Summary Alzheimer’s disease is characterized by the deposition of senile plaques and progressive dementia. The molecular mechanisms that couple plaque deposition to neural system failure, however, are unknown. Using transgenic mouse models of AD together with multiphoton imaging, we measured neuronal calcium in individual neurites and spines in vivo using the genetically-encoded calcium indicator YC3.6. Quantitative imaging revealed elevated [Ca2+]i (calcium overload) in ~20% of neurites in APP mice with cortical plaques, compared to less than 5% in wildtype mice, PS1-mutant mice, or young APP mice (animals without cortical plaques). Calcium overload depended on the existence and proximity to plaques. The downstream consequences included the loss of spino-dendritic calcium compartmentalization (critical for synaptic integration) and a distortion of neuritic morphologies mediated, in part, by the phosphatase calcineurin. Together, these data demonstrate that senile plaques impair neuritic calcium homeostasis in vivo and result in the structural and functional disruption of neuronal networks.

While senile plaques injure neurons focally, the functional response of astrocytes to Alzheimer's disease pathology is unknown. Using multiphoton fluorescence lifetime imaging microscopy in vivo, we quantitatively imaged astrocytic calcium homeostasis in a mouse model of Alzheimer's disease. Resting calcium was globally elevated in the astrocytic network, but was independent of proximity to individual plaques. Time lapse imaging revealed that calcium transients in astrocytes were more frequent, were synchronously coordinated across long distances, and were uncoupled from neuronal activity. Furthermore, rare intercellular calcium waves were observed, but only in mice with amyloid-β plaques, originating near plaques and spreading radially at least 200 µm. Thus, while neurotoxicity is observed near amyloid-β deposits, there exists a more general astrocyte-based network response to focal pathology.Growing evidence supports the hypothesis that in Alzheimer's disease (AD), synapses fail and dendritic spines are lost in the amyloid-β (Aβ) plaque micro-environment through a combination of changes to synaptic drive, calcium overload, and activation of calciumdependent degenerative processes (1-4). Neurons, however, make up only part of the brain's volume, with astrocytes making up the bulk of the remaining volume. Astrocytes form a structurally interconnected network that, in vitro, exhibit unique long-distance signaling properties that might be revealed in vivo only after pathological trauma. The idea that neural network dysfunction and degeneration fully mediates the memory loss in AD also does not reflect the growing in vivo evidence that astrocytes play an important role in cortical circuit function (5-7). In AD, pathological studies of human cases and mouse models have shown that astrocytes surround plaques and might play a critical role in Aβ deposition and clearance (8-10). Given the profound impact of Aβ deposition on nearby neuronal calcium homeostasis and synaptic function, it is reasonable to hypothesize that astrocyte networks would also be perturbed and might contribute to cortical dysfunction (11). We sought to test whether senile plaque deposition would similarly impact astrocyte calcium homeostasis or dynamic signaling in vivo in a mouse model of AD.To answer these questions, we used multiphoton fluorescent lifetime imaging microscopy (FLIM) to measure resting calcium levels in astrocytes of live mice with cortical plaques (12). We multiplexed the fluorescent properties of a small molecule calcium dye, OregonGreen BAPTA-1 AM (OGB), in the same experimental model and for the same group of cells (Fig. 1A); we used OGB both as a relative indicator of astrocytic activity (intensity) and as a quantitative measure of steady-state [Ca]i (lifetime). We used mice that express mutant human Aβ precursor protein (APP, swe) and mutant presenilin 1 (PS1, ΔE9) in neurons. These mutations lead to an increase in Aβ production and plaque deposition beginning at ~4.5 months

Amyloid ␤ (A␤)-containing plaques are surrounded by dystrophic neurites in the Alzheimer's disease (AD) brain, but whether and how plaques induce these neuritic abnormalities remain unknown. We tested the hypothesis that soluble oligomeric assemblies of A␤, which surround plaques, induce calcium-mediated secondary cascades that lead to dystrophic changes in local neurites. We show that soluble A␤ oligomers lead to activation of the calcium-dependent phosphatase calcineurin (CaN) (PP2B), which in turn activates the transcriptional factor nuclear factor of activated T cells (NFAT). Activation of these signaling pathways, even in the absence of A␤, is sufficient to produce a virtual phenocopy of A␤-induced dystrophic neurites, dendritic simplification, and dendritic spine loss in both neurons in culture and in the adult mouse brain. Importantly, the morphological deficits in the vicinity of A␤ deposits in a mouse model of AD are ameliorated by CaN inhibition, supporting the hypothesis that CaN-NFAT are aberrantly activated by A␤ and that CaN-NFAT activation is responsible for disruption of neuronal structure near plaques. In accord with this, we also detect increased levels of an active form of CaN and NFATc4 in the nuclear fraction from the cortex of patients with AD. Thus, A␤ appears to mediate the neurodegeneration of AD, at least in part, by activation of CaN and subsequent NFAT-mediated downstream cascades.

