24 research outputs found

    Using SuperClomeleon to measure changes in intracellular chloride during development and after early life stress

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    Intraneuronal chloride concentrations ([Cl- ]i) decrease during development resulting in a shift from depolarizing to hyperpolarizing γ-aminobutyric acid (GABA) responses via chloride-permeable GABAA receptors. This GABA shift plays a pivotal role in postnatal brain development, and can be strongly influenced by early life experience. Here, we assessed the applicability of the recently developed fluorescent SuperClomeleon (SClm) sensor to examine changes in [Cl- ]i using two-photon microscopy in brain slices. We used SClm mice of both sexes to monitor the developmental decrease in neuronal chloride levels in organotypic hippocampal cultures. We could discern a clear reduction in [Cl-]i between DIV3 and DIV9 (equivalent to the second postnatal week in vivo) and a further decrease in some cells until DIV22. In addition, we assessed alterations in [Cl- ]i in the medial prefrontal cortex (mPFC) of P9 male SClm mouse pups after early life stress (ELS). ELS was induced by limiting nesting material between P2 and P9. ELS induced a shift towards higher (i.e. immature) chloride levels in layer 2/3 cells in the mPFC. Although conversion from SClm fluorescence to absolute chloride concentrations proved difficult, our study underscores that the SClm sensor is a powerful tool to measure physiological changes in [Cl- ]i in brain slices

    Knock-in models related to Alzheimer’s disease: synaptic transmission, plaques and the role of microglia

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    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

    Knock-in models related to Alzheimer’s disease: synaptic transmission, plaques and the role of microglia

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    Funder: Cure Alzheimer's Fund; doi: http://dx.doi.org/10.13039/100007625Funder: UK Dementia Research Institute (GB)Funder: Censejo Nacional de Ciencia Tecnilogia (MX)Funder: Alzheimerfonden; doi: http://dx.doi.org/10.13039/501100008599Funder: Dolby Family FundAbstract: 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

    Waterdragers

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    In het waterbeheer en de ruimtelijke ordening wordt steeds meer gebruik gemaakt van de watersysteembenadering. Dit rapport behandelt in vier essays de aspecten die bijdragen aan een goede afstemming en communicatie tussen deze beleidsvelden. Het betreft het omgaan met definities, het proces van planvorming naar uitvoering, het gebruik van modellen, en ontwerp als instrumenten voor planvorming. Geconcludeerd wordt dat maatwerk, draagvlak en visualisatie sleutelbegrippen zijn voor een succesvolle integratie van waterbeheer en ruimtelijke ordening

    Waterdragers

    No full text
    In het waterbeheer en de ruimtelijke ordening wordt steeds meer gebruik gemaakt van de watersysteembenadering. Dit rapport behandelt in vier essays de aspecten die bijdragen aan een goede afstemming en communicatie tussen deze beleidsvelden. Het betreft het omgaan met definities, het proces van planvorming naar uitvoering, het gebruik van modellen, en ontwerp als instrumenten voor planvorming. Geconcludeerd wordt dat maatwerk, draagvlak en visualisatie sleutelbegrippen zijn voor een succesvolle integratie van waterbeheer en ruimtelijke ordening

    Using SuperClomeleon to measure changes in intracellular chloride during development and after early life stress

    No full text
    Intraneuronal chloride concentrations ([Cl- ]i) decrease during development resulting in a shift from depolarizing to hyperpolarizing γ-aminobutyric acid (GABA) responses via chloride-permeable GABAA receptors. This GABA shift plays a pivotal role in postnatal brain development, and can be strongly influenced by early life experience. Here, we assessed the applicability of the recently developed fluorescent SuperClomeleon (SClm) sensor to examine changes in [Cl- ]i using two-photon microscopy in brain slices. We used SClm mice of both sexes to monitor the developmental decrease in neuronal chloride levels in organotypic hippocampal cultures. We could discern a clear reduction in [Cl-]i between DIV3 and DIV9 (equivalent to the second postnatal week in vivo) and a further decrease in some cells until DIV22. In addition, we assessed alterations in [Cl- ]i in the medial prefrontal cortex (mPFC) of P9 male SClm mouse pups after early life stress (ELS). ELS was induced by limiting nesting material between P2 and P9. ELS induced a shift towards higher (i.e. immature) chloride levels in layer 2/3 cells in the mPFC. Although conversion from SClm fluorescence to absolute chloride concentrations proved difficult, our study underscores that the SClm sensor is a powerful tool to measure physiological changes in [Cl- ]i in brain slices
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