T. Cowen

Pontamine sky blue: A counterstain for background autofluorescence in fluorescence and immunofluorescence histochemistry

SummaryThe stain pontamine sky blue (PSB) has been shown to reduce background autofluorescence in catecholamine fluorescence and immunofluorescence histochemical preparations. Using PSB as a counterstain on whole-mount stretch preparations of human mesenteric blood vessels, a medium dense noradrenergic nerve plexus is clearly revealed, which previously had been only partially visible because of background autofluorescence. Image analysis of nerve densities in whole-mount stretch preparations of guinea-pig arteries containing noradrenergic, substance P-, and vasoactive intestinal polypeptide (VIP)-positive nerve plexuses shows that PSB staining does not alter the specific neuronal fluorescence and that it improves image definition.

Restricted diet rescues rat enteric motor neurones from age related cell death

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 effects of age on the overall population and on subpopulations of myenteric neurons in the rat small intestine

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.

Perivascular noradrenergic and peptide-containing nerves show different patterns of changes during development and ageing in the guinea-pig

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.

Development and ageing of perivascular adrenergic nerves in the rabbit. A quantitative fluorescence histochemical study using image analysis

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.

Neurotoxic and neurotrophic roles of proNGF and the receptor sortilin in the adult and ageing nervous system

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.

5-HT-containing nerves to major cerebral arteries of the gerbil originate in the superior cervical ganglia

This study has shown that over 95% of the 5-hydroxytryptamine (5-HT)-containing nerves to major cerebral arteries in the gerbil share a common origin with noradrenergic cerebrovascular nerves in the superior cervical ganglia. A small group of 5-HT-containing and noradrenergic nerves to the vessels of the posterior brain circulation had a different origin, which may be either central or peripheral. The pial blood vessels of the gerbil appeared to have no 5-HT-containing nerve supply. The effects of unilateral superior cervical ganglionectomy on 5-HT-containing and noradrenergic nerves to the arteries of the circle of Willis were different. Our results showed total loss of 5-HT-containing nerves on the vessels ipsilateral to the ganglionectomy combined with ca. 50% reductions of nerve density on the vessels of the contralateral side. There were no contralateral reductions of noradrenergic nerve density in parallel experiments although again the ipsilateral side was totally denervated. We suggest that 5-HT-containing cerebrovascular nerves are differently distributed as well as being in some way more sensitive to nerve damage compared to noradrenergic cerebrovascular nerves. The relationship between a combined serotonergic and noradrenergic vasoconstrictor control of large cerebral blood vessels and serotonergic vasodilation of small pial blood vessels is discussed.

Target-specific differences in the dendritic morphology and neuropeptide content of neurons in the rat SCG during development and aging

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.

Regulation of Rat Sympathetic Nerve Density by Target Tissues and NGF in Maturity and Old Age

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 ‐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 ‐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.

ProNGF, Sortilin, and Age‐related Neurodegeneration

Abstract:  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 β‐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 Alzheimers 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.