Physical features of sensory stimuli are fixed, but sensory perception is context-dependent. The precise mechanisms that govern contextual modulation remain unknown. Here, we trained mice to switch between two contexts: passively listening to pure tones vs. performing a recognition task for the same stimuli. Two-photon imaging showed that many excitatory neurons in auditory cortex were suppressed, while some cells became more active during behavior. Whole-cell recordings showed that excitatory inputs were only modestly affected by context, but inhibition was more sensitive, with PV, SOM+, and VIP+ interneurons balancing inhibition/disinhibition within the network. Cholinergic modulation was involved in context-switching, with cholinergic axons increasing activity during behavior and directly depolarizing inhibitory cells. Network modeling captured these findings, but only when modulation coincidently drove all three interneuron subtypes, ruling out either inhibition or disinhibition alone as sole mechanism for active engagement. Parallel processing of cholinergic modulation by cortical interneurons therefore enables context-dependent behavior.

Neuronal chloride concentration [Cl−]i is an important determinant of GABAA receptor (GABAAR)-mediated inhibition and cytoplasmic volume regulation. Equilibrative cation-chloride cotransporters (CCC) move Cl− across the membrane, but accumulating evidence suggests factors other than the bulk concentrations of transported ions determine [Cl−]i. Measurement of [Cl−]i in murine brain slice preparations expressing the transgenic fluorophore Clomeleon demonstrated that cytoplasmic impermeant anions ([A]i) and polyanionic extracellular matrix glycoproteins ([A]o) constrain the local [Cl−]. CCC inhibition had modest effects on [Cl−]i and neuronal volume, but substantial changes were produced by alterations of the balance between [A]i and [A]o. Therefore, CCC are important elements of Cl− homeostasis, but local impermeant anions determine the homeostatic set-point for [Cl−], and hence, neuronal volume and the polarity of local GABAAR signaling.

Electroclinical dissociation of neonatal seizures refers to electrographic seizure activity that is not clinically manifest. Dissociation increases after treatment with Phenobarbital, which increases the GABAA receptor (GABAAR) conductance. The effects of GABAAR activation depend on the intracellular Cl− concentration ([Cl−]i) that is determined by the inward Cl− transporter NKCC1 and the outward Cl− transporter KCC2. Differential maturation of Cl− transport observed in cortical vs. subcortical regions should alter the efficacy of GABA-mediated inhibition. In perinatal rat pups, most thalamic neurons maintained low [Cl−]i, and were inhibited by GABA. Phenobarbital suppressed thalamic seizure activity. Most neocortical neurons maintained higher [Cl−]i, and were excited by GABAAR activation. Phenobarbital had insignificant anticonvulsant responses in the neocortex until NKCC1 was blocked. Regional differences in the ontogeny of Cl− transport may thus explain why seizure activity in the cortex is not suppressed by anticonvulsants that block the transmission of seizure activity through subcortical networks.

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Alzheimer's disease (AD) is pathologically characterized by the deposition of extracellular amyloid-β plaques and intracellular aggregation of tau protein in neurofibrillary tangles (NFTs) (1, 2). Progression of NFT pathology is closely correlated with both increased neurodegeneration and cognitive decline in AD (3) and other tauopathies, such as frontotemporal dementia (4,5). The assumption that mislocalization of tau into the somatodendritic compartment (6) and accumulation of fibrillar aggregates in NFTs mediates neurodegeneration underlies most current therapeutic strategies aimed at preventing NFT formation or disrupting existing NFTs (7,8). Although several disease-associated mutations cause both aggregation of tau and neurodegeneration, whether NFTs per se contribute to neuronal and network dysfunction in vivo is unknown (9). Here we used awake in vivo two-photon calcium imaging to monitor neuronal function in adult rTg4510 mice that overexpress a human mutant form of tau (P301L) and develop cortical NFTs by the age of 7-8 mo (10). Unexpectedly, NFT-bearing neurons in the visual cortex appeared to be completely functionally intact, to be capable of integrating dendritic inputs and effectively encoding orientation and direction selectivity, and to have a stable baseline resting calcium level. These results suggest a reevaluation of the common assumption that insoluble tau aggregates are sufficient to disrupt neuronal function.paired helical filaments | tau pathology | neuronal networks N eurofibrillary tangles (NFTs) containing aggregated tau protein (1) have long been considered key players in the progressive neural dysfunction and neurodegeneration observed in Alzheimer's disease (AD) (2, 3) and other tauopathies (4, 5). It is commonly assumed that NFT-bearing neurons exhibit deficits in synaptic integration and eventually lead to neurodegeneration (11,12). However, the actual functional properties of NFT-bearing neurons in intact neural circuits have not been explored previously (13). We addressed this question directly using awake in vivo two-photon calcium imaging in a mouse model of NFT formation (rTg4510) by applying recently developed imaging approaches allowing for single-neuron-level and population-level assessment of neural activity in awake mice (14). Because two-photon calcium imaging allows for measurement of response properties in many neurons simultaneously, we were able to directly isolate the impact of NFT deposition in a neuronal microcircuit by evaluating population-level network dynamics and, more specifically, by differentiating the function of individual NFT-bearing and neighboring non-NFT-bearing neurons.To assess the functional properties of neurons in the visual cortex, we used a genetically encoded ratiometric calcium indicator, yellow cameleon 3.6 (YC3.6), packaged in an adenoassociated viral vector (15,16). To assess functional responses, we exploited the well-characterized functional architecture of visual cortex whereby neurons in mouse visual cortex modulate their activity d...