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The brainstem is a neglected brain area in neurodegenerative diseases, including Alzheimer's and Parkinson's disease, frontotemporal lobar degeneration and autonomic dysfunction. In Depression, several observations have been made in relation to changes in one particular the Dorsal Raphe Nucleus (DRN) which also points toward as key area in various age-related and neurodevelopmental diseases. The DRN is further thought to be related to stress regulated processes and cognitive events. It is involved in neurodegeneration, e.g., amyloid plaques, neurofibrillary tangles, and impaired synaptic transmission in Alzheimer's disease as shown in our autopsy findings. The DRN is a phylogenetically old brain area, with projections that reach out to a large number of regions and nuclei of the central nervous system, particularly in the forebrain. These ascending projections contain multiple neurotransmitters. One of the main reasons for the past and current interest in the DRN is its involvement in depression, and its main transmitter serotonin. The DRN also points toward the increased importance and focus of the brainstem as key area in various age-related and neurodevelopmental diseases. This review describes the morphology, ascending projections and the complex neurotransmitter nature of the DRN, stressing its role as a key research target into the neural bases of depression., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Amyloid-β (Aβ) plaques are a prominent pathological hallmark of Alzheimer's disease (AD). They consist of aggregated Aβ peptides, which are generated through sequential proteolytic processing of the transmembrane protein amyloid precursor protein (APP) and several Aβ-associated factors. Efficient clearance of Aβ from the brain is thought to be important to prevent the development and progression of AD. The ubiquitin-proteasome system (UPS) is one of the major pathways for protein breakdown in cells and it has been suggested that impaired UPS-mediated removal of protein aggregates could play an important role in the pathogenesis of AD. To study the effects of an impaired UPS on Aβ pathology in vivo, transgenic APP Swe /PS1ΔE9 mice (APPPS1) were crossed with transgenic mice expressing mutant ubiquitin (UBB +1 ), a protein-based inhibitor of the UPS. Surprisingly, the APPPS1/UBB +1 crossbreed showed a remarkable decrease in Aβ plaque load during aging. Further analysis showed that UBB +1 expression transiently restored PS1-NTF expression and γ-secretase activity in APPPS1 mice. Concurrently, UBB +1 decreased levels of β-APP-CTF, which is a γ-secretase substrate. Although UBB +1 reduced Aβ pathology in APPPS1 mice, it did not improve the behavioral deficits in these animals., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of the present study was to assess alterations in DNA methylation and hydroxymethylation during aging in cerebellar Purkinje cells and to determine the effects of putatively preventative measures to such age-related changes. Using immunohistochemical techniques, 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) immunoreactivity in cerebellar Purkinje cells of 12-month- and 24-month-old mice was interrogated. Additionally, the modulatory effects of caloric restriction (CR) and normal human Cu/Zn super oxide dismutase 1 overexpression on these changes were assessed. We show that aging is associated with an increase of 5-mC and 5-hmC immunoreactivity in mouse cerebellar Purkinje cells. These age-related increases were mitigated by CR but not super oxide dismutase 1 overexpression. Additionally, the ratio between 5-mC and 5-hmC decreased with age and CR treatment, suggesting that CR has a stronger effect on DNA methylation than DNA hydroxymethylation. These findings enforce the notion that aging is closely connected to marked epigenetic changes, affecting multiple brain regions, and that CR is an effective means to prevent or counteract deleterious age-related epigenetic alterations., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is a complex neurodegenerative disorder involving dysregulation of many biological pathways at multiple levels. Classical epigenetic mechanisms, including DNA methylation and histone modifications, and regulation by microRNAs (miRNAs), are among the major regulatory elements that control these pathways at the molecular level, with epigenetic modifications regulating gene expression transcriptionally and miRNAs suppressing gene expression posttranscriptionally. Epigenetic mechanisms and miRNAs have recently been shown to closely interact with each other, thereby creating reciprocal regulatory circuits, which appear to be disrupted in neuronal and glial cells affected by AD. Here, we review those miRNAs implicated in AD that are regulated by promoter DNA methylation and/or chromatin modifications and, which frequently direct the expression of constituents of the epigenetic machinery, concluding with the delineation of a complex epigenetic-miRNA regulatory network and its alterations in AD., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Bifunctional drug therapy targeting distinct receptor signalling systems can generate increased efficacy at lower concentrations compared to monofunctional therapy. Non-competitive blockade of the NMDA receptors or the potentiation of AMPA receptors is well documented to result in memory enhancement. Here, we compared the efficacy of the low-affinity NMDA receptor blocker memantine or the positive modulator of AMPA receptor QXX (in C57BL/6J at 1 or 5mg/kg, ip) with new derivatives of isothiourea (0.5-1 mg/kg, ip) that have bifunctional efficacy. Low-affinity NMDA blockade by these derivatives was achieved by introducing greater flexibility into the molecule, and AMPA receptor stimulation was produced by a sulfamide-containing derivative of isothiourea. Contextual learning was examined in a step-down avoidance task and extinction of contextual memory was studied in a fear-conditioning paradigm. Memantine enhanced contextual learning while QXX facilitated memory extinction; both drugs were effective at 5 mg/kg. The new derivative IPAC-5 elevated memory scores in both tasks at the dose 0.5 mg/kg and exhibited the lowest IC₅₀ values of NMDA receptor blockade and highest potency of AMPA receptor stimulation. Thus, among the new drugs tested, IPAC-5 replicated the properties of memantine and QXX in one administration with increased potency. Our data suggest that a concomitant manipulation of NMDA- and AMPA-receptors results in pro-cognitive effects and supports the concept bifunctional drug therapy as a promising strategy to replace monofunctional therapies with greater efficacy and improved compliance., (Copyright © 2013 Elsevier B.V. and ECNP. All rights reserved.)

