Cerebral amyloid induces aberrant axonal sprouting and ectopic terminal formation in amyloid precursor protein transgenic mice.

A characteristic feature of Alzheimer's disease (AD) is the formation of amyloid plaques in the brain. Although this hallmark pathology has been well described, the biological effects of plaques are poorly understood. To study the effect of amyloid plaques on axons and neuronal connectivity, we have examined the axonal projections from the entorhinal cortex in aged amyloid precursor protein (APP) transgenic mice that exhibit cerebral amyloid deposition in plaques and vessels (APP23 mice). Here we report that entorhinal axons form dystrophic boutons around amyloid plaques in the entorhinal termination zone of the hippocampus. More importantly, entorhinal boutons were found associated with amyloid in ectopic locations within the hippocampus, the thalamus, white matter tracts, as well as surrounding vascular amyloid. Many of these ectopic entorhinal boutons were immunopositive for the growth-associated protein GAP-43 and showed light and electron microscopic characteristics of axonal terminals. Our findings suggest that (1) cerebral amyloid deposition has neurotropic effects and is the main cause of aberrant sprouting in AD brain; (2) the magnitude and significance of sprouting in AD have been underestimated; and (3) cerebral amyloid leads to the disruption of neuronal connectivity which, in turn, may significantly contribute to AD dementia.

Spontaneous hemorrhagic stroke in a mouse model of cerebral amyloid angiopathy.

A high risk factor for spontaneous and often fatal lobar hemorrhage is cerebral amyloid angiopathy (CAA). We now report that CAA in an amyloid precursor protein transgenic mouse model (APP23 mice) leads to a loss of vascular smooth muscle cells, aneurysmal vasodilatation, and in rare cases, vessel obliteration and severe vasculitis. This weakening of the vessel wall is followed by rupture and bleedings that range from multiple, recurrent microhemorrhages to large hematomas. Our results demonstrate that, in APP transgenic mice, the extracellular deposition of neuron-derived beta-amyloid in the vessel wall is the cause of vessel wall disruption, which eventually leads to parenchymal hemorrhage. This first mouse model of CAA-associated hemorrhagic stroke will now allow development of diagnostic and therapeutic strategies.

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.

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.

Hippocampal neuron and synaptophysin-positive bouton number in aging C57BL/6 mice.

A loss of hippocampal neurons and synapses had been considered a hallmark of normal aging and, furthermore, to be a substrate of age-related learning and memory deficits. Recent stereological studies in humans have shown that only a relatively minor neuron loss occurs with aging and that this loss is restricted to specific brain regions, including hippocampal subregions. Here, we investigate these age-related changes in C57BL/6J mice, one of the most commonly used laboratory mouse strains. Twenty-five mice (groups at 2, 14, and 28-31 months of age) were assessed for Morris water-maze performance, and modern stereological techniques were used to estimate total neuron and synaptophysin-positive bouton number in hippocampal subregions at the light microscopic level. Results revealed that performance in the water maze was largely maintained with aging. No age-related decline was observed in number of dentate gyrus granule cells or CA1 pyramidal cells. In addition, no age-related change in number of synaptophysin-positive boutons was observed in the molecular layer of the dentate gyrus or CA1 region of hippocampus. We observed a significant correlation between dentate gyrus synaptophysin-positive bouton number and water-maze performance. These results demonstrate that C57BL/6J mice do not exhibit major age-related deficits in spatial learning or hippocampal structure, providing a baseline for further study of mouse brain aging.

Activity-regulated cytoskeletal-associated protein is localized to recently activated excitatory synapses.

Activity-regulated, cytoskeletal-associated protein (Arc) is an immediate early gene induced in excitatory circuits following behavioral episodes. Arc mRNA is targeted to activated regions of the dendrite after long-term potentiation (LTP) of the dentate gyrus, a process dependent on NMDA receptor activation. We used post-embedding immunogold electron microscopy (EM) to test whether synaptic Arc expression patterns are selectively modified by plasticity. Consistent with previous light microscopic observations, Arc protein was rapidly induced in the dentate gyrus following LTP-producing stimulation of the perforant path and was detectable in granule cell nuclei, somata and dendrites after two hours of high frequency stimulation. Post-embedding EM revealed Arc immunogold labeling in three times as many spines in the middle molecular layer of the stimulated dentate gyrus than in either the ipsilateral outer molecular layer or the contralateral middle and outer molecular layers. This upregulation did not occur with low frequency stimulation of the perforant path. Therefore Arc protein localization may be a powerful tool to isolate recently activated dendritic spines.

