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Multielectrode arrays (MEAs) are electrical devices that transduce (record/deliver) cellular voltage signals. Commercially available MEAs are expensive and here we provide proof of concept for the application of an additive manufacturing approach to prepare inexpensive MEAs and demonstrate their ability to interact with brain tissue ex vivo.

Using spatial cell-type-enriched transcriptomics, we compare plaque-induced gene (PIG) expression in microglia-touching plaques, neighboring plaques, and far from plaques in an aged Alzheimer's mouse model with late plaque development. In 18-month-old APP

Using spatial cell-type-enriched transcriptomics, we compare plaque-induced gene (PIG) expression in microglia touching plaques, neighboring plaques, and far from plaques in 18-month-oldAPPNLF/NLFknock-in mice with and without the Alzheimer’s disease risk mutationTrem2R47H/R47H. We report that, inAppNLF/NLFmice, expression of 35/55 PIGs, is exclusively upregulated in microglia that are touching plaques. In 7 PIGs includingTrem2this upregulation is prevented by theTrem2R47H/R47Hmutation. Unlike in young mice, knockin of theTrem2R47H/R47Hmutation does not significantly decrease theTrem2expression but decreases protein levels by 20% in the absence of plaques. On plaques, despite the mutation preventing increased gene expression, TREM2 protein levels increased by 1.6-fold (compared to 3-fold withTrem2WT/WT) and microglial density increased 20-fold compared to 30-fold. Hence microglia must touch plaques beforeTrem2gene expression is increased but small changes in protein expression can increase microglia density without a change in gene expression.

Funder: Cure Alzheimer's Fund; doi: http://dx.doi.org/10.13039/100007625, Funder: UK Dementia Research Institute (GB), Funder: Censejo Nacional de Ciencia Tecnilogia (MX), Funder: Alzheimerfonden; doi: http://dx.doi.org/10.13039/501100008599, Funder: Dolby Family Fund, Background: Microglia are active modulators of Alzheimer’s disease but their role in relation to amyloid plaques and synaptic changes due to rising amyloid beta is unclear. We add novel findings concerning these relationships and investigate which of our previously reported results from transgenic mice can be validated in knock-in mice, in which overexpression and other artefacts of transgenic technology are avoided. Methods: AppNL-F and AppNL-G-F knock-in mice expressing humanised amyloid beta with mutations in App that cause familial Alzheimer’s disease were compared to wild type mice throughout life. In vitro approaches were used to understand microglial alterations at the genetic and protein levels and synaptic function and plasticity in CA1 hippocampal neurones, each in relationship to both age and stage of amyloid beta pathology. The contribution of microglia to neuronal function was further investigated by ablating microglia with CSF1R inhibitor PLX5622. Results: Both App knock-in lines showed increased glutamate release probability prior to detection of plaques. Consistent with results in transgenic mice, this persisted throughout life in AppNL-F mice but was not evident in AppNL-G-F with sparse plaques. Unlike transgenic mice, loss of spontaneous excitatory activity only occurred at the latest stages, while no change could be detected in spontaneous inhibitory synaptic transmission or magnitude of long-term potentiation. Also, in contrast to transgenic mice, the microglial response in both App knock-in lines was delayed until a moderate plaque load developed. Surviving PLX5266-depleted microglia tended to be CD68-positive. Partial microglial ablation led to aged but not young wild type animals mimicking the increased glutamate release probability in App knock-ins and exacerbated the App knock-in phenotype. Complete ablation was less effective in altering synaptic function, while neither treatment altered plaque load. Conclusions: Increased glutamate release probability is similar across knock-in and transgenic mouse models of Alzheimer’s disease, likely reflecting acute physiological effects of soluble amyloid beta. Microglia respond later to increased amyloid beta levels by proliferating and upregulating Cd68 and Trem2. Partial depletion of microglia suggests that, in wild type mice, alteration of surviving phagocytic microglia, rather than microglial loss, drives age-dependent effects on glutamate release that become exacerbated in Alzheimer’s disease.