Epigenetic dysregulation of gene expression is thought to be critically involved in the pathophysiology of Alzheimer's disease (AD). Recent studies indicate that DNA methylation and DNA hydroxymethylation are 2 important epigenetic mechanisms that regulate gene expression in the aging brain. However, very little is known about the levels of markers of DNA methylation and hydroxymethylation in the brains of patients with AD, the cell-type specificity of putative AD-related alterations in these markers, as well as the link between epigenetic alterations and the gross pathology of AD. The present quantitative immunohistochemical study investigated the levels of the 2 most important markers of DNA methylation and hydroxymethylation, that is, 5-methylcytidine (5-mC) and 5-hydroxymethylcytidine (5-hmC), in the hippocampus of AD patients (n = 10) and compared these to non-demented, age-matched controls (n = 10). In addition, the levels of 5-hmC in the hippocampus of a pair of monozygotic twins discordant for AD were assessed. The levels of 5-mC and 5-hmC were furthermore analyzed in a cell-type and hippocampal subregion-specific manner, and were correlated with amyloid plaque load and neurofibrillary tangle load. The results showed robust decreases in the hippocampal levels of 5-mC and 5-hmC in AD patients (19.6% and 20.2%, respectively). Similar results were obtained for the twin with AD when compared to the non-demented co-twin. Moreover, levels of 5-mC as well as the levels of 5-hmC showed a significant negative correlation with amyloid plaque load in the hippocampus (r(p) = -0.539, p = 0.021 for 5-mC and r(p) = -0.558, p = 0.016 for 5-hmC). These human postmortem results thus strengthen the notion that AD is associated with alterations in DNA methylation and hydroxymethylation, and provide a basis for further epigenetic studies identifying the exact genetic loci with aberrant epigenetic signatures., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A chronic stress paradigm comprising exposure to predation, tail suspension and restraint induces a depressive syndrome in C57BL/6J mice that occurs in some, but not all, animals. Here, we sought to extend our behavioural studies to investigate how susceptibility (sucrose preference<65%) or resilience (sucrose preference>65%) to stress-induced anhedonia affects the 5HT system and the expression of inflammation-related genes. All chronically stressed animals, displayed increased level of anxiety, but susceptible mice exhibited an increased propensity to float in the forced swim test and demonstrate hyperactivity under stressful lighting conditions. These changes were not present in resilient or acutely stressed animals. Compared to resilient animals, susceptible mice showed elevated expression of tumour necrosis factor alpha (TNF) and the 5-HT transporter (SERT) in the pre-frontal area. Enhanced expression of 5HT(2A) and COX-1 in the pre-frontal area was observed in all stressed animals. In turn, indoleamine-2,3-dioxygenase (IDO) was significantly unregulated in the raphe of susceptible animals. At the cellular level, increased numbers of Iba-1-positive microglial cells were also present in the prefrontal area of susceptible animals compared to resilient animals. Consequently, the susceptible animals display a unique molecular profile when compared to resilient, but anxious, animals. Unexpectedly, this altered profile provides a rationale for exploring anti-inflammatory, and possibly, TNF-targeted therapy for major depression., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Deep brain stimulation (DBS) is currently being evaluated as a potential therapy in improving memory functions in Alzheimer's disease. The target for DBS and the stimulation parameters to be used are unknown. Here, we implanted bilateral electrodes in the vicinity of the fornix, a key element of the memory circuitry, and applied DBS with different stimulation frequencies and amplitudes in an experimental model of dementia. Rats received scopolamine, a muscarinic acetylcholine receptor antagonist, to mimic memory impairment. Rats were then