No hippocampal neuron or synaptic bouton loss in learning-impaired aged beta-amyloid precursor protein-null mice.

Aged beta-amyloid precursor protein-null mice were used to investigate the relationship between beta-amyloid precursor protein, hippocampal neuron and synaptic bouton number, and cognitive function. Learning and memory performance of aged beta-amyloid precursor protein-null mice and age-matched controls were assessed in the Morris water maze. Beta-amyloid precursor protein-null mice demonstrated impaired task acquisition as measured by significantly longer swim path lengths, a higher percentage of failed trials, and more frequent thigmotaxis behavior than controls. In a subsequent probe trial, beta-amyloid precursor protein-null mice spent significantly less time in the old goal quadrant, and made fewer crossings over the old platform location than did controls. No differences in motor or visual skills were observed which could account for the performance differences. In light of these findings and previous evidence for a role of beta-amyloid precursor protein in neuronal maintenance and synaptogenesis, we pursued the hypothesis that the learning impairment of beta-amyloid precursor protein-null mice may be a reflection of differences in neuron or synaptophysin-positive presynaptic bouton number. Thus, unbiased stereological analysis was used to estimate neuron and synaptic bouton number in dentate gyrus and hippocampal CA1 of the behaviorally characterized mice. No difference in neuron or synaptophysin-positive presynaptic bouton number was found between the beta-amyloid precursor protein-null mice and age-matched controls. Our results suggest that the learning impairment of beta-amyloid precursor protein-null mice is not mediated by a loss of hippocampal neurons or synaptic boutons.

Structural brain aging in inbred mice: potential for genetic linkage.

To identify genetic factors involved in brain aging, we have initiated studies assessing behavioral and structural changes with aging among inbred mouse strains. Cognitive performance of C57BL/6J mice is largely maintained with aging, and stereological analysis revealed no significant age-related change in neuron number, synaptic bouton number or glial number in the hippocampus. Moreover, no change in cortical neuron number and cholinergic basal forebrain neuron number has been found in this strain. 129Sv/J mice have more pronounced age-related cognitive deficits, although hippocampal and basal cholinergic forebrain neuron number also appear unchanged with aging. Differences in neurogenesis and neuron vulnerability in the adult CNS of C57BL/6, 129/Sv and other inbred strains have been reported, which in turn may have important consequences for brain aging. Age-related lesions, such as thalamic eosinophilic inclusions and hippocampal clusters of polyglucosan bodies also vary greatly among inbred strains although the functional significance of these lesions is not clear. The continued assessment of such age-related structural and behavioral changes among inbred mouse strains offers the potential to identify genes that control age-related changes in brain structure and function.

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.

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.

Mechanisms of cerebrovascular amyloid deposition. Lessons from mouse models.

Cerebrovascular deposition of amyloid is a frequent observation in Alzheimer's disease patients. It can also be detected sporadically in normal aged individuals and is further found in familial diseases linked to specific gene mutations. The source and mechanism of this pathology are still unknown. It has been suggested that amyloidogenic proteins are derived from blood, the vessel wall itself, or from the central nervous system. In this article evidence is reviewed for and against each of these hypotheses, including new data obtained from transgenic mouse models. In APP23 transgenic mice that develop cerebral amyloid angiopathy (CAA) in addition to amyloid plaques, the transport and drainage of neuronally produced amyloid-beta (A beta) seem to be responsible for CAA rather than vascular A beta production or blood uptake. Although a number of mechanisms may contribute to CAA in humans, these results suggest that a neuronal source of A beta is sufficient to induce vascular amyloid deposition. The possibility to cross genetically defined mouse models of CAA with other mutant mice now has the potential to identify molecular mechanisms of CAA.

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.

Region and task-specific activation of Arc in primary motor cortex of rats following motor skill learning.

Motor learning requires protein synthesis within the primary motor cortex (M1). Here, we show that the immediate early gene Arc/Arg3.1 is specifically induced in M1 by learning a motor skill. Arc mRNA was quantified using a fluorescent in situ hybridization assay in adult Long-Evans rats learning a skilled reaching task (SRT), in rats performing reaching-like forelimb movement without learning (ACT) and in rats that were trained in the operant but not the motor elements of the task (controls). Apart from M1, Arc expression was assessed within the rostral motor area (RMA), primary somatosensory cortex (S1), striatum (ST) and cerebellum. In SRT animals, Arc mRNA levels in M1 contralateral to the trained limb were 31% higher than ipsilateral (p<0.001), 31% higher than in the contralateral M1 of ACT animals (p<0.001) and 48% higher than in controls (p<0.001). Arc mRNA expression in SRT was positively correlated with learning success between two sessions (r=0.52; p=0.026). For RMA, S1, ST or cerebellum no significant differences in Arc mRNA expression were found between hemispheres or across behaviors. As Arc expression has been related to different forms of cellular plasticity, these findings suggest a link between M1 Arc expression and motor skill learning in rats.