Mouse models of Alzheimer's disease (AD) are valuable but do not fully recapitulate human AD pathology, such as spontaneous Tau fibril accumulation and neuronal loss, necessitating the development of new AD models. The transgenic (TG) TgF344-AD rat has been reported to develop age-dependent AD features including neuronal loss and neurofibrillary tangles, despite only expressing APP and PSEN1 mutations, suggesting an improved modelling of AD hallmarks. Alterations in neuronal networks as well as learning performance and cognition tasks have been reported in this model, but none have combined a longitudinal, multimodal approach across multiple centres, which mimics the approaches commonly taken in clinical studies. We therefore aimed to further characterise the progression of AD-like pathology and cognition in the TgF344-AD rat from young-adults (6 months (m)) to mid- (12 m) and advanced-stage (18 m, 25 m) of the disease. Methods: TgF344-AD rats and wild-type (WT) littermates were imaged at 6 m, 12 m and 18 m with [18F]DPA-714 (TSPO, neuroinflammation), [18F]Florbetaben (Aβ) and [18F]ASEM (α7-nicotinic acetylcholine receptor) and with magnetic resonance spectroscopy (MRS) and with (S)-[18F]THK5117 (Tau) at 15 and 25 m. Behaviour tests were also performed at 6 m, 12 m and 18 m. Immunohistochemistry (CD11b, GFAP, Aβ, NeuN, NeuroChrom) and Tau (S)-[18F]THK5117 autoradiography, immunohistochemistry and Western blot were also performed. Results: [18F]DPA-714 positron emission tomography (PET) showed an increase in neuroinflammation in TG vs wildtype animals from 12 m in the hippocampus (+11%), and at the advanced-stage AD in the hippocampus (+12%), the thalamus (+11%) and frontal cortex (+14%). This finding coincided with strong increases in brain microgliosis (CD11b) and astrogliosis (GFAP) at these time-points as assessed by immunohistochemistry. In vivo [18F]ASEM PET revealed an age-dependent increase uptake in the striatum and pallidum/nucleus basalis of Meynert in WT only, similar to that observed with this tracer in humans, resulting in TG being significantly lower than WT by 18 m. In vivo [18F]Florbetaben PET scanning detected Aβ accumulation at 18 m, and (S)-[18F]THK5117 PET revealed subsequent Tau accumulation at 25m in hippocampal and cortical regions. Aβ plaques were low but detectable by immunohistochemistry from 6 m, increasing further at 12 and 18 m with Tau-positive neurons adjacent to Aβ plaques at 18 m. NeuroChrom (a pan neuronal marker) immunohistochemistry revealed a loss of neuronal staining at the Aβ plaques locations, while NeuN labelling revealed an age-dependent decrease in hippocampal neuron number in both genotypes. Behavioural assessment using the novel object recognition task revealed that both WT & TgF344-AD animals discriminated the novel from familiar object at 3 m and 6 m of age. However, low levels of exploration observed in both genotypes at later time-points resulted in neither genotype successfully completing the task. Deficits in social interaction were only observed at 3 m in the TgF344-AD animals. By in vivo MRS, we showed a decrease in neuronal marker N-acetyl-aspartate in the hippocampus at 18 m (-18% vs age-matched WT, and -31% vs 6 m TG) and increased Taurine in the cortex of TG (+35% vs age-matched WT, and +55% vs 6 m TG). Conclusions: This multi-centre multi-modal study demonstrates, for the first time, alterations in brain metabolites, cholinergic receptors and neuroinflammation in vivo in this model, validated by robust ex vivo approaches. Our data confirm that, unlike mouse models, the TgF344-AD express Tau pathology that can be detected via PET, albeit later than by ex vivo techniques, and is a useful model to assess and longitudinally monitor early neurotransmission dysfunction and neuroinflammation in AD.

For our understanding of Alzheimer’s disease (AD) pathophysiology, it is of critical importance to determine how key pathological factors, specifically amyloid β (Aβ) plaque formation, are interconnected and implicated in neurodegeneration, disease progression and the development of clinical symptoms. Exactly how Aβ plaque formation is initiated and how the ongoing plaque deposition proceeds is not well understood. This is partly because we can only examine details of the molecular pathology after death in humans, and in mice, we can only examine a particular point in time without any longitudinal information on the fate of individually formed deposits. Herein, we used metabolic labelling of proteins with stable isotopes, together with multimodal imaging mass spectrometry (IMS) for imaging stable isotope labelling kinetics (iSILK) in the APPNL-G-F knock-in mouse model of AD. The aim was to monitor the earliest seeds of Aβ deposition through ongoing plaque development and track the deposition of Aβ that is produced later in relation to already deposited plaques. This allowed us to visualize Aβ peptide aggregation dynamics within individual deposits across different brain regions. We identified the cortex as a primary site of deposition in precipitating plaque pathology. Further, our data show that structural plaque heterogeneity is associated with differential peptide deposition. Specifically, Aβ1-42 is forming an initial core seed followed by radial outgrowth and late secretion and deposition of Aβ1-38.Together these data prove the potential of iSILK for probing amyloid protein secretion, processing and aggregation dynamics in AD pathology.