tested in the object location task with the following conditions: (i) with attachment of stimulation cable (off stimulation), and (ii) with DBS at various amplitudes (50 μA, 100 μA and 200 μA), 100 μs pulse width and 100 Hz or 10 Hz stimulation frequency. DBS reversed the memory impairing effects of scopolamine when compared to sham rats. We found that the fornix is not sensitive to the frequency of stimulation, but rather to current levels. With the most optimal stimulation parameter, we found no side-effects on anxiety levels and general motor activity. These findings identify the fornix as a key region in controlling spatial memory functions. DBS of this region, using tailored stimulation parameters, has the potential to improve memory functions in conditions characterised by memory impairment., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Epidemiological evidence implies a role for chronic stress and stress-related disorders in the etiopathogenesis of sporadic Alzheimer's disease (AD). Although chronic stress exposure during various stages of life has been shown to exacerbate AD-related cognitive deficits and neuropathology in AD mouse models, the role of stress exposure during the prenatal period on AD development and progression remained to be investigated. The present study therefore explored the effects of prenatal maternal stress (PMS) in both male and female APPswe/PS1dE9 mouse offspring in terms of cognition, affect, and AD-related neuropathology. As prenatal perturbations are likely to mediate their effects via alterations in epigenetic regulation, changes in hippocampal DNA methyltransferase 3a, 5-methylcytosine and 5-hydroxymethylcytosine levels were assessed as underlying mechanisms. Repetitive restraint stress during the first week of gestation exerted a sex-dependent effect, with male PMS mice showing spatial memory deficits and a blunted hypothalamus-pituitary-adrenal axis response, while female PMS mice showed improved spatial memory performance, increased depressive-like behavior, as well as a decrease in hippocampal plaque load. In addition, sex differences were observed among APPswe/PS1dE9 mice, independent of PMS (i.e., female mice showed impaired spatial memory performance, higher hippocampal plaque load, altered amyloid precursor protein processing in the CA3 and lower DNA methyltransferase 3a immunoreactivity in the dentate gyrus when compared with male mice of the same age). In conclusion, PMS exposure impacts on the behavioral phenotype and neuropathology of APPswe/PS1dE9 mice. Moreover, given the remarkable sex differences observed, one should not overlook the impact of sex-specific responses to environmental exposures when investigating gene-environment interactions in AD., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aberrant DNA methylation patterns have been linked to molecular and cellular alterations in the aging brain. Caloric restriction (CR) and upregulation of antioxidants have been proposed as interventions to prevent or delay age-related brain pathology. Previously, we have shown in large cohorts of aging mice, that age-related increases in DNA methyltransferase 3a (Dnmt3a) immunoreactivity in the mouse hippocampus were attenuated by CR, but not by overexpression of superoxide dismutase 1 (SOD1). Here, we investigated age-related alterations of 5-methylcytidine (5-mC), a marker of DNA methylation levels, in a hippocampal subregion-specific manner. Examination of 5-mC immunoreactivity in 12- and 24-month-old wild type (WT) mice on control diet, mice overexpressing SOD1 on control diet, wild type mice on CR, and SOD1 mice on CR, indicated an age-related increase in 5-mC immunoreactivity in the hippocampal dentate gyrus, CA3, and CA1-2 regions, which was prevented by CR but not by SOD1 overexpression. Moreover, positive correlations between 5-mC and Dnmt3a immunoreactivity were observed in the CA3 and CA1-2. These findings suggest a crucial role for DNA methylation in hippocampal aging and in the mediation of the beneficial effects of CR on aging., (Copyright © 2012 Elsevier Inc. All rights reserved.)

