The study was performed to determine the molecular mechanisms that regulate plasticity at the Action Initial Segment within voltage gated sodium channels. (Fréal et al., 2023) The action initial segment is at the base of the axon where it extends from the soma, or cell body of the neuron. This beginning part of the axon is where action potential is generated and shaped before it moves outward from the cell body down the axon. (Leterrier 2018) The study Fréal et al. (2023) conducted, was focused on determining how the structure of the axon initial segment changes during plasticity.
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Knowledge and New Research Advancement
A new green fluorescent protein (AnkG) and the ORANGE CRISPR-Cas9 system were used to illuminate sodium channels so that they would be visible at the molecular level to identify plasticity at the axon initial segment. ORANGE stands for Open Resource for the Application of Neuronal Genome Editing and allows for quantifying the expression, distribution and dynamical properties of proteins within neurons. (Willems et. al., 2020) This new technology allows for researchers to actually see specific components of neurons and how those parts change. Researchers' observations are greatly enhanced due to this technology and can distinguish parts of the neuron that they wish to observe apart from other phenomena. This technology allows researchers to measure the nature of neuroplasticity, for example as seen in the study by Fréal et al. (2023) where they look at properties of the axon initial segment.
The main gap in knowledge and research that the study by Fréal et al. (2023) makes contributions towards is the lack of a molecular model for how plasticity occurs within the axon initial segment. The main limitation to understanding plasticity beginning in the axon initial segment has to do with studying axon initial segments among many neurons and not individual observations. Fréal et al. (2023) focused on observing loss of the axon initial segment during plasticity.
Methods, Findings, and Results
Fréal et al. (2023) used a real time imaging technique to observe neurons in slices of hippocampal neurons of genetically modified mice. These mice had a fluorescence property to their neurons which made the action initial segments observable. They used a form of synaptic depression by exposing the slices to N-methyl-D-aspartate (NMDA). NMDA are receptors that are involved in the process of excitatory synaptic transmission and regulate plasticity. (Hanson et. al., 2024)
The first finding in Fréal et al.'s (2023) study was by comparing NMDA to a control, only the NMDA condition depolarized which caused an action potential to fire. They were able to observe a decrease of axon initial segment length using the fluorescence imaging technique while NMDA was applied. Here, they observed that NMDA induced shortening of the axon initial segment and observed it was a site for plasticity.
The second finding was determined by using the ORANGE CRISPR-Cas9 system that fluorescently tags properties of the neuron; in this case the sodium channel. Fréal et al. (2023) were able to determine that a reduction of sodium channels occurs in the distal axon initial segment specifically and not sodium channels globally. This means that the shortening of the axon initial segment from a reduction of sodium channels showed how the mechanism functioned for plasticity.
Thirdly, Fréal et al. (2023) found that NMDAR triggers axon initiated segment plasticity. To test if it was the NMDAR sites specifically at the axon initiated segment, they used MK-801, a NMDA receptor antagonist (Janus et. al., 2023), that blocks the glutamate NMDA. They used the MK-801 before and while the NMDA was being used to observe that activating the synaptic NMDAR is necessary to trigger plasticity in the axon initial segment. The MK-801 acted as a control to test, experimentally, the influence of the NMDA on plasticity.
The fourth finding from the study Fréal et al. (2023) conducted was to determine what the mechanism was that removed the sodium channels from the axon initial segment when NDMA was facilitating plasticity. Endocytosis was the main mechanism that was involved in the shortening of the axon initial segment. Endocytosis is the process where cells internalize molecules and proteins. (Khan and Steeg, 2021) They tested a calcium based protease, an enzyme that breaks down proteins, by looking at its involvement in axon initial segment disassembly. When a blocker to the protease was introduced, the shortening through the NMDA channels was eliminated. Therefore, they determined that endocytosis is necessary for NMDAR axon initial segment plasticity.
The fifth finding from the Fréal et al. (2023) study was that the sodium channels at the axon initial segment are reduced by the endocytic structures. The specific structure, clathrin, is responsible for carrying materials from the surface of the cell to its interior. (Zeno et. al., 2021) To test this, Fréal et al. (2023) applied Dynasore, which blocks other endocytic structures but not clathrin, before and after the application of NMDA. They were able to see the endocytic structures that formed during plasticity at the axon initial segments. When NMDA was applied, they observed an increase in endocytic structures, specifically clathrin, at the axon initial segment.