Background: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might be curtailed by vaccination. We assessed the safety, reactogenicity, and immunogenicity of a viral vectored coronavirus vaccine that expresses the spike protein of SARS-CoV-2. Methods: We did a phase 1/2, single-blind, randomised controlled trial in five trial sites in the UK of a chimpanzee adenovirus-vectored vaccine (ChAdOx1 nCoV-19) expressing the SARS-CoV-2 spike protein compared with a meningococcal conjugate vaccine (MenACWY) as control. Healthy adults aged 18–55 years with no history of laboratory confirmed SARS-CoV-2 infection or of COVID-19-like symptoms were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 at a dose of 5 × 10 10 viral particles or MenACWY as a single intramuscular injection. A protocol amendment in two of the five sites allowed prophylactic paracetamol to be administered before vaccination. Ten participants assigned to a non-randomised, unblinded ChAdOx1 nCoV-19 prime-boost group received a two-dose schedule, with the booster vaccine administered 28 days after the first dose. Humoral responses at baseline and following vaccination were assessed using a standardised total IgG ELISA against trimeric SARS-CoV-2 spike protein, a muliplexed immunoassay, three live SARS-CoV-2 neutralisation assays (a 50% plaque reduction neutralisation assay [PRNT 50]; a microneutralisation assay [MNA 50, MNA 80, and MNA 90]; and Marburg VN), and a pseudovirus neutralisation assay. Cellular responses were assessed using an ex-vivo interferon-γ enzyme-linked immunospot assay. The co-primary outcomes are to assess efficacy, as measured by cases of symptomatic virologically confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were done by group allocation in participants who received the vaccine. Safety was assessed over 28 days after vaccination. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. The study is ongoing, and was registered at ISRCTN, 15281137, and ClinicalTrials.gov, NCT04324606. Findings: Between April 23 and May 21, 2020, 1077 participants were enrolled and assigned to receive either ChAdOx1 nCoV-19 (n=543) or MenACWY (n=534), ten of whom were enrolled in the non-randomised ChAdOx1 nCoV-19 prime-boost group. Local and systemic reactions were more common in the ChAdOx1 nCoV-19 group and many were reduced by use of prophylactic paracetamol, including pain, feeling feverish, chills, muscle ache, headache, and malaise (all p80 and in 35 (100%) participants when measured in PRNT 50. After a booster dose, all participants had neutralising activity (nine of nine in MNA 80 at day 42 and ten of ten in Marburg VN on day 56). Neutralising antibody responses correlated strongly with antibody levels measured by ELISA (R 2=0·67 by Marburg VN; p

Genome-wide association studies have reported that, amongst other microglial genes, variants in TREM2 can profoundly increase the incidence of developing Alzheimer’s disease (AD). We have investigated the role of TREM2 in primary microglial cultures from wild type mice by using siRNA to decrease Trem2 expression, and in parallel from knock-in mice heterozygous or homozygous for the Trem2 R47H AD risk variant. The prevailing phenotype of Trem2 R47H knock-in mice was decreased expression levels of Trem2 in microglia, which resulted in decreased density of microglia in the hippocampus. Overall, primary microglia with reduced Trem2 expression, either by siRNA or from the R47H knock-in mice, displayed a similar phenotype. Comparison of the effects of decreased Trem2 expression under conditions of lipopolysaccharide (LPS) pro-inflammatory or IL-4 anti-inflammatory stimulation revealed the importance of Trem2 in driving a number of the genes up-regulated in the anti-inflammatory phenotype. RNA-seq analysis showed that IL-4 induced the expression of a program of genes including Arg1 and Ap1b1 in microglia, which showed an attenuated response to IL-4 when Trem2 expression was decreased. Genes showing a similar expression profile to Arg1 were enriched for STAT6 transcription factor recognition elements in their promoter, and Trem2 knockdown decreased levels of STAT6. LPS-induced pro-inflammatory stimulation suppressed Trem2 expression, thus preventing TREM2’s anti-inflammatory drive. Given that anti-inflammatory signaling is associated with tissue repair, understanding the signaling mechanisms downstream of Trem2 in coordinating the pro- and anti-inflammatory balance of microglia, particularly mediating effects of the IL-4-regulated anti-inflammatory pathway, has important implications for fighting neurodegenerative disease., Graphical Abstract Graphical Abstract

We report the application of multiphoton microfabrication to prepare conducting polymer (CP)-based biomaterials that were capable of drug delivery and interacting with brain tissue ex vivo, thereby highlighting the potential of multiphoton lithography to prepare electroactive biomaterials which may function as implantable neural biointerfaces (e.g. electrodes).

Negative regulator of ubiquitin-like protein 1 (NUB1) and its longer isoform NUB1L are ubiquitin-like (UBL)/ubiquitin-associated (UBA) proteins that facilitate the targeting of proteasomal substrates, including tau, synphilin-1 and huntingtin. Previous data revealed that NUB1 also mediated a reduction in tau phosphorylation and aggregation following proteasome inhibition, suggesting a switch in NUB1 function from targeted proteasomal degradation to a role in autophagy. Here, we delineate the mechanisms of this switch and show that NUB1 interacted specifically with p62 and induced an increase in p62 levels in a manner facilitated by inhibition of the proteasome. NUB1 moreover increased autophagosomes and the recruitment of lysosomes to aggresomes following proteasome inhibition. Autophagy flux assays revealed that NUB1 affected the autophagy–lysosomal pathway primarily via the UBA domain. NUB1 localized to cytosolic inclusions with pathological forms of tau, as well as LAMP1 and p62 in the hippocampal neurons of tauopathy mice. Finally, NUB1 facilitated the extracellular release of tau following proteasome inhibition. This study thus shows that NUB1 plays a role in regulating the autophagy–lysosomal pathway when the ubiquitin proteasome system is compromised, thus contributing to the mechanisms targeting the removal of aggregation-prone proteins upon proteasomal impairment.