To understand how microglial cell function may change with aging, various protocols have been developed to isolate microglia from the young and aged central nervous system (CNS). Here we report modification of an existing protocol that is marked by less debris contamination and improved yields and demonstrate that microglial functions are varied and dependent on age. Specifically, we found that microglia from aged mice constitutively secrete greater amounts of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) relative to microglia from younger mice and are less responsive to stimulation. Also, microglia from aged mice have reduced glutathione levels and internalize less amyloid beta peptide (Aβ) while microglia from mice of all ages do not retain the amyloid beta peptide for a significant length of time. These studies offer further support for the idea that microglial cell function changes with aging. They suggest that microglial Aβ phagocytosis results in Aβ redistribution rather than biophysical degradation in vivo and thereby provide mechanistic insight to the lack of amyloid burden elimination by parenchymal microglia in aged adults and those suffering from Alzheimer's disease., (Copyright © 2012. Published by Elsevier Inc.)

Prenatal stress (PS) has been associated with an increased incidence of numerous neuropsychiatric disorders, including depression, anxiety, schizophrenia, and autism. To determine the effects of PS on hippocampal-dependent behaviour hippocampal morphology, we examined behavioural responses and hippocampal cytoarchitecture of a maternal restraint stress paradigm of PS in C57BL6 mice. Female offspring only showed a reduction in hippocampal glial count in the pyramidal layer following PS. Additionally, only PS females showed increased depressive-like behaviour with cognitive deficits predominantly in female offspring when compared to males. This data provides evidence for functional female-specific glial deficits within the hippocampus as a consequence of PS., (Copyright © 2010 Elsevier B.V. and ECNP. All rights reserved.)

Acute tryptophan depletion (ATD) is extensively used to investigate the implication of serotonin (5-hydroxytryptamine; 5-HT) in the onset and treatment of depression and cognitive disorders. Brain-derived neurotrophic factor (BDNF) is strongly linked to the 5-HT system and plays an essential role in mood and memory processes. The present study investigated the effects of ATD upon BDNF in serum, hippocampus and prefrontal cortex in the rat to further explore the underlying mechanism of ATD. ATD significantly decreased peripheral tryptophan (TRP) levels and moderately interrupted 5-HT metabolism 4h after administration of the nutritional mixture. Although no direct effects of ATD upon serum or brain BDNF concentrations were found, a stress-mediated, decrease in BDNF was observed in the prefrontal cortex. Moreover, brain TRP levels correlated positively with BDNF in both the prefrontal cortex and hippocampus. Thus, BDNF-mediated mechanisms due to ATD and/or its application stress might underlie ATD-induced neurochemical and behavioural alterations.

