- Blood–brain barrier
- Review ARTICLE
- Gap junctions in the nervous system.
- Astrocytic Kir channels and gap junctions account for spontaneous epileptic seizure
Advertisement Hide. This process is experimental and the keywords may be updated as the learning algorithm improves. This is a preview of subscription content, log in to check access. Intercellular Communication through Gap Junctions. Prog Cell Res Vol.
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EMBO J ; 7: — Gap junction structures. Variation and conservation in connexon conformation and packing. Stauffer KA, Unwin N. Structure of gap junction channels. The scientific community is just now starting to consider the possible role of these membrane channels in the pathophysiology of epilepsy. In summary, although there is no doubt that GJs, connexins and pannexins are intimately related to epilepsy and seizure generation, the specific details of exactly how they are involved and how we can modulate their function for therapeutic purposes remain to be elucidated.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Allen, N. Nature , — Andrade-Rozental, A. Brain Res. Aronica, E. Expression of connexin 43 and connexin 32 gap-junction proteins in epilepsy-associated brain tumors and in the perilesional epileptic cortex. Acta Neuropathol. Badaut, J. Brain water mobility decreases after astrocytic aquaporin-4 inhibition using RNA interference. Blood Flow Metab. Bao, L. Pannexin membrane channels are mechanosensitive conduits for ATP.
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The role of gap junctions in seizures. Chang, M.
Gap junctions in the nervous system.
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Astrocytic Kir channels and gap junctions account for spontaneous epileptic seizure
Upregulation of gap junction connexin 32 with epileptiform activity in the isolated mouse hippocampus. Lin, J. Gap-junction-mediated propagation and amplification of cell injury. Meanwhile the receiver and the actuator can both designed to be turned on in a cre-dependent manner. This design could make PARIS more versatile in detecting GJC between specific cells labeled by cre-lines without the contamination of the autonomous signal. Future refinements to PARIS include the use of the new actuators we have screened combining a receiver with higher pH sensitivity, thereby increasing both the signal-to-noise ratio and temporal resolution, allowing for an even wider range of in vitro and in vivo applications.
All sequences were verified using Sanger sequencing in our in-house facility sequencing platform in the School of Life Sciences of the Peking University. For cultured cell expression experiments, genes were cloned into the pcDNA3. The cells were negative for mycoplasma. Alternatively, in some experiments, we used sequential transfection, in which the cells were first transfected with the pHluorinCAAX construct; 6 hr later, the medium was changed and the cells were transfected with the ArchT-BFP2 construct.
The medium was changed 6 hr later, and the cells were incubated for 24 hr prior to imaging. The medium was replaced 10 hr later, and the cells were incubated for an additional 24 hr prior to imaging. Laser light was delivered via a Sutter DG-4 equipped with a xenon lamp. The recording electrodes were filled with an internal solution containing in mM : K-gluconate, 10 KCl, 2 MgCl 2 , 2. Currents were smoothed using a 20 ms moving average in order to minimize 50 Hz AC noise.
For simultaneous optical and electrophysiology recordings, the Sutter DG-4 light source was triggered by the HEKA EPC10 amplifier in order to synchronize the electrophysiological recording with the light simulation. All recordings were performed at room temperature. Cardiomyocytes CMs were enzymatically dissociated from the ventricles of neonatal P0 Sprague-Dawley rats, and 0. Beating rate was measured using ImageJ analysis of the white-field images. Liqun Luo. Krasavietz-Gal4 Dubnau et al. Donggen Luo which has been verified by genotyping and sequencing the mutated site.
Yi Rao. All the transgenic flies have been genotype verified by sequencing in our in-house facility sequencing platform in the School of Life Sciences of the Peking University. The brain was then transferred to a glass-bottom chamber containing ALH for confocal imaging. The brain was held in place using a custom-made platinum frame and positioned with the anterior surface of the brain toward the objective for imaging and stimulation of the antennal lobe , or with the posterior surface toward the objective for imaging and stimulation of the lateral horn.
ArchT was photostimulated using a nm scanning laser at 0. The nm imaging was continued 2—5 s intervals for 1—2 min after the nm stimulation in order to record the fluorescence recovery of the receiver. Fly brains were stimulated and imaged in ALH using the same laser configuration described above at RT.
Genotypes of samples were verified by both the presence and the pattern of green pHluorin or red ArchT fluorescence. The antennal lobe AL and lateral horn LH were identified by the green fluorescence of pHluorin and were stimulated with 0. A mean background value obtained from regions away from the pHluorin was subtracted in order to correct for fluorescence intensity F. The raw data of each cell or brain sample are presented in the graphs and the sample size are indicated in the legends. All data analyses were performed using Origin 9. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses.