Mouse models of Alzheimer's disease have commonly used transgenic overexpression of genes involved in production of amyloid β (APP and/or PSEN1/2) or Tau (MAPT) with mutations that result in familial forms of dementia. We discuss possible improvements that may create full models while avoiding the problems of overexpression and report synaptic results in APPKI models. We stress use of inappropriate controls without overexpression of the normal human protein and the mismatch between the learning deficits reported in mice with plaques but no tangles and the human condition. We focus on Tau overexpression, including new data that support previous reports of the grossly nonlinear relationship between Tau overexpression and neurofibrillary tangle load, with a twofold increase in Tau protein, resulting in a 100-fold increase in tangle density. These data also support the hypothesis that a high concentration of soluble Tau, in overexpression models, plays an important direct role in neurodegeneration, rather than only via aggregation. Finally, we hypothesize that there is an optimal concentration range over which Tau can bind to microtubules and a threshold beyond which much of the overexpressed protein is unable to bind. The excess thus causes toxicity in ways not necessarily related to the process in human dementias.

BackgroundProgression of Alzheimer’s disease is thought initially to depend on rising amyloidβ and its synaptic interactions. Transgenic mice (TASTPM; APPSwe/PSEN1M146V) show altered synaptic transmission, compatible with increased physiological function of amyloidβ, before plaques are detected. Recently, the importance of microglia has become apparent in the human disease. Similarly, TASTPM show a close association of plaque load with upregulated microglial genes.MethodsCA1 Synaptic transmission and plasticity were investigated using in vitro electrophysiology. Migroglial relationship to plaques was examined with immunohistochemistry. Behaviour was assessed with a forced-alternation T-maze, open field, light/dark box and elevated plus maze.FindingsThe most striking finding is the increase in microglial numbers in TASTPM, which, like synaptic changes, begins before plaques are detected. Further increases and a reactive phenotype occur later, concurrent with development of larger plaques. Long-term potentiation is initially enhanced at pre-plaque stages but decrements with the initial appearance of plaques. Finally, despite altered plasticity, TASTPM have little cognitive deficit, even with a heavy plaque load, although they show altered non-cognitive behaviours.InterpretationThe pre-plaque synaptic changes and microglial proliferation are presumably related to low, non-toxic amyloidβ levels in the general neuropil and not directly associated with plaques. However, as plaques grow, microglia proliferate further, clustering around plaques and becoming phagocytic. Like in humans, even when plaque load is heavy, without development of neurofibrillary tangles and neurodegeneration, these alterations do not result in cognitive deficits. Behaviours are seen that could be consistent with pre-diagnosis changes in the human condition.Research in contextEvidence before this studyThere is a large body of research examining many aspects of phenotypes associated with mouse models of Alzheimer’s disease – a PubMed search for the terms Alzheimer* AND mouse returns in excess of 21000 articles. However, there are few systematic articles pulling together pathological, functional (electrophysiological), and behavioural analyses across the life-span of such models. There is also a number of conflicting outcomes, for example reports of impaired versus enhanced synaptic plasticity; cognitive impairments or not.Recently, the importance of microglia in Alzheimer’s disease has come to the fore in human Genome Wide Association Studies (GWAS), with variants of a number of microglial genes identified as risk-factors for developing the disease. Interestingly, we have recently reported that Trem2 and other genes identified as risk-factors in humans are strongly up regulated in close association to plaque development in the mouse model used in this study. Moreover, this previous study predicted two of the most recently identified genes that were identified in GWAS since the publication of our paper.We have previously used this model to identify the earliest synaptic changes and shown changes in release of glutamate, the primary excitatory neurotransmitter in the brain, to occur even before plaques are detectable.Added value of this studyBy studying this transgenic mouse model of Alzheimer’s disease, throughout the development of plaques, from prior to detection through to heavy plaque loads, we have been able to identify a clear time course of key phenotypic changes associated with early disease. In particular, this study identifies the very early changes in microglia and can separate the time course of the microglial phenotype. In addition, we detail the changes in synaptic plasticity over time and importantly identify that, like in humans in the absence of Tau tangles or neurodegeneration, considerable synaptic changes can occur and a heavy plaque load without resulting in substantial cognitive loss.Implications of all the available evidenceOur data indicate that rising amyloid beta prior to detectable plaque deposition results in changes in synaptic function that likely reflects an enhanced physiological effect of amyloid beta. At this stage, microglia proliferate but do not activate. Once plaques begin to appear, microglia migrate to surround the plaque and become phagocytic, likely targeting dystrophic synapses and neurites caused by the cloud of highly-toxic amyloid beta around the plaque. Similarly to humans, who have plaques but no tangles and have yet to develop substantial neurodegeneration, cognitive deficits are not seen, even with a heavy plaque load; behavioural changes are limited to anxiety-like effects.This investigation of the parallel time-course of events highlights the probability that, if progression of disease can be reversed or slowed early enough, before Tau tangles and substantial neurodegeneration occur, the symptoms of cognitive decline could be very largely avoided. Moreover, it suggests that the substantial increases in microglia number and upregulation of their specific gene expression in association with plaques, is not associated with cognitive loss and may indeed be protective.