According to a long-standing hypothesis, aging is mainly caused by accumulation of nuclear (n) DNA damage in differentiated cells such as neurons due to insufficient nDNA repair during lifetime. In line with this hypothesis it was until recently widely accepted that neuron loss is a general consequence of normal aging, explaining some degree of decline in brain function during aging. However, with the advent of more accurate procedures for counting neurons, it is currently widely accepted that there is widespread preservation of neuron numbers in the aging brain, and the changes that do occur are relatively specific to certain brain regions and types of neurons. Whether accumulation of nDNA damage and decline in nDNA repair is a general phenomenon in the aging brain or also shows cell-type specificity is, however, not known. It has not been possible to address this issue with the biochemical and molecular-biological methods available to study nDNA damage and nDNA repair. Rather, it was the introduction of autoradiographic methods to study quantitatively the relative amounts of nDNA damage (measured as nDNA single-strand breaks) and nDNA repair (measured as unscheduled DNA synthesis) on tissue sections that made it possible to address this question in a cell-type-specific manner under physiological conditions. The results of these studies revealed a formerly unknown inverse relationship between age-related accumulation of nDNA damage and age-related impairment in nDNA repair on the one hand, and the age-related, selective, loss of neurons on the other hand. This inverse relation may not only reflect a fundamental process of aging in the central nervous system but also provide the molecular basis for a new approach to understand the selective neuronal vulnerability in neurodegenerative diseases, particularly Alzheimer's disease.

The dorsal raphe nucleus (DRN) is a heterogeneous brainstem nucleus located in the midbrain and pons. Via widespread projections, which target a multitude of brain areas, its neurons utilize many transmitters to control various physiological functions, including learning, memory and affect. Accordingly, the DRN has been strongly associated with brain dysfunction, especially mood disorders such as depression, but also Alzheimer's disease. The DRN's most abundant transmitter, serotonin, has received the most attention in studies on both normal brain function and disease, and lately its involvement in the regulation of neuroplasticity has been under particular scrutiny. This chapter begins with a systematic overview of what we currently know about the anatomy of the DRN and its neurons, including their ascending projections. It continues with a review of the transmitters of the DRN, followed by a discussion on the connection between the DRN and neuroplasticity. Special emphasis is put on serotonin and its central role in neuroplasticity, which is proving to be of high priority in unraveling the full picture of the cellular mechanisms and their interconnections in the etiology of major depression and Alzheimer's disease.

Inspired by the suggested associations between neurological diseases and infections, we determined the susceptibility of brain cells to Chlamydia pneumoniae (Cpn). Murine astrocyte (C8D1A), neuronal (NB41A3) and microglial (BV-2) cell lines were inoculated with Cpn. Infection was established by immunofluorescence and real-time PCR at various time points. Productive infection was assessed by transferring medium of infected cells to a detection layer. Finally, apoptosis and necrosis post-infection was determined. Our data demonstrate that the neuronal cell line is highly sensitive to Cpn, produces viable progeny and is prone to die after infection by necrosis. Cpn tropism was similar in an astrocyte cell line, apart from the higher production of extracellular Cpn and less pronounced necrosis. In contrast, the microglial cell line is highly resistant to Cpn as the immunohistochemical signs almost completely disappeared after 24 h. Nevertheless, significant Cpn DNA amounts could be detected, suggesting Cpn persistence. Low viable progeny and hardly any necrotic microglial cells were observed. Further research is warranted to determine whether these cell types show the same sensitivity to Cpn in an in vivo setting.

Age-related molecular and cellular alterations in the central nervous system are known to show selectivity for certain cell types and brain regions. Among them age-related accumulation of nuclear (n) DNA damage can lead to irreversible loss of genetic information content. In the present study on the aging mouse brain, we observed a substantial increase in the amount of nDNA single-strand breaks in hippocampal pyramidal and granule cells as well as in cerebellar granule cells but not in cerebellar Purkinje cells. The reverse pattern was found for age-related reductions in total numbers of neurons. Only the total number of cerebellar Purkinje cells was significantly reduced during aging whereas the total numbers of hippocampal pyramidal and granule cells as well as of cerebellar granule cells were not. This formerly unknown inverse relation between age-related accumulation of nDNA damage and age-related loss of neurons may reflect a fundamental process of aging in the central nervous system.

We investigated the effects of a single course of antenatal betamethasone on neonatal somatic and brain development. On day 20 of gestation, pregnant rats were injected with either with 170 microg kg(-1) body weight of betamethasone ("clinically-equivalent dose," equivalent to 12 mg twice, 24 hours apart) or half this dose or vehicle. Pups (8-11 animals per experimental group per timepoint per gender) were analyzed at 1 (P1), 2, and 21 days after birth. We report that betamethasone induced a significant dose-dependent decrease of somatic measurements in both genders. At P1 cell proliferation was affected by the "clinically equivalent dose" only in the subventricular zone in both genders and in the hippocampus in males. In summary, we show for the first time that a lower dose (equivalent to 6 mg) induces fewer and less severe effects on somatic growth, whereas it does not affect cell proliferation within the brain.

