When most people hear the word “Botox”, the first thing that comes to mind are images of Hollywood stars getting facial injections for tight, youthful skin. However, Botox (the commercial name for Botulinum toxin A or BTX-A) also has a reputation as a clinical treatment for chronic pain. This powerful neurotoxin is produced by the bacteria Clostridium botulinum and can cause paralysis by acting on the neuromuscular junction, the border between a motor neuron (a type of nerve cell controlling movement) and a muscle fiber. Here, the toxin blocks the neurotransmitter acetylcholine which is essential for muscle contraction. Small, membrane-bound structures called synaptic vesicles which store acetylcholine cannot release it into the neuromuscular junction because BTX-A cleaves the SNAP-25 protein, which acts like a hook and allows the vesicle to attach to the wall of the neuron and release the neurotransmitter. Now, researchers from Zhejiang University in China have discovered that BTX-A may act in a similar way to relieve pain.
Previous studies have shown that stimulating pain in mice causes more molecules called AMPA receptors to be inserted in the membranes of spinal cord neurons, particularly those of the spinal dorsal horn (SDH). AMPA receptors are activated by the neurotransmitter glutamate, a key neurotransmitter in the central nervous system. When glutamate binds to these receptors, they open like pores, allowing charged atoms to rush into neurons and causing the neuron to fire. The SDH is a region of the spinal cord where sensory neurons insert themselves, allowing sensory information from the environment, such as pain sensations, to travel to the brain. By potentially cleaving the SNAP-25 protein in the SDH neurons, BTX-A could prevent glutamate from being transported in vesicles, released from these sensory neurons, and binding to AMPA receptors, thus preventing the neurons from firing to give the sensation of pain.
By injecting mice in the foot with a formaldehyde solution, researchers were able to produce a pain response which could be measured by watching the number of times a mouse licked or lifted its paw. These mice were then injected with either BTX-A or a salt solution. Compared to mice injected with salt solution, the BTX-A mice had fewer pain responses 10-45 minutes after injection. Researchers also confirmed the presence of the cleaved SNAP-25 protein in the SDH neurons of mice treated with BTX-A, but not in those treated with salt solution. Using immunohistochemistry, a method in which fluorescent proteins which bind specifically to cleaved SNAP-25 are applied to slices of the spinal cord, the scientists were able to see a fluorescent green glow were cleaved SNAP-25 was located in the SDH neurons. Researchers also wanted to determine if levels of the GluR1 receptor, which makes up part of the AMPA receptor, would decrease after BTX-A treatment. The scientists were able to confirm that 3 days after BTX-A treatment, levels of the GluR1 receptor in the SDH neurons decreased significantly compared to mice treated with salt solution. For the next part of the experiment, the scientists not only treated some mice with formalin and BTX-A combined, but also some with either BTX-A, formalin, or salt solution alone. Experiments which measured the flow of charged atoms in SDH neurons in these four groups showed a significant reduction in the current passing through the AMPA receptors in the mice treated with BTX-A. In addition, mini excitatory postsynaptic currents (mEPSCs), small currents causing the release of glutamate from vesicles, were also measured between these groups. Although BTX-A treatment did not reduce the mEPSCs in mice that had not been injected with formalin, it did reduce mEPSCs in mice treated with both BTX-A and formalin compared to mice treated with formalin alone, indicating that BTX-A only reduced glutamate release when formalin had been injected to cause a pain response. Finally, the levels of glutamate in the SDH neurons were measured between these groups. This experiment showed that glutamate concentration in the SDH neurons decreased 3 days after BTX-A treatment significantly compared to untreated mice.
In summary, the researchers confirmed that injection of BTX-A reduces pain by causing the cleavage of the SNAP-25 protein in SDH sensory neurons, thus preventing both the release of AMPA receptors and glutamate from vesicles, resulting in reduced AMPA current and mEPSCs. This demonstrates that BTX-A can be injected at sites far from the central nervous system (which consists of the brain and spinal cord) yet still affect it due to retrograde axonal transport, a phenomenon in which a molecule is transported over long-distances in neurons from the injection site to the spinal cord. These findings provide greater insight into the mechanism by which BTX-A acts, confirming that it can be a promising treatment for chronic pain. This exciting discovery may also lead to the development of new pain therapies targeting glutamate and its receptors.
References
Bear, M. F., Connors, B. W., & Paradiso, M. A. (2016). Neuroscience: exploring the brain (Fourth edition). Philadelphia: Wolters Kluwer
Galan, A., Laird, J. M. A., & Cervero, F. (2004). In vivo recruitment by painful stimuli of AMPA receptor subunits to the plasma membrane of spinal cord neurons: Pain, 112(3), 315–323. https://doi.org/10.1016/j.pain.2004.09.011
Hong, B., Yao, L., Ni, L., Wang, L., & Hu, X. (2017). Antinociceptive effect of botulinum toxin A involves alterations in AMPA receptor expression and glutamate release in spinal dorsal horn neurons. Neuroscience, 357, 197–207. https://doi.org/10.1016/j.neuroscience.2017.06.004
Park, J., & Chung, M. (2018). Botulinum Toxin for Central Neuropathic Pain. Toxins, 10(6), 224. https://doi.org/10.3390/toxins10060224