Genome-wide association studies have reported that, amongst other microglial genes, variants inTREM2can profoundly increase the incidence of developing Alzheimer’s disease (AD). We have investigated the role of TREM2 in primary microglial cultures from wild type mice by using siRNA to decreaseTrem2expression, and in parallel from knock-in mice heterozygous or homozygous for theTrem2R47H AD risk variant. The prevailing phenotype ofTrem2R47H knock-in mice was decreased expression levels ofTrem2in microglia, which resulted in decreased density of microglia in the hippocampus. Overall, primary microglia with reducedTrem2expression, either by siRNA or from the R47H knock-in mice, displayed a similar phenotype. Comparison of the effects of decreasedTrem2expression under conditions of LPS pro-inflammatory or IL-4 anti-inflammatory stimulation revealed the importance ofTrem2in driving a number of the genes up-regulated in the anti-inflammatory phenotype. RNA-seq analysis showed that IL-4 induced the expression of a programme of genes includingArg1andAp1b1in microglia, which showed an attenuated response to IL-4 whenTrem2expression was decreased. Genes showing a similar expression profile toArg1were enriched for STAT6 transcription factor recognition elements in their promoter, andTrem2knockdown decreased levels of the transcription factor STAT6. LPS-induced pro-inflammatory stimulation suppressedTrem2expression, thus preventing TREM2’s anti-inflammatory drive. Given that anti-inflammatory signaling is associated with tissue repair, understanding the signaling mechanisms downstream ofTrem2in coordinating the pro- and anti-inflammatory balance of microglia, particularly mediating effects of the IL-4-regulated anti-inflammatory pathway, has important implications for fighting neurodegenerative disease.Graphical abstract

Genetic analysis of late-onset Alzheimer’s disease risk has previously identified a network of largely microglial genes that form a transcriptional network. In transgenic mouse models of amyloid deposition we have previously shown that the expression of many of the mouse orthologs of these genes are co-ordinately up-regulated by amyloid deposition. Here we investigate whether systematic analysis of other members of this mouse amyloid-responsive network predicts other Alzheimer’s risk loci. This statistical comparison of the mouse amyloid-response network with Alzheimer’s disease genome-wide association studies identifies 5 other genetic risk loci for the disease (OAS1, CXCL10, LAPTM5, ITGAM and LILRB4). This work suggests that genetic variability in the microglial response to amyloid deposition is a major determinant for Alzheimer’s risk.One Sentence SummaryIdentification of 5 new risk loci for Alzheimer’s by statistical comparison of mouse Aβ microglial response with gene-based SNPs from human GWAS

BackgroundLate stage Alzheimer’s disease and other dementias are associated with neurofibrillary tangles and neurodegeneration. Here we describe a mouse (TauD35) carrying human Tau with the P301L mutation that results in Tau hyperphosphorylation and tangles. Previously we have compared gene expression in TauD35 mice to mice which develop plaques but no tangles. A similar comparison of other pathological features throughout disease progression is made here between amyloidβ and Tau mice described in Parts I and II of this study.MethodsIn vitro CA1 patch clamp and field recordings were used to investigate synaptic transmission and plasticity. Plaque load and microglia were investigated with immunohistochemistry. Cognition, locomotor activity and anxiety-related behaviours were assessed with a forced-alternation T-maze, open field and light/dark box.ResultsTransgene copy number in TauD35 mice fell into two groups (HighTAU and LowTAU), allowing assessment of dose-dependent effects of overexpression and resulting in tangle load increasing 100-fold for a 2-fold change in protein levels. Tangles were first detected at 8 (HighTAU) or 13 months (LowTAU) but the effects on synaptic transmission and plasticity and behaviour were subtle. However, severe neurodegeneration occurred in HighTAU mice at around 17 months, preceded by considerable proliferation and activation of microglia at 13 months of age; both increasing further at 17 months. LowTAU mice at 24 months of age showed a comparable tangle load and microglial proliferation to that occurring at 13 months in HighTAU mice. However, LowTAU mice showed no neurodegeneration at this stage and considerable microglial activation, stressing the dependence of these effects on overexpression and/or age.ConclusionsComparison of the effects of amyloidβ and plaques without tangles in a model of preclinical Alzheimer’s disease to the effects of tangles without amyloidβ plaques in the late stage model described here may clarify the progressive stages of Alzheimer’s disease. While Tau hyperphosphorylation and neurofibrillary tangles are eventually sufficient to cause severe neurodegeneration, initial effects on synaptic transmission and the immune response are subtle. In contrast while even with a heavy plaque load little if any neurodegeneration occurs, considerable effects on synaptic transmission and the immune system result, even before plaques are detectable.

Evidence suggests that amyloid β is highly toxic to synapses in a phospho-Tau-dependent manner. Here, I present a hypothesis that links previous evidence from the first rise of amyloid β through to Tau tangles and neurodegeneration. In the immediate vicinity of plaques, concentrated soluble amyloid β occurs in equilibrium with deposited forms. Initially, plaques cover only a small percentage of brain volume. Microglia, by efficiently removing damaged synapses, may prevent spread of damage along the axon, restricting damage to the immediate vicinity of plaques. However, as plaque load increases, as seen in Alzheimer's disease, an individual axon may suffer multiple points of damage, leading to dissociation of Tau, formation of a tangle, and loss of the axon. As more axons suffer this fate, the network eventually degenerates. According to this hypothesis, the degree of plaque load that an individual can tolerate would depend on the efficiency of their microglia in removing amyloid-β-damaged synapses and the distribution of plaques, relative to axon trajectories, would determine the eventual cognitive symptoms.

