High-Resolution Imaging and Evaluation of Spines in Organotypic Hippocampal Slice Cultures

Sündermann, Frederik; Golovyashkina, Nataliya; Tackenberg, Christian; Brandt, Roland; Bakota, Lidia

doi:10.1007/978-1-61779-536-7_24

Cited by 14 publications

(10 citation statements)

References 13 publications

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“…The method employed confocal high-resolution imaging of fixed slices followed by algorithm-based image analysis with the program 3DMA neuron. We and others had previously shown that this approach allows sensitive and robust evaluation of dendritic spines (Santuccione et al, 2013; Sundermann et al, 2012; Tackenberg et al, 2013). We observed that the density of spines was reduced in most regions and time points in APP SDL transgenic cultures compared to control cultures (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In short, one or two week old APP SDL transgenic or non-transgenic C57BL/6 mice were decapitated, brains were removed, both hippocampi isolated and cut into 400 μm thick slices using McIllwain tissue chopper (Gabler, Bad Schwalbach, Germany). Slices were cultured as described previously (Sundermann et al, 2012; Tackenberg and Brandt, 2009). On day 12 or day 17 in vitro slice cultures were infected with Sindbis virus using droplet method (Shahani et al, 2006).…”

Section: Methodsmentioning

confidence: 99%

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Aβ-mediated spine changes in the hippocampus are microtubule-dependent and can be reversed by a subnanomolar concentration of the microtubule-stabilizing agent epothilone D

et al. 2016

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Dendritic spines represent the major postsynaptic input of excitatory synapses. Loss of spines and changes in their morphology correlate with cognitive impairment in Alzheimer’s disease (AD) and are thought to occur early during pathology. Therapeutic intervention at a preclinical stage of AD to modify spine changes might thus be warranted. To follow the development and to potentially interfere with spine changes over time, we established a long term ex vivo model from organotypic cultures of the hippocampus from APP transgenic and control mice. The cultures exhibit spine loss in principal hippocampal neurons, which closely resembles the changes occurring in vivo, and spine morphology progressively changes from mushroom-shaped to stubby. We demonstrate that spine changes are completely reversed within few days after blocking amyloid-β (Aβ) production with the gamma-secretase inhibitor DAPT. We show that the microtubule disrupting drug nocodazole leads to spine loss similar to Aβ expressing cultures and suppresses DAPT-mediated spine recovery in slices from APP transgenic mice. Finally, we report that epothilone D (EpoD) at a subnanomolar concentration, which slightly stabilizes microtubules in model neurons, completely reverses Aβ-induced spine loss and increases thin spine density. Taken together the data indicate that Aβ causes spine changes by microtubule destabilization and that spine recovery requires microtubule polymerization. Moreover, our results suggest that a low, subtoxic concentration of EpoD is sufficient to reduce spine loss during the preclinical stage of AD.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Aβ-mediated spine changes in the hippocampus are microtubule-dependent and can be reversed by a subnanomolar concentration of the microtubule-stabilizing agent epothilone D

et al. 2016

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“…Hippocampal slice cultures were prepared from mice of either sex and processed as described previously [ 5 , 52 ]. On day 12 in vitro slice cultures were infected with Sindbis virus.…”

Section: Methodsmentioning

confidence: 99%

Region-specific dendritic simplification induced by Aβ, mediated by tau via dysregulation of microtubule dynamics: a mechanistic distinct event from other neurodegenerative processes

