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P R Mouton

Publications and source records attributed to P R Mouton.

At least 19 recordsLinked to original sources
PubMed2002

Stereological length estimation using spherical probes.

Lineal structures in biological tissue support a wide variety of physiological functions, including membrane stabilization, vascular perfusion, and cell-to-cell communication. In 1953, Smith and Guttman demonstrated a stereological method to estimate the total length density (Lv) of linear objects based on random intersections with a two-dimensional sampling probe. Several methods have been developed to ensure the required isotropy of object-probe intersections, including isotropic-uniform-random (IUR) sections, vertical-uniform-random (VUR) slices, and isotropic virtual planes. The disadvantages of these methods are the requirements for inconvenient section orientations (IUR, VUR) or complex counting rules at multiple focal planes (isotropic virtual planes). To overcome these limitations we report a convenient and straightforward approach to estimate Lv and total length, L, for linear objects on tissue sections cut at any arbitrary orientation. The approach presented here uses spherical probes that are inherently isotropic, combined with unbiased fractionator sampling, to demonstrate total L estimation for thin nerve fibres in dorsal hippocampus of the mouse brain.

Animals↗
PubMed2001

Estrogens modulate experimentally induced apoptosis of granule cells in the adult hippocampus.

Estrogens are known to have broad effects on neuronal plasticity, but their specific role in neuronal cell death has not been determined. In the present study, we investigated the effects of beta-estradiol on an experimental model of apoptosis of granule cells of the dentate gyrus, i.e., apoptosis induced by intraventricular injection of the microtubule polymerization inhibitor colchicine. Cell death was characterized with multiple methods, including TUNEL and DNA electrophoresis. Nonrandom digestion of DNA was observed within 8-10 hours after colchicine injection, followed by condensation and fragmentation of granule cell nuclei and extensive anterograde degeneration of mossy fibers/terminals in 2 days. We compared the outcomes of the above-described manipulation in ovariectomized or sham-operated rats and animals treated daily with beta-estradiol or vehicle. Animals were lesioned with colchicine or vehicle 2 weeks after ovariectomy or sham operation. Beta-estradiol or vehicle was administered for 1 week prior to lesion and was continued for a further 2 weeks. Total numbers and densities of granule cells in different animal groups were counted by stereology in various anteroposterior levels of the hippocampus. Our results show that ovariectomy intensifies colchicine-induced granule cell apoptosis, which is ameliorated by exogenous beta-estradiol. In doses that ameliorate the adverse effect of ovariectomy, exogenous beta-estradiol appears to have no effect of preventing granule cell death in animals with intact ovaries; i.e., an estrogen excess is not more neuroprotective than physiological levels of these hormones. Taken together, our results indicate that estrogen deprivation increases the vulnerability of hippocampal neurons to injury and may predispose to neurological diseases occurring after menopause.

Alzheimer Disease↗
PubMed2001

Length measurement: new developments in neurostereology and 3D imagery.

Quantification of linear biological structures has important applications in neuroscience; for example, the length of neurotransmitter-specific axonal innervation or length of dendritic processes within particular brain structures. Until recently, however, there have been practical limitations in the application of stereological tools for the unbiased estimation of object length on tissue sections. The recent development of efficient new approaches allows for the wider application of theoretically unbiased sampling and estimation techniques that are devoid of the assumptions and models of earlier methods. In this review, we outline the historical background and recent advances in the estimation of total length for biological objects on tissue sections, including a practical method to estimate the length of cholinergic fibers using newly developed methods. These newer methods also take advantage of three-dimensional image datasets and virtual probes, techniques that may have wider application in quantitative morphometry.

Acetylcholinesterase↗
PubMed2000

Length measurement: new developments in neurostereology and 3D imagery.

Quantification of linear biological structures has important applications in neuroscience; for example, the length of neurotransmitter-specific axonal innervation or length of dendritic processes within particular brain structures. Until recently, however, there have been practical limitations in the application of stereological tools for the unbiased estimation of object length on tissue sections. The recent development of efficient new approaches allows for the wider application of theoretically unbiased sampling and estimation techniques that are devoid of the assumptions and models of earlier methods. In this review, we outline the historical background and recent advances in the estimation of total length for biological objects on tissue sections, including a practical method to estimate the length of cholinergic fibers using newly developed methods. These newer methods also take advantage of three-dimensional image datasets and virtual probes, techniques that may have wider application in quantitative morphometry.

