Microglia cells are essential for brain homeostasis and have essential roles in neurodegenerative diseases. Aging is the main risk factor for most neurodegenerative diseases, and age-related changes in microglia may contribute to the susceptibility of the aging brain to dysfunction and neurodegeneration. We have analyzed morphology and dynamic behavior of neocortical microglia in their physiological environment in young adult (3-month-old), adult (11- to 12-month-old), and aged (26- to 27-month-old) C57BL/6J-Iba1-eGFP mice using in vivo 2-photon microscopy. Results show that surveying microglial cells in the neocortex exhibit age-related soma volume increase, shortening of processes, and loss of homogeneous tissue distribution. Furthermore, microglial process speed significantly decreased with age. While only a small population of microglia showed soma movement in adult mice, the microglia population with soma movement was increased in aged mice. However, in response to tissue injury, the dynamic microglial response was age-dependently diminished. These results provide novel insights into microglial behavior and indicate that microglial dysfunction in the aging brain may contribute to age-related cognitive decline and neurodegenerative diseases.

Microglia are morphologically dynamic cells that rapidly extend their processes in response to various stimuli including extracellular ATP. In this study, we tested the hypothesis that stimulation of neuronal NMDARs trigger ATP release leading to communication with microglia. We used acute mouse hippocampal brain slices and two-photon laser scanning microscopy to study microglial dynamics and developed a novel protocol for fixation and immunolabeling of microglia processes. Similar to direct topical ATP application in vivo, short multiple applications of NMDA triggered transient microglia process outgrowth that was reversible and repeatable indicating that this was not due to excitotoxic damage. Stimulation of NMDAR was required as NMDAR antagonists, but not blockers of AMPA/kainate receptors or voltage-gated sodium channels, prevented microglial outgrowth. We report that ATP release, secondary to NMDAR activation, was the key mediator of this neuron-microglia communication as both blocking purinergic receptors and inhibiting hydrolysis of ATP to prevent locally generated gradients abolished outgrowth. Pharmacological and genetic analyses showed that the NMDA-triggered microglia process extension was independent of Pannexin 1, the ATP releasing channels, ATP release from astrocytes via connexins, and nitric oxide generation. Finally, using whole-cell patch clamping we demonstrate that activation of dendritic NMDAR on single neurons is sufficient to trigger microglia process outgrowth. Our results suggest that dendritic neuronal NMDAR activation triggers ATP release via a Pannexin 1-independent manner that induces outgrowth of microglia processes. This represents a novel uncharacterized form of neuron-microglial communication mediated by ATP.

To clarify the role of microglia in brain homeostasis and disease, an understanding of their maintenance, proliferation and turnover is essential. The lifespan of brain microglia, however, remains uncertain, and reflects confounding factors in earlier assessments that were largely indirect. We genetically labeled single resident microglia in living mice and then used multiphoton microscopy to monitor these cells over time. Under homeostatic conditions, we found that neocortical resident microglia were long-lived, with a median lifetime of well over 15 months; thus, approximately half of these cells survive the entire mouse lifespan. While proliferation of resident neocortical microglia under homeostatic conditions was low, microglial proliferation in a mouse model of Alzheimer's β-amyloidosis was increased threefold. The persistence of individual microglia throughout the mouse lifespan provides an explanation for how microglial priming early in life can induce lasting functional changes and how microglial senescence may contribute to age-related neurodegenerative diseases.

Article Nanoscale Surveillance of the Brain by Microglia via cAMP-Regulated Filopodia Graphical Abstract Highlights d Microglia use actin-dependent filopodia to efficiently sample the brain parenchyma d Intracellular cAMP drives filopodia growth but induces large process retraction d Norepinephrine and nitric oxide contribute to cAMP-driven filopodia extension d Fine filopodia and large processes establish dual-scale surveillance by microglia This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). relative to variations in the brain parenchyma environment. STAR+METHODSDetailed methods are provided in the online version of this paper and include the following:TABLE d CONTACT FOR REAGENTS AND RESOURCE SHARING d EXPERIMENTAL MODEL AND SUBJECT DETAILS B Animals B Acute slice preparation and maintenance B Isolation of rat microglia d METHOD DETAILS B Open cranial window surgery and in vivo two-photon imaging B Brain perfusion, immunofluorescence and confocal imaging B Acute slice two-photon imaging B Slice fixation and immunolabelling B Rat primary microglia live imaging B Western blotting d QUANTIFICATION AND STATISTICAL ANALYSIS B Image processing and analysis B Statistical analysis SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.

