Dataset Information

Ex Vivo Recording of Axonal Transport Dynamics on Postnatal Organotypic Cortical Slices.

ABSTRACT: Axonal transport is a physiological process adopted by neurons to transport organelles, proteins, and other molecules along their axonal projections. Here, we describe a step-by-step protocol to record the dynamics of axonal transport along the projections of callosal neurons by combining the in utero electroporation technique with the preparation of postnatal organotypic cortical slices. This ex vivo protocol has been developed to investigate axonal transport in a physiological setting closely reproducing the in vivo environment. For complete details on the use and execution of this protocol, please refer to Even et al. (2019).

SUBMITTER: Turchetto S

PROVIDER: S-EPMC7757112 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Ex Vivo Recording of Axonal Transport Dynamics on Postnatal Organotypic Cortical Slices.

Turchetto Silvia S Broix Loic L Nguyen Laurent L

STAR protocols 20201017 3

Axonal transport is a physiological process adopted by neurons to transport organelles, proteins, and other molecules along their axonal projections. Here, we describe a step-by-step protocol to record the dynamics of axonal transport along the projections of callosal neurons by combining the in utero electroporation technique with the preparation of postnatal organotypic cortical slices. This ex vivo protocol has been developed to investigate axonal transport in a physiological se ...[more]

PMID: 33377025

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Project description:BACKGROUND:Epilepsy is a prevalent neurological disorder worldwide. It is characterized by an enduring predisposition to generate seizures and its development is accompanied by alterations in many cellular processes. Organotypic slice cultures represent a multicellular environment with the potential to assess biological mechanisms, and they are used as a starting point for refining molecules for in vivo studies. Here, we investigated organotypic slice cultures as a model of epilepsy. METHODS:We assessed, by electrophysiological recordings, the spontaneous activity of organotypic slices maintained under different culture protocols. Moreover, we evaluated, through molecular-based approaches, neurogenesis, neuronal death, gliosis, expression of proinflammatory cytokines, and activation of NLRP3 inflammasome (nucleotide-binding, leucine-rich repeat, pyrin domain) as biomarkers of neuroinflammation. RESULTS:We demonstrated that organotypic slices, maintained under a serum deprivation culture protocol, develop epileptic-like activity. Furthermore, throughout a comparative study with slices that do not depict any epileptiform activity, slices with epileptiform activity were found to display significant differences in terms of inflammation-related features, such as (1) increased neuronal death, with higher incidence in CA1 pyramidal neurons of the hippocampus; (2) activation of astrocytes and microglia, assessed through western blot and immunohistochemistry; (3) upregulation of proinflammatory cytokines, specifically interleukin-1? (IL-1?), interleukin-6, and tumor necrosis factor ?, revealed by qPCR; and (4) enhanced expression of NLRP3, assessed by western blot, together with increased NLRP3 activation, showed by IL-1? quantification. CONCLUSIONS:Thus, organotypic slice cultures gradually deprived of serum mimic the epileptic-like activity, as well as the inflammatory events associated with in vivo epilepsy. This system can be considered a new tool to explore the interplay between neuroinflammation and epilepsy and to screen potential drug candidates, within the inflammatory cascades, to reduce/halt epileptogenesis.

Dataset Information

Ex Vivo Recording of Axonal Transport Dynamics on Postnatal Organotypic Cortical Slices.

Publications

<i>Ex Vivo</i> Recording of Axonal Transport Dynamics on Postnatal Organotypic Cortical Slices.

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