A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. Thank you for submitting your article "PARIS, an optogenetic method for functionally mapping gap junctions" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Eve Marder as the Senior Editor.
The following individuals involved in review of your submission have agreed to reveal their identity: David J. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. During the consultation session, the three reviewers were in agreement that no new experiments are required, but that there are issues that will require editorial changes and some rewriting. I am taking the unusual tactic of including all three complete reviews so that you have the benefit of the context for the concerns as you prepare your revisions.
Strengths of this new method include non-invasiveness, subcellular resolution, and the ability to apply this through transgenic means that allow for targeting to specific defined cell types. Overall, I find the work novel and innovative and potentially interesting to a growing field of researchers focused on these underappreciated cellular connections.
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The manuscript itself is very well crafted — and the figures of very high quality throughout. In many ways this was a pleasure to read. Of particular note is the authors' care to appropriately discuss the benefits and limitations of the new technique. If I have one major concern with the utility of PARIS, it is that it seems to be at this point a largely qualitative tool.
What would truly be exceptional is if this could be used to measure not only the presence of coupling, but the relative strength of coupling as well. To do so, the authors would need to measure coupling strength with a traditional approach e. While I do not think this is required for this study to be ready for publication, it would greatly enhance the utility of this approach. My only other concern, which is touched on by the authors, is that PARIS will manipulate intracellular pH — and invoke homeostatic mechanisms to maintain cellular pH.
If this occurs, there may be physiological questions that alter cellular properties as a result. This may limit the utility of PARIS for longitudinal studies where multiple measurements of coupling are taken over the time course of some manipulation. Taking advantage of genetics the method pairs an actuator a light gated outward proton pump ArchT in one cell with a receiver the pH sensitive GFP pHluorin in an adjacent cell, a combination that has the ability of to detect gap junctional communication between these two cells. The development of this method represents a significant contribution to the current toolbox of techniques available to study intercellular communication via gap junctions because:.
The authors provide evidence of the sensitivity of this method that results from extensive testing under different conditions. Every possible source of confound was explored and addressed. Thus, this method certainly adds to the palette of methods available for studying gap junctional communication and it could be used in combination with other approaches.
A concern of the technique is the modification of intracellular pH resulting from changes in the concentration of protons. As the authors discuss, gap junction channels were shown to be gated by pH. However, the authors document the changes in pH resulting from different activation strengths and conclude that it is possible to operate with sufficient sensitivity and minimal variation in intracellular pH.
It is anyway a limitation of the technique that should be used with caution, as the sensitivity for pH might be different for gap junction channels made of different isoforms and the pH buffering capability although highly conserved could be different between cells types.
While the authors discuss the effect of pH on connexin-based gap junction channels there is not reference to the effect of pH on innexin-based gap junctions, at which this novel approach was also tested and will most likely be used. Here are a few papers on the effect of pH on invertebrate gap junctions that the authors should include: Giaume, Spira and Korn, ; Obaid, Socolar and Rose, ; Moreno et al. Anyway, while a limitation inherent to any method reminding the experimenter to use this method with caution, it does not weaken the many virtues of the approach.
Overall, this is a new exciting approach that provides a new tool to study the role of electrical synapses in neuronal circuits. The presence, distribution and functional role of electrical synapses has recently received a great deal of attention in C. Together with the future technical improvements discussed by the authors its likely immediate use in these genetically tractable invertebrate species will promote the necessary improvements to make possible its use in the vertebrate brain. Dev Biol 1 DOI: J Cell Sci Pt 14 Organ ablation studies. Am J Physiol 5 Pt 1 :C This study describes a new optical methodology, PARIS, for functionally mapping gap junction synapses between neurons.
The approach involves expression of an actuator in one cell or cell type and a receiver in another cell or cell type. Photoactivation of ArchT lets protons flow out of the actuator cell, which are then replenished by protons flowing in from another cell, through gap-junctions. If the gap-junction-coupled cell expresses pHluorinCAAX, then the loss of protons changes the pH, which yields a change in fluorescence.
The authors demonstrate that this methodology works in HEK cells, cardiomyocytes, and Drosophila neurons ex vivo. They validate it against other established methodologies for detecting gap junctions, specifically with paired patch-clamp recordings or with fluorescence recovery after photobleaching FRAP. Because PARIS is an optical method, it is easier to perform than paired electrophysiological recordings and has the advantage of subcellular localization of signals. Overall, I see this as a useful new methodology in Drosophila. I feel that this tool is appropriate for publication in eLife , since it promises to facilitate the detection of gap junctions, which could easily enable novel insights into neural circuit functions.