Algorithms↗
PubMed1999

What counts in brain aging? Design-based stereological analysis of cell number.

The advent and implementation of new design-based stereological techniques allows the quantification of cell number without the assumptions required when obtaining areal densities. These new techniques are rapidly becoming the standard for quantifying cell number, particularly in aging studies. Recently, studies using stereological techniques have failed to confirm earlier findings regarding age-associated neural loss. This newly emerging view of retained cell number during aging is having a major impact on biogerontology, prompting revaluation of long-standing hypotheses of age-related cell loss as causal for age-related impairments in brain functioning. Rather than focus on neuronal loss as the end-result of a negative cascade of neuronal injury, research has begun to consider that age-related behavioral declines may reflect neuronal dysfunction (e.g., synaptic or receptor loss, signal transduction deficits) instead of neuronal death. Here we discuss design-based stereology in the context of age-related change in brain cell number and its impact on consideration of structural change in brain aging. Emergence of this method of morphometrics, however, can have relevance to many areas of gerontological research.

Aging↗
PubMed1998

Stereological estimation of total microglia number in mouse hippocampus.

Microglia are brain cells of considerable interest because of their role in CNS inflammatory responses and strong association with neuritic plaques in Alzheimer's disease (AD). In the present study, immunocytochemistry was combined with unbiased stereology to estimate the mean total number of microglia in dentate gyrus and CA1 regions of the mouse hippocampus. Systematic-uniform-random (SUR) sections were cut through the hippocampal formation of male C57BL/6J mice (n = 7, 4-5 months) and immunostained with Mac-1, an antibody to the complement subunit 3 receptor (CR3). The total number of Mac-1 immunopositive cells was determined using the optical fractionator method. The mean total number of microglia in the mouse dentate gyrus was estimated to be 20,300 (CV = 0.21) with a mean coefficient of error (CE) = 0.09. The mean total number of microglia in the mouse CA1 was estimated to be 43,200 (CV = 0.24) with a CE = 0.11. Comparison of total number estimates, derived from fraction- or volume-based methods, supported stereological theory regarding the equivalence of the two techniques. The time required to determine total microglia number in both hippocampal sub-regions was approximately 6 h per mouse from stained sections. The combination of immunocytochemistry and stereology provides a reliable means to assess microglia number that can easily be adopted for studies of transgenic and lesion-based models of aging and neurodegenerative diseases.

Animals↗
PubMed1997

Deafferentation causes apoptosis in cortical sensory neurons in the adult rat.

The present study provides an experimental model of the apoptotic death of pyramidal neurons in rat olfactory cortex after total bulbectomy. Terminal transferase (TdT)-mediated deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL), DNA electrophoresis, and neuronal ultrastructure were used to provide evidence of apoptosis; neurons in olfactory cortex were counted by stereology. Maximal TUNEL staining occurred in the piriform cortex between 18 and 26 hr postbulbectomy. Within the survival times used in the present study (up to 48 hr postlesion), cell death was observed exclusively in the piriform cortex; there was no evidence of cell death in any other areas connected with the olfactory bulb. Neurons undergoing apoptosis were pyramidal cells receiving inputs from, but not projecting to, the olfactory bulb. The apical dendrites of these neurons were contacted by large numbers of degenerating axonal terminals. Gel electrophoresis of DNA purified from lesioned olfactory cortex showed a ladder pattern of fragmentation. Inflammatory cells or phagocytes were absent in the environment of degenerating neurons in the early stages of the apoptotic process. The present model suggests that deafferentation injury in sensory systems can cause apoptosis. In addition, olfactory bulbectomy can be used for investigating molecular mechanisms that underlie apoptosis in mature mammalian cortical neurons and for evaluating strategies to prevent the degeneration of cortical neurons.

Afferent Pathways↗
PubMed1997

Empirical assessment of synapse numbers in primate neocortex.