Neuronal pentraxin 1 (NPTX1) has been implicated in Alzheimer's disease, being present in and around dystrophic neurons in plaques, affecting glutamatergic transmission postsynaptically and mediating effects of amyloidβ. Here, we confirm the presence of NPTX1 around plaques in postmortem Alzheimer's disease brain and report that acutely applied human NPTX1 increases paired-pulse ratio at mouse CA3-CA1 hippocampal synapses, indicating a decrease in glutamate release. In contrast, chronic exposure to NPTX1, NPTX2, or NPTX receptor decreases paired-pulse ratio, mimicking some of the earliest changes in mice expressing familial Alzheimer's disease genes. The peripheral pentraxin, serum amyloid P component (SAP), causes similar synaptic effects to NPTX1. The presence of SAP on amyloid plaques in Alzheimer's disease confirms that it can enter the brain. We show that SAP and neuronal pentraxins can interact and that SAP can enter the brain if the blood-brain barrier is compromised, suggesting that peripheral pentraxins could affect central synaptic transmission via this interaction, especially in the event of blood-brain barrier breakdown.

Objective: To identify communication needs and evaluate the effectiveness of alternative communication strategies for bioterrorism responses.Methods: We provide a framework for evaluating communication needs during a bioterrorism response. Then, using a simulation model of a hypothetical response to anthrax bioterrorism in a large metropolitan area, we evaluate the costs and benefits of alternative strategies for communication during a response.Results: Expected mortality increases significantly with increases in the time for attack detection and announcement; decreases in the rate at which exposed individuals seek and receive prophylaxis; increases in the number of unexposed people seeking prophylaxis; and increases in workload imbalances at dispensing centers. Thus, the timeliness, accuracy, and precision of communications about the mechanisms of exposure and instructions for obtaining prophylaxis and treatment are critical. Investment in strategies that improve adherence to prophylaxis is likely to be highly cost effective, even if the improvement in adherence is modest, and even if such strategies reduce the prophylaxis dispensing rate.Conclusions: Communication during the response to a bioterror attack must involve the right information delivered at the appropriate time in an effective manner from trusted sources. Because the response system for bioterror communication is only fully operationalized once an attack has occurred, tabletop planning and simulation exercises, and other up-front investments in the design of an effective communication strategy, are critical for effective response planning.

αCaMKIIT286A mutant mice lack long-term potentiation (LTP) in the hippocampal CA1 region and are impaired in spatial learning. In situ hybridization confirms that the mutant mice show the same developmental expression of αCaMKII as their wild-type littermates. A simple hypothesis would suggest that if LTP is a substrate for learning, then enriching the environment should cause learning-dependent changes in wild-type mice that have LTP. Such changes would not be seen in LTP-deficient αCaMKIIT286A mutants. Excitatory synaptic currents in CA1 neurons, recorded with patch clamp in brain slices, revealed that enrichment induces an increase in glutamate release probability and a decreased miniature current amplitude. Confocal microscopy also showed dendritic spine density to be reduced. However, contrary to the hypothesis above, these enrichment-induced changes occur only in the mutant mice and are not detectable in wild-type littermates. We suggest that enrichment induces αCaMKII-independent changes in both wild-type and mutant mice. Such changes may be subsequently reversed in wild-type animals via αCaMKII-dependent mechanisms, such as LTP. Reversal of plasticity has long been hypothesized to be essential for the hippocampus to maintain its role in memory processing. The inability to reverse plasticity in αCaMKIIT286A mutant mice would then result in impairment of spatial learning.

Hippocampal pathology is likely to contribute to cognitive disability in Down syndrome, yet the neural network basis of this pathology and its contributions to different facets of cognitive impairment remain unclear. Here we report dysfunctional connectivity between dentate gyrus and CA3 networks in the transchromosomic Tc1 mouse model of Down syndrome, demonstrating that ultrastructural abnormalities and impaired short-term plasticity at dentate gyrus-CA3 excitatory synapses culminate in impaired coding of new spatial information in CA3 and CA1 and disrupted behavior in vivo. These results highlight the vulnerability of dentate gyrus-CA3 networks to aberrant human chromosome 21 gene expression and delineate hippocampal circuit abnormalities likely to contribute to distinct cognitive phenotypes in Down syndrome.

