to indicate that the degeneration of sympathetic nerves in late pregnancy, as well as their slow regrowth to the uterus after delivery, may be due to changes in uterine smooth muscle rather than a particular sensitivity of short adrenergic neurons to hormonal changes.

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.

We have investigated the contribution of the acellular component (i.e., the extracellular matrix, or ECM) in determining the innervation density of peripheral target tissue, and the possible involvement of ECM molecules in age-related axonal atrophy. We addressed this issue using cerebral blood vessels where both the overall density and the density of sympathetic innervation decrease by 50% in aged rats. In order to study the capacity of the ECM to influence pattern and density of innervation, cerebral blood vessels from young (6 weeks) and old (24 months) rats were repeatedly freeze-thawed to kill the cellular component of the vessel wall before transplantation in oculo into young host rats. After 4 weeks, the nature and degree of reinnervation of transplants by host nerves was studied by quantitative immunohistochemistry using PGP9.5 (a general neuronal marker) or CGRP (calcitonin gene-related peptide, a sensory marker), or catecholamine histochemistry using the glyoxylic acid technique to visualise noradrenergic sympathetic nerves. The density of reinnervation was measured by computerized image analysis. The PGP-immunoreactive reinnervation on old transplanted cerebral blood vessels was significantly less dense compared to young ones. However, nerve density on both young and old transplants was lower than on control, ex vivo blood vessels. CGRP-positive nerves on both young and old transplants were sparse, but similar to control tissues, whereas catecholamine-containing nerves were very sparse on all transplants, and significantly less than on control blood vessels. These results suggest that alterations occur in the aged ECM, that make it less receptive to innervation. However, they also indicate that, along with the ECM, factors produced by the cellular components of the blood vessel wall are essential, at least for the maintenance of the sympathetic innervation.

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 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.

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 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.

The aim of this study was to investigate the possibility that inadequate venous return to the heart in diabetes is the result of a neuropathy which affects autonomic nerves supplying the splanchnic vasculature. Mesenteric veins from rats with streptozotocin-induced diabetes were markedly dilated in vivo compared to veins from control animals. Dilation appeared to be the result of loss of muscle tone rather than hypertrophy or hyperplasia of the vessel wall. Using quantification by image analysis and double-labeling immunohistochemistry on mesenteric veins, significant reductions in the density of nerve plexuses staining for 5-hydroxytryptamine (5-HT) and tyrosine hydroxylase (TH) were shown in vessels from diabetic rats compared to controls. No reductions were observed in the density of nerve plexuses stained for the neuronal marker, PGP 9.5, or for substance P (SP), a marker for afferent nerve fibers. These results indicate neurochemical deficits in experimentally induced diabetes which are specific to perivascular noradrenergic nerves and which, within the time-scale of our experiments, do not involve loss of nerve fibers. These deficits may contribute to an increase in venous pooling of blood in the splanchnic vasculature of diabetic rats and thus to inadequate venous return to the heart.

Nerve plexuses showing 5-hydroxytryptamine (5-HT)-like immunoreactivity (5-HTLI) have been demonstrated in all the major cerebral arteries of the rat using fluorescence immunohistochemistry on whole-mount stretch preparations. Following bilateral superior cervical ganglionectomy, nerves disappeared from all the vessels studied with the exception of the vertebral and basilar arteries where fibres remained. Using image analysis, the density of nerves containing 5-HTLI showed little change between vessels from 3 to 4-week-old and 6 to 7-week-old animals, in contrast to another study which has shown marked reductions in 5-HT-immunoreactive neuronal cell bodies in the superior cervical ganglion over the same period of early development. It was concluded that while the terminals of cerebrovascular nerves retain the capacity to synthesise and/or store 5-HT throughout life, their cell bodies may lose this function soon after birth.

Automated quantitative image analysis (QIAF) was used to measure and compare the adrenergic nerve plexuses of 4 blood vessels from the guinea pig, demonstrated by glyoxylic acid fluorescence (GAF). The results showed considerable quantitative variation of plexus density, size of bundles, and numbers of varicosities. A range of alternative procedural and anatomical sources of variability were investigated and assessed. The carotid artery was found to have a dense plexus with more nerves than that of the mesenteric artery; the mesenteric vein and abdominal aorta had sparse plexuses. The carotid artery plexus, despite the density of its nerves, possessed only half the number of varicosities of the mesenteric artery plexus. This sparse varicosity population was shown to have a similar density to the varicosities demonstrated by QIAF in the scattered nerves of the mesenteric vein and abdominal aorta. QIAF confirmed visual estimates of adrenergic plexus density, and was able to demonstrate less obvious differences of nerve density and size, and varicosity populations, between the different plexuses studied. The method is applicable to stretch preparations and transverse sections of many adrenergically innervated tissues.

The origin of serotonergic nerves supplying the large cerebral vessels of the gerbil has been investigated after bilateral superior cervical ganglionectomy. Immunohistochemical techniques were applied to whole-mount stretch preparations of the cerebral vessels. Removal of both ganglia resulted in the complete loss of immunoreactive fibres in the vessels supplied by the internal carotid artery and in a marked reduction of fibres innervating vessels of the vertebro-basilar system, indicating that most of the cerebrovascular serotonergic nerves have a peripheral sympathetic origin in the gerbil. The contrast with the central origin of serotonergic perivascular nerves claimed in the rat is discussed.

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. [Copyright &y& Elsevier]