Reliable methods are needed to assess the impact of synaptic loss on brain function. In this empirical study we demonstrate a novel and efficient method using immunocytochemistry (ICC) and modern stereological techniques to quantify synapses in neocortex of adult primates (Macaca fascicularis). Systematic-uniform-random sections through forebrain from two 10-year-old monkeys were immunostained for estimation of synaptophysin-immunoreactive (synaptophysin-IR) presynaptic boutons (synapses). Adjacent sections were stained with cresyl violet for estimation of total number of neuronal cell bodies. The unbiased Cavalieri method was used to estimate total forebrain and neocortical volumes to a high level of precision (coefficient of error (CE) < or = 0.10)). Synapse-to-neuron ratios varied from 860 in frontal cortex to 2300 in parietal-temporal cortex. The combination of Cavalieri and optical disector methods provided a direct means of estimating approximately 1.25 trillion (x 10(12)) total synaptophysin-immunopositive boutons and approximately 1.01 billion (x 10(9)) cell bodies in neocortex, with low CEs (0.12). Time required to make precise estimates of total neocortical and forebrain volumes and total numbers of synapses and neurons in neocortex was approximately 2-3 h per case from stained sections. The approach is a direct and efficient technique to quantify total synapse and neuron numbers within a defined brain structure.

Animals↗
PubMed1996

An unbiased method for estimation of total epidermal nerve fibre length.

It is of interest to quantify accurately lineal biological features such as nerve fibres, capillaries, and tubules for studies of development and diseases such as sensory neuropathy, and for evaluation of therapeutic regimens in humans and animal models. An unbiased stereological method to sample and estimate total length of immunostained epidermal nerve fibres by using vertical sections of punch skin biopsies from two human volunteers is presented. The essential steps in the procedure are as follows: (1) serially section the skin punch in a random plane perpendicular to the cutaneous surface; (2) immunostain a known fraction of total sections with antibody specific for nerve fibres; (3) orient a test line-grid over the epidermis; and (4) count intersections between test lines and immunostained epidermal nerve fibres. The optical fractionator method is employed to estimate total length of immunostained epidermal nerve fibres in the biopsy. By using these techniques the total length of nerve fibre in a defined region can be determined without methodological bias, assumptions or correction factors.

Adult↗
PubMed1996

Comparative evaluation of synaptophysin-based methods for quantification of synapses.

Development, ageing, and a variety of neurological disorders are characterized by selective alterations in specific populations of nerve cells which are, in turn, associated with changes in the numbers of synapses in the target fields of these neurons. To begin to delineate the significance of changes in synapses in development, ageing, and disease, it is first essential to quantify the number of synapses in defined regions of the CNS. In the past, investigators have used EM methods to assess synapse numbers or density, but these approaches are costly, labour intensive, and technically difficult, particularly in autopsy material. To begin to define reliable strategies useful for studies of both animals and humans, we used three techniques to measure synaptophysin-immunoreactivity in rat brain. The levels of synaptophysin protein were determined by Western blots of five hippocampal subregions; the intensity of synaptophysin-immunoreactivity in dentate gyrus and stratum oriens was determined by optical densitometry of immunocytochemically stained sections; and the total number of synaptophysin-immunoreactivity presynaptic boutons in dentate gyrus and stratum oriens was assessed by unbiased stereology. Each approach has advantages and disadvantages. Western blotting is the least time-consuming of the three methods and allows simultaneous processing of multiple samples. In systematically sampled histological sections, both densitometry and stereology allow precise definition of the region of interest, and the stereological optical dissector method allows quantitation of the numbers of synaptophysin-immunoreactive boutons. Stereology was the only method that clearly demonstrated greater synaptophysin-immunoreactivity in the dentate gyrus as compared to stratum oriens. The use of systematic sampling and the dissector technique offer a high degree of anatomical resolution (lacking in Western blot methods) and has quantitative advantage over the greyscale-based density approach. Thus, at present, stereology is the most useful method for estimating synaptic numbers in defined regions of the brain.

Animals↗
PubMed1994

Absolute number and size of pigmented locus coeruleus neurons in young and aged individuals.