Golovyashkina

Penazzi

Ballatore

et al. 2015

Mol Neurodegeneration

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BackgroundDendritic simplification, a key feature of the neurodegenerative triad of Alzheimer’s disease (AD) in addition to spine changes and neuron loss, occurs in a region-specific manner. However, it is unknown how changes in dendritic complexity are mediated and how they relate to spine changes and neuron loss.ResultsTo investigate the mechanisms of dendritic simplification in an authentic CNS environment we employed an ex vivo model, based on targeted expression of enhanced green fluorescent protein (EGFP)-tagged constructs in organotypic hippocampal slices of mice. Algorithm-based 3D reconstruction of whole neuron morphology in different hippocampal regions was performed on slices from APPSDL-transgenic and control animals. We demonstrate that induction of dendritic simplification requires the combined action of amyloid beta (Aβ) and human tau. Simplification is restricted to principal neurons of the CA1 region, recapitulating the region specificity in AD patients, and occurs at sites of Schaffer collateral input. We report that γ-secretase inhibition and treatment with the NMDA-receptor antagonist, CPP, counteract dendritic simplification. The microtubule-stabilizing drug epothilone D (EpoD) induces simplification in control cultures per se. Similar morphological changes were induced by a phosphoblocking tau construct, which also increases microtubule stability. In fact, low nanomolar concentrations of naturally secreted Aβ decreased phosphorylation at S262 in a cellular model, a site which is known to directly modulate tau-microtubule interactions.ConclusionsThe data provide evidence that dendritic simplification is mechanistically distinct from other neurodegenerative events and involves microtubule stabilization by dendritic tau, which becomes dephosphorylated at certain sites. They imply that treatments leading to an overall decrease of tau phosphorylation might have a negative impact on neuronal connectivity.

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“…Imaging of the dissociated cells was performed after 24 h of EGFP expression (Malinow et al, 2010). In organotypic hippocampal slice culture, virus infection was performed as described previously (Sündermann et al, 2012). Briefly, 12 DIV slices were infected with the pSinRep5 EGFP virus following the droplet method.…”

Section: Sindbis Virus Infectionmentioning

confidence: 99%

Early Effects of Aβ Oligomers on Dendritic Spine Dynamics and Arborization in Hippocampal Neurons

Ortiz-Sanz

Gaminde-Blasco

Valero

et al. 2020

Front. Synaptic Neurosci.

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Alzheimer's disease (AD) is a neurodegenerative disorder that leads to impaired memory and cognitive deficits. Spine loss as well as changes in spine morphology correlates with cognitive impairment in this neurological disorder. Many studies in animal models and ex vivo cultures indicate that amyloid β-peptide (Aβ) oligomers induce synaptic damage early during the progression of the disease. Here, in order to determine the events that initiate synaptic alterations, we acutely applied oligomeric Aβ to primary hippocampal neurons and an ex vivo model of organotypic hippocampal cultures from a mouse after targeted expression of EGFP to allow high-resolution imaging and algorithmbased evaluation of spine changes. Dendritic spines were classified as thin, stubby or mushroom, based on morphology. In vivo, time-lapse imaging showed that the three spine types were relatively stable, although their stability significantly decreased after treatment with Aβ oligomers. Unexpectedly, we observed that the density of total dendritic spines increased in organotypic hippocampal slices treated with Aβ compared to control cultures. Specifically, the fraction of stubby spines significantly increased, while mushroom and thin spines remained unaltered. Pharmacological tools revealed that acute Aβ oligomers induced spine changes through mechanisms involving CaMKII and integrin β1 activities. Additionally, analysis of dendritic complexity based on a 3D reconstruction of the whole neuron morphology showed an increase in the apical dendrite length and branching points in CA1 organotypic hippocampal slices treated with Aβ. In contrast to spines, the morphological changes were affected by integrin β1 but not by CaMKII inhibition. Altogether, these data indicate that the Aβ oligomers exhibit early dual effects by acutely enhancing dendritic complexity and spine density.

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High-Resolution Imaging and Evaluation of Spines in Organotypic Hippocampal Slice Cultures

Cited by 14 publications

References 13 publications

Aβ-mediated spine changes in the hippocampus are microtubule-dependent and can be reversed by a subnanomolar concentration of the microtubule-stabilizing agent epothilone D

Aβ-mediated spine changes in the hippocampus are microtubule-dependent and can be reversed by a subnanomolar concentration of the microtubule-stabilizing agent epothilone D

Region-specific dendritic simplification induced by Aβ, mediated by tau via dysregulation of microtubule dynamics: a mechanistic distinct event from other neurodegenerative processes

Early Effects of Aβ Oligomers on Dendritic Spine Dynamics and Arborization in Hippocampal Neurons

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