Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

Ippolito, Dominic; Eroğlu, Çağla

doi:10.3791/2270-v

Cited by 31 publications

(32 citation statements)

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“…To estimate the co-localization between the spots of the pre-synaptic marker VGlut1 and the post-synaptic marker homer 1b/c, the plugin puncta analyzer was applied, as previously described. 102 Briefly, both channels were converted using the maximum intensity of the Z projection and merged. Following the identification of a specific ROI corresponding to all the neurites of the cell, and keeping default parameters, the plugin quantifies the puncta in each channel and the co-localized puncta between the two channels.…”

Section: Methodsmentioning

confidence: 99%

Axonal plasticity in response to active forces generated through magnetic nano-pulling

et al. 2023

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Section: Methodsmentioning

confidence: 99%

Axonal plasticity in response to active forces generated through magnetic nano-pulling

et al. 2023

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“…During the post processing of captured images, each z-stack was converted into 5 maximum projection images (MPI) by combining three optical sections using ImageJ software. The number of co-localized excitatory thalamocortical (VGluT2/PSD95) synaptic puncta were obtained using the ImageJ plugin Puncta Analyzer (Ippolito and Eroglu 2010).…”

Section: Methodsmentioning

confidence: 99%

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates

Chen

Wolfe

Bindu

et al. 2023

Preprint

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Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds and rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and ex vivo human brain slices although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial-specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. Vasculature-secreted Hevin (a synaptogenic protein) rescued synaptic deficits in a mouse model.

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“…These analyses were done blind to the individual genotypes. We used the using the ImageJ Puncta Analyzer plugin (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) for analyses of synapse density in brain sections as described 71,72 . Multiple studies have used this approach providing supporting as an accurate estimator of synapse number [73][74][75][76] , including validation of synapse numbers previously detected ultrastructurally and by electrophysiologically 72,[77][78][79] .…”

Section: Methodsmentioning

confidence: 99%

“…We used the using the ImageJ Puncta Analyzer plugin (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) for analyses of synapse density in brain sections as described 71,72 . Multiple studies have used this approach providing supporting as an accurate estimator of synapse number [73][74][75][76] , including validation of synapse numbers previously detected ultrastructurally and by electrophysiologically 72,[77][78][79] . For this, 20 μm thick cryosections from 2-4 mo old Khsrp −/− , Khsrp +/+ and Khsrp fl/fl × Syn1:Cre mice were immunostained with antisynaptophysin and anti-PSD95 as above.…”

Section: Methodsmentioning

confidence: 99%

KHSRP loss increases neuronal growth and synaptic transmission and alters memory consolidation through RNA stabilization

et al. 2022

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The KH-type splicing regulatory protein (KHSRP) is an RNA-binding protein linked to decay of mRNAs with AU-rich elements. KHSRP was previously shown to destabilize Gap43 mRNA and decrease neurite growth in cultured embryonic neurons. Here, we have tested functions of KHSRP in vivo. We find upregulation of 1460 mRNAs in neocortex of adult Khsrp−/− mice, of which 527 bind to KHSRP with high specificity. These KHSRP targets are involved in pathways for neuronal morphology, axon guidance, neurotransmission and long-term memory. Khsrp−/− mice show increased axon growth and dendritic spine density in vivo. Neuronal cultures from Khsrp−/− mice show increased axon and dendrite growth and elevated KHSRP-target mRNAs, including subcellularly localized mRNAs. Furthermore, neuron-specific knockout of Khsrp confirms these are from neuron-intrinsic roles of KHSRP. Consistent with this, neurons in the hippocampus and infralimbic cortex of Khsrp−/− mice show elevations in frequency of miniature excitatory postsynaptic currents. The Khsrp−/− mice have deficits in trace conditioning and attention set-shifting tasks compared Khsrp+/+ mice, indicating impaired prefrontal- and hippocampal-dependent memory consolidation with loss of KHSRP. Overall, these results indicate that deletion of KHSRP impairs neuronal development resulting in alterations in neuronal morphology and function by changing post-transcriptional control of neuronal gene expression.

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Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

Cited by 31 publications

References 0 publications

Axonal plasticity in response to active forces generated through magnetic nano-pulling

Axonal plasticity in response to active forces generated through magnetic nano-pulling

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates

KHSRP loss increases neuronal growth and synaptic transmission and alters memory consolidation through RNA stabilization

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