The sixth finding that Fréal et al. (2023) discovered was that sodium channels have higher mobility in the distal axon initial segment. They tested this by using a photobleaching technique that removes the fluorescence in the distal and proximal areas of the axon initial segment. This allowed observations of the difference in recovery of the sodium channels at different points of the axon initial segment.
The final finding of the Fréal et al. (2023) study was NMDA increased the threshold of action potentials by reducing the axon initial segment length. They measured the length of axon initial segments which were correlated with action potential voltage thresholds. In NMDA conditions, there was a reduction in the probability of action potentials firing, but not in the control condition.
Significance, Implications and Importance
The main significance of the study was the implementation of the genetic tools that labeled endogenous sodium channels and proteins that allowed for nanoscale, longitudinal imaging of their organization during plasticity. The implications of the findings show the possibility that NMDA spikes create plasticity within the axon initial segment. (Fréal et al., 2023) These observations lead to new understandings and knowledge about plasticity and support previous theories stating that neural circuits are more efficient by increasing synaptic strength and excitability. (Xu et. al., 2005)
Conclusion: Research Strengths
Each of the seven findings had robust testing to rule out alternative hypotheses. The integrity of the research is very high because Fréal et al. (2023) took the initiative to conduct multiple tests after conceptualizing alternative theories that could account for the results. This results in using the Popperian principle of falsification within their own research to narrow down the nature of the phenomena they were observing. There is very high scientific and intellectual honesty in an approach as robust as this.
The endogenous labeling of sodium channels and proteins gave Fréal et al. (2023) the ability to longitudinally image the axon initial segment’s plasticity. The technology of using genetic fluorescence to identify individual axons allowed the researchers to observe the molecular mechanisms involved in plasticity.
References
Fréal, A., Jamann, N., Ten Bos, J., Jansen, J., Petersen, N., Ligthart, T., Hoogenraad, C. C., & Kole, M. H. P. (2023) Sodium channel endocytosis drives axon initial segment plasticity. Science Advances, 9(37), 1-15. http://doi:10.1126/sciadv.adf3885
Hanson, J.E., Yuan, H., Perszyk, R.E., Banke, T. G., Xing, H., Tsai, M. C., Menniti, F., & Traynelis, S. (2024) Therapeutic potential of N-methyl-D-aspartate receptor modulators in psychiatry. Neuropsychopharmacology, 49, 51–66. https://doi.org/10.1038/s41386-023-01614-3
Janus, A., Lustyk, K. & Pytka, K. (2023) MK-801 and cognitive functions: Investigating the behavioral effects of a non-competitive NMDA receptor antagonist. Psychopharmacology, 240, (2435–2457). https://doi.org/10.1007/s00213-023-06454-z
Khan, I., Steeg, P. S. (2021) Endocytosis: a pivotal pathway for regulating metastasis. British Journal of Cancer. 124(1), 66-75. https://doi:10.1038/s41416-020-01179-8
Leterrier, C. (2018) The axon initial segment: an updated viewpoint. The Journal of Neuroscience, 38(9), 2135-2145. http://doi:10.1523/JNEUROSCI.1922-17.2018
Willems, J., de Jong, A. P. H., Scheefhals, N., Mertens, E., Catsburg, L. A. E., Poorthuis, R. B., de Winter, F., Verhaagen, J., Maye, F. J., & MacGillavry, H. D. (2020) ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons. PLOS Biology, 18(4), 1-41. https://doi.org/10.1371/journal.pbio.3000665
Xu, J., Kang, N., Jiang, L., Nedergaard, M., & Kang, J., (2005). Activity-dependent long-term potentiation of intrinsic excitability in hippocampal CA1 pyramidal neurons. Journal of Neuroscience. 25, 1750–1760. https://doi:10.1523/JNEUROSCI.4217-04.2005
Zeno, W. F., Hochfelder, J. B., Thatte, A. S., Wang, L., Gadok, A. K., Hayden, C. C., Lafer, E. M., Stachowiak, J. C. (2021) Clathrin senses membrane curvature. Biophysical Journal. 120(5), (818-828). https://doi:10.1016/j.bpj.2020.12.035
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