In Parkinson's disease (PD), several neurotransmitter systems, such as the dopaminergic and serotonergic system, show signs of degeneration. This led to the suggestion that alterations in the serotonergic system play a role in the pathophysiology of PD. Partial bilateral dopaminergic lesions of the caudate putamen complex (CPu) of rats induced by 6-hydroxydopamine (6-OHDA) produce behavioral symptoms mimicking PD. In the present study, the role of serotonin and dopamine was investigated both behaviorally and neuroanatomically. In a reaction time task, motor initiation and motor performance were impaired in the lesioned animals compared to controls. The performance of rats treated with d-amphetamine or serotonergic ligands (DOI and ketanserin) in the reaction time task indicated that 5-HT and DA appear to be agonistically related in the CPu. The relation was the same in both control and 6-OHDA lesioned rats. 12 weeks after lesioning, motor initiation recovered, whereas motor performance did not. Parallel to the behavioral study, a second group of animals was lesioned and, at 3 days, 6 weeks and 12 weeks after lesioning, a subgroup was killed to obtain a qualitative indication of the degree of 6-OHDA lesion. Over the three time points, a substantial recovery of tyrosine hydroxylase staining in the CPu was visible. Taken together, since serotonergic ligands have the same effect as dopaminergic ligands on reaction time responding indicated that 5-HT and DA release are agonistically linked in control and 6-OHDA lesioned rats.

Neuron and synapse loss are important features of the neuropathology of Alzheimer's disease (AD). Recently, we observed substantial age-related hippocampal neuron loss in APP751SL/PS1M146L transgenic mice but not in PS1M146L mice. Here, we investigated APP751SL mice, PS1M146L mice, and APP751SL/PS1M146L mice for age-related alterations in synaptic integrity within hippocampal stratum moleculare of the dentate gyrus (SM), stratum lucidum of area CA3 (SL), and stratum radiatum of area CA1-2 (SR) by analyzing densities and numbers of synaptophysin-immunoreactive presynaptic boutons (SIPBs). Wild-type mice, APP751SL mice and PS1M146L mice showed similar amounts of age-related SIPB loss within SM, and no SIPB loss within SL. Both APP751SL mice and PS1M146L mice showed age-related SIPB loss within SR. Importantly, APP751SL/PS1M146L) mice displayed the severest age-related SIPB loss within SM, SL, and SR, even in regions free of extracellular Abeta deposits. Together, these mouse models offer a unique framework to study the impact of several molecular and cellular events caused by mutant APP and/or mutant PS1 on age-related alterations in synaptic integrity. The observation of age-related SIPB loss within SR of PS1M146L mice supports a role of mutant PS1 in neurodegeneration apart from its contribution to alterations in Abeta generation.

According to the "amyloid hypothesis of Alzheimer's disease," beta-amyloid is the primary driving force in Alzheimer's disease pathogenesis. Despite the development of many transgenic mouse lines developing abundant beta-amyloid-containing plaques in the brain, the actual link between amyloid plaques and neuron loss has not been clearly established, as reports on neuron loss in these models have remained controversial. We investigated transgenic mice expressing human mutant amyloid precursor protein APP751 (KM670/671NL and V717I) and human mutant presenilin-1 (PS-1 M146L). Stereologic and image analyses revealed substantial age-related neuron loss in the hippocampal pyramidal cell layer of APP/PS-1 double-transgenic mice. The loss of neurons was observed at sites of Abeta aggregation and surrounding astrocytes but, most importantly, was also clearly observed in areas of the parenchyma distant from plaques. These findings point to the potential involvement of more than one mechanism in hippocampal neuron loss in this APP/PS-1 double-transgenic mouse model of Alzheimer's disease.