A potential synaptic mechanism to maintain muscle function during periods of increased activity.
In some interesting work into synaptic plasticity, Zhu and colleagues tackled the complex mechanisms behind presynaptic homeostatic potentiation (PHP) at the neuromuscular junction (NMJ) (1). PHP is a synaptic way of keeping neurotransmission going in response to a decrease in postsynaptic sensitivity; in this case, partial blockage of the nicotinic acetylcholine receptors (nAChRs). The precise mechanism behind PHP remains somewhat of a mystery, but this study takes significant steps forward by focusing on the role of extracellular acidification and acid-sensing ion channels (ASICs)
Probing PHP with Venom-Derived
Toxins
The researchers relied heavily on a suite of venom-derived toxins to tease apart PHP. The peptide toxin, µ-Conotoxin GIIIB (#C-270) (μ-Ctx), was essential for isolating the synapse by inhibiting sodium channels in muscle fibers, effectively eliminating background noise from muscle contractions. This was crucial for studying synaptic changes in response to partial nAChR blockage.
To directly test the role of ASICs, Psalmotoxin-1 (#STP-200) (PcTx1) and Mambalgin-3 (#STM-500) (Mamb3) – toxin blockers of ASICs – were brought into play. PcTx1, targeting ASIC1a with nanomolar precision, or Mamb3, a broader ASICs inhibitor, completely abolished PHP. On the other hand, acidification of the extracellular saline from pH 7.4 to 7.2 triggered a reversible increase in quantal content (QC), and PcTx1 blocked the acidification-induced QC increase. These findings strongly linked ASICs activation – driven by a small drop in synaptic cleft pH – to the QC upregulation central to PHP.
Mapping ASICs to the NMJ – Multiplexing Antibodies with a Fluorescently Labeled Toxin
To complement the functional data, Zhu et al. used a range of ASICs antibodies to confirm channels presence at the NMJ. Alomone’s antibodies against ASIC1a (Anti-ASIC1 Antibody (#ASC-014)), ASIC2a (Anti-ASIC2a Antibody (#ASC-012)), and ASIC1 (Guinea pig Anti-ASIC1 Antibody(#ASC-014-GP)) were key for their immunofluorescence studies (Figure 1), allowing the team to identify ASICs subunits within the synapse. Interestingly, for NMJ visualizing, the researchers used a fluorescently labeled α-bungarotoxin (α-BTX). This spatial mapping reinforced the idea that ASICs are strategically positioned to modulate presynaptic activity. The combination of functional inhibition and ion channel
localization created a cohesive picture of ASICs involvement in the mechanism
of PHP.
Figure 1. ASIC1 and ASIC2a are located at the mouse NMJ. A. All of the images shown are maximum projections of 16 images collected at 0.5 μm increments vertically through the field containing the NMJ. α-bungarotoxin (α -BTX) is shown in Red. ASIC2a antibodies are in Green. Arrows point to clusters of ASIC2a staining that are outside the area defining the motor nerve terminal but near perisynaptic Schwann cell nuclei, indicated by the DAPI stain (Blue). B. All of the images shown are maximum projections of 18 images collected at 0.5 μm increments vertically through the field containing the NMJ. α-bungarotoxin (α -BTX) is shown in Red. ASIC1 antibodies are in Green. Arrows point to clusters of ASIC2a staining that are outside the area defining the motor nerve terminal but near perisynaptic Schwann cell nuclei, indicated by the DAPI stain (Blue). Calibration bar, 10 μm. Image and legend form Zhu et al. (2021) (1).
Closing the Loop
The study proposes a novel model where partial nAChR blockage leads to a slight acidification at the synaptic cleft, activating presynaptic ASICs. This, in
turn, upregulates neurotransmitter release to maintain synaptic function. While
the full PHP mechanism remains unresolved, this study identifies ASICs as central players in synaptic plasticity at the NMJ. The reliance on venom-derived toxins – µCtx, PcTx1, Mamb3 and α-BTX – was a major contributor to unraveling these pathways, while the inclusion of ASICs antibodies and advanced imaging enriched the analysis. By focusing on how extracellular protons and ASICs drive QC upregulation, Zhu et al. take a significant step toward understanding how synapses adapt under stress, like during periods of increased physical activity. It’s an impressive example of how specialized tools can illuminate complex biological systems.
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Reference
1. Y. Zhu, C. I. C. Warrenfelt, J. C. Flannery, C. A. Lindgren, Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction. Neuroscience 467, 188–200 (2021). DOI: https://doi.org/10.1016/j.neuroscience.2021.01.036
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