The uptake of gamma-aminobutyrate by organotypic cultures of chick spinal cord.
Ontology highlight
ABSTRACT:
SUBMITTER: Tunnicliff G
PROVIDER: S-EPMC1177784 | biostudies-other | 1973 May
REPOSITORIES: biostudies-other
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Project description:BackgroundSynaptic dysfunction, named synaptopathy, due to inflammatory status of the central nervous system (CNS) is a recognized factor potentially underlying both motor and cognitive dysfunctions in neurodegenerative diseases. To gain knowledge on the mechanistic interplay between local inflammation and synapse changes, we compared two diverse inflammatory paradigms, a cytokine cocktail (CKs; IL-1?, TNF-?, and GM-CSF) and LPS, and their ability to tune GABAergic current duration in spinal cord cultured circuits.MethodsWe exploit spinal organotypic cultures, single-cell electrophysiology, immunocytochemistry, and confocal microscopy to explore synaptic currents and resident neuroglia reactivity upon CK or LPS incubation.ResultsLocal inflammation in slice cultures induced by CK or LPS stimulations boosts network activity; however, only CKs specifically reduced GABAergic current duration. We pharmacologically investigated the contribution of GABAAR ?-subunits and suggested that a switch of GABAAR ?1-subunit might have induced faster GABAAR decay time, weakening the inhibitory transmission.ConclusionsLower GABAergic current duration could contribute to providing an aberrant excitatory transmission critical for pre-motor circuit tasks and represent a specific feature of a CK cocktail able to mimic an inflammatory reaction that spreads in the CNS. Our results describe a selective mechanism that could be triggered during specific inflammatory stress.
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Project description:The objective of this paper is to describe in detail the method of organotypic longitudinal spinal cord slice culture and the scientific basis for its potential utility. The technique is based on the interface method, which was described previously and thereafter was modified in our laboratory. The most important advantage of the presented model is the preservation of the intrinsic spinal cord fiber tract and the ventrodorsal polarity of the spinal cord. All the processes occurring during axonal growth, regeneration, synapse formation, and myelination could be visualized while being cultured in vitro for up to 4-5 weeks after the slices had been isolated. Both pups and adult animals can undergo the same, equally efficient procedures when going by the protocol in question. The urgent need for an appropriate in vitro model for spinal cord regeneration results from a greater number of clinical trials concerning regenerative medicine in the spinal cord injury and from still insufficient knowledge of the molecular mechanisms involved in the neuroreparative processes. The detailed method of organotypic longitudinal spinal cord slice culture is accompanied by examples of its application to studying biological processes to which both the CNS inhabiting and grafted cells are subjected.
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Project description:Graphene-based nanomaterials are increasingly engineered as components of biosensors, interfaces or drug delivery platforms in neuro-repair strategies. In these developments, the mostly used derivative of graphene is graphene oxide (GO). To tailor the safe development of GO nanosheets, we need to model in vitro tissue responses, and in particular the reactivity of microglia, a sub-population of neuroglia that acts as the first active immune response, when challenged by GO. Here, we investigated central nervous system (CNS) tissue reactivity upon long-term exposure to GO nanosheets in 3D culture models. We used the mouse organotypic spinal cord cultures, ideally suited for studying long-term interference with cues delivered at controlled times and concentrations. In cultured spinal segments, the normal presence, distribution and maturation of anatomically distinct classes of neurons and resident neuroglial cells are preserved. Organotypic explants were developed for 2 weeks embedded in fibrin glue alone or presenting GO nanosheets at 10, 25 and 50 ?g/mL. We addressed the impact of such treatments on premotor synaptic activity monitored by patch clamp recordings of ventral interneurons. We investigated by immunofluorescence and confocal microscopy the accompanying glial responses to GO exposure, focusing on resident microglia, tested in organotypic spinal slices and in isolated neuroglia cultures. Our results suggest that microglia reactivity to accumulation of GO flakes, maybe due to active phagocytosis, may trim down synaptic activity, although in the absence of an effective activation of inflammatory response and in the absence of neuronal cell death.
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Project description:In vertebrate embryos, most spinal commissural axons cross the ventral midline (VM) and project either alongside or significant distances away from the floor plate (FP). The upregulation of repulsive Robo1/2 receptors on postcrossing commissural axons, in mammals, presumably allows these axons to respond to the midline-associated repellents, Slit1-3, facilitating their expulsion from, and prohibiting their reentry into, the FP. Compelling data suggest that Robo3 represses Robo1/2 function on precrossing axons and that Robo1/2 inhibit attractive guidance receptors on postcrossing axons, thereby ensuring that decussated axons are selectively responsive to midline Slits. However, whether Robo1/2 expel decussated commissural axons from the VM and/or prevent their reentry into the FP has not been explicitly established in vivo. Furthermore, some commissural axons do not require Robo1/2 to elaborate appropriate contralateral projections in the mouse spinal cord. Here, we use unilateral in ovo electroporation together with Atoh1 and Neurog1 enhancer elements to visualize, and assess the consequences of manipulating Robo expression on, dl1 and dl2 chick commissural axons. In response to misexpressing a cytoplasmic truncation of Robo1 and/or Robo2, which should block all Robo-ligand interactions, postcrossing commissural axons extend alongside, but do not project away from or reenter the FP. In contrast, misexpression of full-length Robo2 prevents many commissural axons from crossing the VM. Together, these findings support key and selective in vivo roles for Robo receptors in presumably altering the responsiveness of decussated commissural axons and facilitating their expulsion from the VM within the chick spinal cord.
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