Significant loss of noradrenergic neurons of the locus coeruleus in aging and Alzheimer's disease has been reported. The interpretation of these analyses, however, is problematic because of the model- and assumption-based nature of conventional sampling and estimation techniques. In the present study, unbiased stereological methods were used to estimate the total number and mean cell volume of pigmented neurons of the locus coeruleus in the brains of young and aged nondemented persons. No side-to-side differences are seen, and there is no change in pigmented cell number or size in the locus coeruleus of nondemented older persons as compared with that of young individuals. In light of previous studies that show severe locus coeruleus cell loss in Alzheimer's disease, these data support further critical investigations into the possible protective role of noradrenaline in normal cognitive functions and emphasize the importance of avoiding methodological bias in quantitative neuroanatomical studies.

Adult↗
PubMed1993

Nerve growth factor increases the size of intracortical cholinergic transplants.

The effects of continuous intracortical mouse Nerve Growth Factor on fetal rat basal forebrain transplants in denervated adult rat neocortex were investigated. Enzyme-linked immunoassay (ELISA) was used to measure the time course of endogenous NGF protein production in neocortex, hippocampus, and basal forebrain in a cohort of animals receiving unilateral ibotenic acid (IBO) lesions of the nucleus basalis magnocellularis (nBM). A second cohort of IBO-nBM lesioned animals received transplants of fetal basal forebrain followed by two to four weeks of continuous NGF or cytochrome-C infusion into the ipsilateral frontoparietal neocortex. To study the effects of abnormally high NGF doses on transplanted and host tissue, the cumulative dose of intracortical NGF was on the order of micrograms, compared with maximum picogram levels of neocortical NGF produced following IBO-nBM lesions. A four-fold increase in transplant size, and greater cell and fiber densities were observed in NGF-treated compared with NGF-untreated transplants. No adverse histological effects of long-term, high-dose NGF treatment were observed on transplanted basal forebrain or host neocortical tissue. These data indicate that cholinergic-rich mammalian brain tissue and intrinsic host tissue can be stimulated by high doses exogenous NGF without obvious deleterious effects.

Acetylcholinesterase↗
PubMed1990

Atrophy of cholinergic neurons within the rat nucleus basalis magnocellularis following intracortical AF64A infusion.

The rat nucleus basalis magnocellularis (nBM) was morphometrically analyzed following multiple intracortical AF64A infusions. At 3 weeks post-infusion, brains were histochemically double-stained for acetyl-cholinesterase and Nissl substance following diisopropylfluorophosphate pretreatment. Intracortical AF64A induced significant atrophy, but not degeneration, of nucleus basalis cholinergic cell bodies. These results suggest that retrograde cellular atrophy is associated with inhibition of presynaptic high-affinity choline transport on cortical terminals of nBM cholinergic neurons.

Acetylcholinesterase↗
PubMed1989

Intracortical AF64A: memory impairments and recovery from cholinergic hypofunction.

The long-term effects of intracortical AF64A (ethylcholine mustard aziridinium ion) treatment on presynaptic cortical cholinergic markers and cognitive function in the rat were investigated. Two 1.0 microliter infusions of AF64A (1 nmole/microliter) or vehicle were placed bilaterally into the fronto-parietal cortex. At 3 weeks postinfusion, AF64A-treated animals were found to be deficient in passive avoidance memory retention. During weeks 4 through 10, AF64A-treated animals were markedly deficient in the extinction (memory) phase, but not the acquisition (learning) phase of 2-way active avoidance behavior. Cortical acetylcholine synthesis and high-affinity choline uptake were significantly decreased in AF64A-treated animals at 24 hours, 3 weeks, and 10 weeks following infusion. At 6 months after AF64A treatment, however, cortical cholinergic markers were not reduced compared to controls. These data indicate that memory deficits are observed during a period of cortical cholinergic hypofunction induced by cortical AF64A infusions and that a recovery from such hypofunction occurs by 6 months after these infusions.

Acetylcholine↗
PubMed1988

Induction of cortical cholinergic hypofunction and memory retention deficits through intracortical AF64A infusions.

