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-1β, TNF-α, 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 α-subunits and suggested that a switch of GABAAR α1-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 aim of the present report was to investigate whether, in the mammalian spinal cord, cell death induced by transient excitotoxic stress could trigger activation and proliferation of endogenous neuroprogenitor cells as a potential source of a lesion repair process and the underlying time course. Because it is difficult to address these issues in vivo, we used a validated model of spinal injury based on rat organotypic slice cultures that retain the fundamental tissue cytoarchitecture and replicate the main characteristics of experimental damage to the whole spinal cord. Excitotoxicity evoked by 1 h kainate application produced delayed neuronal death (40%) peaking after 1 day without further losses or destruction of white matter cells for up to 2 weeks. After 10 days, cultures released a significantly larger concentration of endogenous glutamate, suggesting functional network plasticity. Indeed, after 1 week the total number of cells had returned to untreated control level, indicating substantial cell proliferation. Activation of progenitor cells started early as they spread outside the central area, and persisted for 2 weeks. Although expression of the neuronal progenitor phenotype was observed at day 3, peaked at 1 week and tapered off at 2 weeks, very few cells matured to neurons. Astroglia precursors started proliferating later and matured at 2 weeks. These data show insult-related proliferation of endogenous spinal neuroprogenitors over a relatively brief time course, and delineate a narrow temporal window for future experimental attempts to drive neuronal maturation and for identifying the factors regulating this process.
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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:Cellular differentiation is a tightly regulated process under the control of intricate signaling and transcription factors interaction network working in coordination. These interactions make the systems dynamic, robust and stable but also difficult to dissect. In the spinal cord, recent work has shown that a network of FGF, WNT and Retinoic Acid (RA) signaling factors regulate neural maturation by directing the activity of a transcription factor network that contains CDX at its core. Here we have used partial and ordinary (Hill) differential equation based models to understand the spatiotemporal dynamics of the FGF/WNT/RA and the CDX/transcription factor networks, alone and in combination. We show that in both networks, the strength of interaction among network partners impacts the dynamics, behavior and output of the system. In the signaling network, interaction strength determine the position and size of discrete regions of cell differentiation and small changes in the strength of the interactions among networking partners can result in a signal overriding, balancing or oscillating with another signal. We also show that the spatiotemporal information generated by the signaling network can be conveyed to the CDX/transcription network to produces a transition zone that separates regions of high cell potency from regions of cell differentiation, in agreement with most in vivo observations. Importantly, one emerging property of the networks is their robustness to extrinsic disturbances, which allows the system to retain or canalize NP cells in developmental trajectories. This analysis provides a model for the interaction conditions underlying spinal cord cell maturation during embryonic axial elongation.
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Project description:Development requires tissue growth as well as cell diversification. To address how these processes are coordinated, we analyzed the development of molecularly distinct domains of neural progenitors in the mouse and chick neural tube. We show that during development, these domains undergo changes in size that do not scale with changes in overall tissue size. Our data show that domain proportions are first established by opposing morphogen gradients and subsequently controlled by domain-specific regulation of differentiation rate but not differences in proliferation rate. Regulation of differentiation rate is key to maintaining domain proportions while accommodating both intra- and interspecies variations in size. Thus, the sequential control of progenitor specification and differentiation elaborates pattern without requiring that signaling gradients grow as tissues expand.
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Project description:The genetic dissection of spinal circuits is an essential new means for understanding the neural basis of mammalian behavior. Molecular targeting of specific neuronal populations, a key instrument in the genetic dissection of neuronal circuits in the mouse model, is a complex and time-demanding process. Here we present a circuit-deciphering 'tool box' for fast, reliable and cheap genetic targeting of neuronal circuits in the developing spinal cord of the chick. We demonstrate targeting of motoneurons and spinal interneurons, mapping of axonal trajectories and synaptic targeting in both single and populations of spinal interneurons, and viral vector-mediated labeling of pre-motoneurons. We also demonstrate fluorescent imaging of the activity pattern of defined spinal neurons during rhythmic motor behavior, and assess the role of channel rhodopsin-targeted population of interneurons in rhythmic behavior using specific photoactivation.
| S-EPMC4231727 | biostudies-literature
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 μg/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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Project description:BackgroundWNT1 and WNT3A drive a dorsal to ventral gradient of β-catenin-dependent Wnt signaling in the developing spinal cord. However, the identity of the receptors mediating downstream functions remains poorly understood.ResultsIn this report, we show that the spatiotemporal expression patterns of FZD10 and WNT1/WNT3A are highly correlated. We further show that in the presence of LRP6, FZD10 promotes WNT1 and WNT3A signaling using an 8xSuperTopFlash reporter assay. Consistent with a functional role for FZD10, we demonstrate that FZD10 is required for proliferation in the spinal cord. Finally, by using an in situ proximity ligation assay, we observe an interaction between FZD10 and WNT1 and WNT3A proteins.ConclusionsTogether, our results identify FZD10 as a receptor for WNT1 and WNT3A in the developing chick spinal cord.
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Project description:Hydrogen sulfide (H₂S) is an endogenous gasotransmitter recognized as an essential body product with a dual, biphasic action. It can function as an antioxidant and a cytoprotective, but also as a poison with a high probability of causing brain damage when present at noxious levels. In a previous study, we measured toxic liquoral levels of H₂S in sporadic amyotrophic lateral sclerosis (ALS) patients and in the familial ALS (fALS) mouse model, SOD1G93A. In addition, we experimentally demonstrated that H₂S is extremely and selectively toxic to motor neurons, and that it is released by glial cells and increases Ca2+ concentration in motor neurons due to a lack of ATP. The presented study further examines the effect of toxic concentrations of H₂S on embryonic mouse spinal-cord cultures. We performed a proteomic analysis that revealed a significant H₂S-mediated activation of pathways related to oxidative stress and cell death, particularly the Nrf-2-mediated oxidative stress response and peroxiredoxins. Furthermore, we report that Na₂S (a stable precursor of H₂S) toxicity is, at least in part, reverted by the Bax inhibitor V5 and by necrostatin, a potent necroptosis inhibitor.
| S-EPMC6070951 | biostudies-literature