Extracellular deposition of the amyloid-␤ peptide (A␤) in the brain parenchyma is a hallmark lesion of Alzheimer's disease (AD) and a predictive marker for the progression of preclinical to symptomatic AD. Here, we used multiphoton in vivo imaging to study A␤ plaque formation in the brains of 3-to 4-month-old APPPS1 transgenic mice over a period of 6 months. A novel head fixation system provided robust and efficient long-term tracking of single plaques over time. Results revealed an estimated rate of 35 newly formed plaques per cubic millimeter of neocortical volume per week at 4 -5 months of age. At later time points (i.e., in the presence of increasing cerebral ␤-amyloidosis), the number of newly formed plaques decreased. On average, both newly formed and existing plaques grew at a similar growth rate of 0.3 m (radius) per week. A solid knowledge of the dynamics of cerebral ␤-amyloidosis in mouse models provides a powerful tool to monitor preclinical A␤ targeting therapeutic strategies and eases the interpretation of diagnostic amyloid imaging in humans.

Cramer et al . (Reports, 23 March 2012, p. 1503; published online 9 February 2012) reported that bexarotene rapidly reduces β-amyloid (Aβ) levels and plaque burden in two mouse models of Aβ deposition in Alzheimer’s disease (AD). We now report that, although bexarotene reduces soluble Aβ 40 levels in one of the mouse models, the drug has no impact on plaque burden in three strains that exhibit Aβ amyloidosis.

Amyloid-β (Aβ) plaques, a hallmark of Alzheimer's disease (AD), are surrounded by regions of neuronal and glial hyperactivity. We use in vivo two-photon and wide-field imaging of the glutamate sensor iGluSnFR to determine whether pathological changes in glutamate dynamics in the immediate vicinity of Aβ deposits in APPPS1 transgenic mice could alter neuronal activity in this microenvironment. In regions close to Aβ plaques chronic states of high spontaneous glutamate fluctuations are observed and the timing of glutamate responses evoked by sensory stimulation exhibit slower decay rates in two cortical brain areas. GLT-1 expression is reduced around Aβ plaques and upregulation of GLT-1 expression and activity by ceftriaxone partially restores glutamate dynamics to values in control regions. We conclude that the toxic microenvironment surrounding Aβ plaques results, at least partially, from enhanced glutamate levels and that pharmacologically increasing GLT-1 expression and activity may be a new target for early therapeutic intervention.

Familial Danish dementia (FDD) is a progressive neurodegenerative disease with cerebral deposition of Dan-amyloid (ADan), neuroinflammation, and neurofibrillary tangles, hallmark characteristics remarkably similar to those in Alzheimer's disease (AD). We have generated transgenic (tg) mouse models of familial Danish dementia that exhibit the age-dependent deposition of ADan throughout the brain with associated amyloid angiopathy, microhemorrhage, neuritic dystrophy, and neuroinflammation. Tg mice are impaired in the Morris water maze and exhibit increased anxiety in the open field. When crossed with TauP301S tg mice, ADan accumulation promotes neurofibrillary lesions, in all aspects similar to the Tau lesions observed in crosses between β-amyloid (Aβ)-depositing tg mice and TauP301S tg mice. Although these observations argue for shared mechanisms of downstream pathophysiology for the sequence-unrelated ADan and Aβ peptides, the lack of codeposition of the two peptides in crosses between ADan-and Aβ-depositing mice points also to distinguishing properties of the peptides. Our results support the concept of the amyloid hypothesis for AD and related dementias, and suggest that different proteins prone to amyloid formation can drive strikingly similar pathogenic pathways in the brain.ADan | ABeta | tau | neurodegeneration | mouse model