The mouse mutant ducky and its allele ducky2Jrepresent a model for absence epilepsy characterized by spike-wave seizures and cerebellar ataxia. These mice have mutations inCacna2d2, which encodes the α2δ-2 calcium channel subunit. Of relevance to the ataxic phenotype, α2δ-2 mRNA is strongly expressed in cerebellar Purkinje cells (PCs). TheCacna2d2du2Jmutation results in a 2 bp deletion in the coding region and a complete loss of α2δ-2 protein. Here we show thatdu2J/du2Jmice have a 30% reduction in somatic calcium current and a marked fall in the spontaneous PC firing rate at 22°C, accompanied by a decrease in firing regularity, which is not affected by blocking synaptic input to PCs. At 34°C,du2J/du2JPCs show no spontaneous intrinsic activity.Du2J/du2Jmice also have alterations in the cerebellar expression of several genes related to PC function. At postnatal day 21, there is an elevation of tyrosine hydroxylase mRNA and a reduction in tenascin-C gene expression. Althoughdu2J/+ mice have a marked reduction in α2δ-2 protein, they show no fall in PC somatic calcium currents or increase in cerebellar tryrosine hydroxylase gene expression. However,du2J/+ PCs do exhibit a significant reduction in firing rate, correlating with the reduction in α2δ-2. A hypothesis for future study is that effects on gene expression occur as a result of a reduction in somatic calcium currents, whereas effects on PC firing occur as a long-term result of loss of α2δ-2 and/or a reduction in calcium currents and calcium-dependent processes in regions other than the soma.

The output of the hippocampus is largely determined by interaction of the three excitatory pathways that impinge on CA1 pyramidal neurons. These synapses, formed by axons of: (1) CA3 pyramidal neurons; (2) neurons of the entorhinal cortex (EC); and (3) neighboring CA1 neurons, are all potentially plastic. Here, we take advantage of the accessibility of the organotypic slice preparation to identify the type of spines with which each of these pathways forms synapses, at different developmental stages. Recent reports have shown that morphology of dendritic spines is activity-dependent with large mushroom spines being thought to represent stronger synaptic connections than thin or stubby spines. Although in a wide range of preparations, mushroom spines represent only 15% of spines across the whole dendritic tree, we find that this proportion is highly pathway specific. Thus in organotypic slices, the axons of CA3 neurons form synapses with mushroom spines on CA1 neurons in similar to 50% of cases, whereas this spine type is rare (< 10%) in either of the other two pathways. This high proportion of mushroom spines only occurs after spontaneous excitatory activity in the CA1 cells increases over the second week in vitro. Previous studies suggest that pathway specificity also occurs in vivo. in tissue fixed in vivo, it is the synapses of distal apical dendrites thought to be formed by axons originating in the EC that are richer in mushroom spines. Hence, contrary to previous suggestions, the proportion of mushroom spines is clearly not an intrinsic property of the pathway but rather a characteristic dependent on the environment. We suggest that this is most likely a result of the previous activity of the synapses. The fact that, despite the large differences in pathway specificity between preparations, the overall proportion of different spine types remains unchanged, suggests a strong influence of homeostasis across the network. (c) 2006 Wiley-Liss, Inc.

Despite their wide use, the physiological relevance of organotypic slices remains controversial. Such cultures are prepared at 5 days postnatal. Although some local circuitry remains intact, they develop subsequently in isolation from the animal and hence without plasticity due to experience. Development of synaptic connectivity and morphology might be expected to proceed differently under these conditions than in a behaving animal. To address these questions, patch-clamp techniques and confocal microscopy were used in the CA1 region of the rat hippocampus to compare acute slices from the third postnatal week with various stages of organotypic slices. Acute slices prepared at postnatal days (P) 14, 17 and 21 were found to be developmentally equivalent to organotypic slices cultured for 1, 2 and 3 weeks, respectively, in terms of development of synaptic transmission and dendritic morphology. The frequency of inhibitory and excitatory miniature synaptic currents increased in parallel. Development of dendritic length and primary branching as well as spine density and proportions of different spine types were also similar in both preparations, at these corresponding stages. The most notable difference between organotypic and acute slices was a four- to five-fold increase in the absolute frequency of glutamatergic (but not GABAergic) miniature postsynaptic currents in organotypic slices. This was probably related to an increase in complexity of higher order dendritic branching in organotypic slices, as measured by fractal analysis, resulting in an increased total synapse number. Both increased excitatory miniature synaptic current frequency and dendritic complexity were already established during the first week in culture. The level of complexity then stayed constant in both preparations over subsequent stages, with synaptic frequency increasing in parallel. Thus, although connectivity was greater in organotypic slices, once this was established, development continued in both preparations at a remarkably similar rate. We conclude that, for the parameters studied, changes seem to be preprogrammed by 5 days and their subsequent development is largely independent of environment.

We recorded a novel fast GABAergic synaptic current in cerebellar Purkinje cells in rat brain slices using patch-clamp techniques. Because of a relatively low sensitivity to bicuculline, these currents can be recorded under conditions in which basket and stellate cell inputs are blocked. The observations that the novel synaptic currents occur spontaneously only in the presence of serotonin, and the specific limited positions in the slice from which they can be electrically evoked, suggest that the presynaptic cell is the Lugaro cell. Cell-attached recordings confirm that the Lugaro cell is the only interneuron in the cerebellar cortex with firing behavior consistent with the spontaneous activity recorded in Purkinje cells. The input shows a strong presynaptic modulation mediated by GABAAreceptors, resulting in a dynamic range from almost 0 to >90% release probability. Modeling GABAAreceptor responses to different GABA transients suggests that the relatively low sensitivity of the synaptic currents to bicuculline, compared with the higher affinity GABAAreceptor antagonist SR-95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl) pyridazinium), is attributable to an unusually long GABA dwell time and/or high GABA concentration in the synaptic cleft. The significance of this novel input is discussed in relation to other GABAergic synapses impinging on Purkinje cells. We suggest that the release of GABA onto Purkinje cells from Lugaro cells would primarily occur during motor activity under conditions in which the activity of basket and stellate cells might be inhibited.