Ethylcholine mustard aziridinium ion (AF64A), an irreversible inhibitor of high-affinity choline uptake on cholinergic nerve terminals, appears to selectively decrease presynaptic cholinergic markers after intracerebral injection. To restrict AF64A's action to cholinergic terminals within the frontoparietal (FP) cortex, the present study utilized multiple-site cortical infusions of the agent. Following an extensive histological analysis, a dose of 1 nmol AF64A/1 microliter was selected for determining AF64A's effects on acetylcholinesterase (AChE) staining, cortical cholinergic/non-cholinergic markers, and passive avoidance behavior. Adult rats given two infusions of AF64A into the right FP cortex had reduced AChE staining throughout 75% of the ipsilateral FP cortex at 10 days following infusion, thus suggesting an extensive cortical diffusion of the agent; minimal non-specific damage was seen (totalling only 4% of the ipsilateral FP cortex for both infusion sites) and no effects on AChE staining were observed in the striatum or hippocampus. Three weeks after bilateral AF64A infusions into the FP cortex (two injections on each side), significant frontal cortex deficits were observed in high-affinity choline uptake, acetylcholine synthesis, acetylcholine release, and hemicholinium-3 binding compared to vehicle-infused controls. However, choline acetyltransferase activity within the anterior cortex did not appear to be consistently affected by AF64A infusion. Cortical glutamic acid decarboxylase activity, as well as cortical monoaminergic markers, and neuropeptide levels were also unaffected. Moreover, animals that received bilateral AF64A infusions and were tested two weeks afterwards showed marked memory retention deficits during both the 24-h and 48-h postshock trials of passive avoidance testing. These results indicate that cortical AF64A infusion induces a specific, long-term cholinergic hypofunction of presynaptic markers within the cortex, resulting in a significant long-term memory impairment. Since the primary cholinergic innervation to the FP cortex, originating in the nucleus basalis of Meynert, appears to become dysfunctional (but not totally degenerative) in Alzheimer's disease, cortical AF64A infusions may closely reflect this cholinergic dysfunction by 'functionally' eliminating cortical cholinergic terminals.

Acetylcholine↗
PubMedDate not supplied

Cognitive decline strongly correlates with cortical atrophy in Alzheimer's dementia.

Alzheimer's disease (AD) is characterized by progressive dementia and distinct neuropathology at autopsy. In order to test the relationship between dementia severity and loss of brain volumes, we prospectively documented the neurological/medical health of 26 male and 26 female controls and AD cases, and evaluated a subset of controls and AD cases using the Mini Mental State Examination (MMSE). At autopsy, Consortium to Establish a Registry for Alzheimer's Disease (CERAD) criteria confirmed diagnoses in 33 AD cases and 19 controls, and using unbiased stereology we quantified total volumes of cortical gray matter, subcortical grey matter including white matter, and forebrain. For ages of death between 50 to 100 years, controls showed minor cortical atrophy in the absence of cognitive decline. Cortical atrophy in AD cases was 20 to 25% greater than that in controls; AD patients dying at older ages showed less severe cortical atrophy than those dying at younger ages. Across all AD cases there was a strong correlation between cognitive performance on the Mini Mental State Examination and cortical volume loss. These findings confirm fundamental differences in the temporal patterns of cortical volume loss in aging and AD, and support cortical degeneration as the primary basis for cognitive decline in AD.

Aged↗
PubMedDate not supplied

Stereological analysis of astrocyte and microglia in aging mouse hippocampus.

Recent evidence suggests neuroglia-mediated inflammatory mechanisms may stimulate neurodegenerative processes in mammalian brain during aging. To test the hypothesis that the number of microglia and astrocytes increase in the hippocampus during normal aging, unbiased stereological techniques were used to estimate total cell number in hippocampal subregions (CA1, dentate gyrus and hilus) of male C57BL/6J mice of different ages: 4-5 months, 13-14 months and 27-28 months. Immunocytochemical visualization for microglia and astrocytes were via Mac-1 and GFAP antibody, respectively. Estimates of total microglia and astrocyte number were assessed using the optical fractionator. No statistically significant age differences were found in the numbers of microglia or astrocytes in the hippocampal regions sampled. These findings suggest that age-related increases in the total numbers of hippocampal microglia and astrocytes is not causal for observed age-related increases in cytokine response.

Aging↗