Stereological analysis of the reorganization of the dentate gyrus following entorhinal cortex lesion in mice.

Denervation of the dentate gyrus by entorhinal cortex lesion has been widely used to study the reorganization of neuronal circuits following central nervous system lesion. Expansion of the non-denervated inner molecular layer (commissural/associational zone) of the dentate gyrus and increased acetylcholinesterase-positive fibre density in the denervated outer molecular layer have commonly been regarded as markers for sprouting following entorhinal cortex lesion. However, because this lesion extensively denervates the outer molecular layer and causes tissue shrinkage, stereological analysis is required for an accurate evaluation of sprouting. To this end we have performed unilateral entorhinal cortex lesions in adult C57BL/6J mice and have assessed atrophy and sprouting in the dentate gyrus using modern unbiased stereological techniques. Results revealed the expected increases in commissural/associational zone width and density of acetylcholinesterase-positive fibres on single brain sections. Yet, stereological analysis failed to demonstrate concomitant increases in layer volume or total acetylcholinesterase-positive fibre length. Interestingly, calretinin-positive fibres did grow beyond the border of the commissural/associational zone into the denervated layer and were regarded as sprouting axons. Thus, our data suggest that in C57BL/6J mice shrinkage of the hippocampus rather than growth of fibres underlies the two morphological phenomena most often cited as evidence of regenerative sprouting following entorhinal cortex lesion. Moreover, our data suggest that regenerative axonal sprouting in the mouse dentate gyrus following entorhinal cortex lesion may be best assessed at the single-fibre level.

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.

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.

Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology.

Mutations in the amyloid precursor protein (APP) gene cause early-onset familial Alzheimer disease (AD) by affecting the formation of the amyloid beta (A beta) peptide, the major constituent of AD plaques. We expressed human APP751 containing these mutations in the brains of transgenic mice. Two transgenic mouse lines develop pathological features reminiscent of AD. The degree of pathology depends on expression levels and specific mutations. A 2-fold overexpression of human APP with the Swedish double mutation at positions 670/671 combined with the V717I mutation causes A beta deposition in neocortex and hippocampus of 18-month-old transgenic mice. The deposits are mostly of the diffuse type; however, some congophilic plaques can be detected. In mice with 7-fold overexpression of human APP harboring the Swedish mutation alone, typical plaques appear at 6 months, which increase with age and are Congo Red-positive at first detection. These congophilic plaques are accompanied by neuritic changes and dystrophic cholinergic fibers. Furthermore, inflammatory processes indicated by a massive glial reaction are apparent. Most notably, plaques are immunoreactive for hyperphosphorylated tau, reminiscent of early tau pathology. The immunoreactivity is exclusively found in congophilic senile plaques of both lines. In the higher expressing line, elevated tau phosphorylation can be demonstrated biochemically in 6-month-old animals and increases with age. These mice resemble major features of AD pathology and suggest a central role of A beta in the pathogenesis of the disease.

Neuronal overexpression of mutant amyloid precursor protein results in prominent deposition of cerebrovascular amyloid.

Transgenic mice that overexpress mutant human amyloid precursor protein (APP) exhibit one hallmark of Alzheimer's disease pathology, namely the extracellular deposition of amyloid plaques. Here, we describe significant deposition of amyloid beta (Abeta) in the cerebral vasculature [cerebral amyloid angiopathy (CAA)] in aging APP23 mice that had striking similarities to that observed in human aging and Alzheimer's disease. Amyloid deposition occurred preferentially in arterioles and capillaries and within individual vessels showed a wide heterogeneity (ranging from a thin ring of amyloid in the vessel wall to large plaque-like extrusions into the neuropil). CAA was associated with local neuron loss, synaptic abnormalities, microglial activation, and microhemorrhage. Although several factors may contribute to CAA in humans, the neuronal origin of transgenic APP, high levels of Abeta in cerebrospinal fluid, and regional localization of CAA in APP23 mice suggest transport and drainage pathways rather than local production or blood uptake of Abeta as a primary mechanism underlying cerebrovascular amyloid formation. APP23 mice on an App-null background developed a similar degree of both plaques and CAA, providing further evidence that a neuronal source of APP/Abeta is sufficient to induce cerebrovascular amyloid and associated neurodegeneration.