A novel form of long-term potentiation of glutamatergic synaptic transmission is described in the rat medial habenula nucleus. It occurs when uridine 5'-triphosphate is bath applied at low micromolar concentrations and is prevented by Reactive Blue 2, suggesting that it is mediated by P2Y4 receptors. Uridine 5'-diphosphate can also cause such a Reactive Blue 2-sensitive potentiation, but at higher concentrations (200 microm), suggesting that this might also be an effect on the relatively uridine 5'-diphosphate-insensitive P2Y4 receptor. The potentiation is due to an increase in presynaptic release probability. It requires neither depolarization nor calcium influx postsynaptically and is thus probably non-Hebbian. When potentiation due to low concentrations of uridine 5'-triphosphate is inhibited in the presence of Reactive Blue 2, uridine 5'-triphosphate causes instead a significant inhibition of glutamate release. We suggest that this inhibition may be mediated by a Reactive Blue 2-insensitive P2Y2-like receptor. At higher concentrations of uridine 5'-triphosphate (200 micro m), the inhibitory effect dominates such that even in the absence of Reactive Blue 2 no potentiation is seen.

We provide microarray data comparing genome-wide differential expression and pathology throughout life in four lines of "amyloid" transgenic mice (mutant human APP, PSEN1, or APP/PSEN1) and "TAU" transgenic mice (mutant human MAPT gene). Microarray data were validated by qPCR and by comparison to human studies, including genome-wide association study (GWAS) hits. Immune gene expression correlated tightly with plaques whereas synaptic genes correlated negatively with neurofibrillary tangles. Network analysis of immune gene modules revealed six hub genes in hippocampus of amyloid mice, four in common with cortex. The hippocampal network in TAU mice was similar except that Trem2 had hub status only in amyloid mice. The cortical network of TAU mice was entirely different with more hub genes and few in common with the other networks, suggesting reasons for specificity of cortical dysfunction in FTDP17. This Resource opens up many areas for investigation. All data are available and searchable at http://www.mouseac.org.

Over the past two years, ATP has clearly been shown to act as a co-transmitter with GABA, glycine and probably glutamate in the central nervous system. Our understanding of the ATP-gated P2X receptors is progressing rapidly, and the pharmacology, stoichiometry and subunit combinations of heteropolymeric P2X channels has been substantially elucidated.

1. The effects of the naturally occurring neurosteroid tetrahydrodeoxycorticosterone (THDOC) on GABAA receptor-mediated miniature, spontaneous and evoked IPSCs was tested using patch-clamp techniques in slices of hippocampus and cerebellum from rats at two developmental stages ( approximately 10 and approximately 20 days postnatal). The cells studied were hippocampal granule cells and cerebellar Purkinje and granule cells. 2. Most miniature GABAergic currents (mIPSCs) decayed with two exponentials and neurosteroids caused a approximately 4-fold increase in the decay time constant of the second exponential at the highest concentration used (2 microM). Similar effects were seen at high concentrations of THDOC (1-2 microM) in all cell groups tested. No effects were seen on amplitude or rise time of mIPSCs. 3. The effects of THDOC (1 microM) were shown to be stereoselective and rapidly reversible, indicating that the neurosteroid binds to the GABAA receptor, rather than acting genomically. 4. At concentrations of THDOC likely to occur physiologically (50-100 nM), the decay time of IPSCs was also enhanced (25-50 %) in all cerebellar cell groups tested. In contrast, at 100 nM THDOC, seven of 11 hippocampal granule cells were sensitive from the 10 day group but the 20 day hippocampal granule cells showed no significant enhancement in the presence of these lower concentrations of THDOC. 5. The differences in sensitivity of hippocampal and cerebellar cells to THDOC are compared to data reported in the literature on regional development of expression of different receptor subunits in the brain and it is suggested that the progressive relative insensitivity of the 20 day hippocampal cells may depend on increasing expression of the delta subunit of the GABAA receptor and possibly an increase in the alpha4 subunit.

One of the major controversies in neuroscience concerns whether the expression of long-term potentiation (LTP) is a pre- or postsynaptic phenomenon, with apparently contradictory data being the norm. The model that is outlined in this article combines anatomical and electrophysiological evidence to allow apparently contradictory data to be compatible. Development of LTP involves both influx of Ca 2+ through NMDA receptors, and activation of another factor, perhaps the metabotropic glutamate receptor. These two processes might result, respectively, in the insertion or activation of additional postsynaptic receptors, and the growth of microfilaments that could split simple synapses into perforated synapses, consisting of multiple active zones. Whether the latter occurred, and at what rate, would be likely to depend on multiple factors, such as temperature, the metabolic state of the cell, buffering of Ca 2+ , and the concentration of factors such as nitric oxide. These subtle experimental variables would thus determine whether the dominant effect observed was pre- or postsynaptic.