Without electrodes
Mammalian neurons can be monitored by ultrasound
Taisiya Latypova, PCR.news
About a decade ago, Srikant Chalasani, an associate professor at the Salk Institute for Biological Research (USA), developed a method of using ultrasound to stimulate genetically modified cells. The new approach was called sonogenetics.
In 2015, Chalasani's group showed that the expression of the TRP-4 mechanoreceptor in Caenorhabditis elegans neurons makes neurons sensitive to ultrasound. Expression of TRP-4 in mammalian cells did not produce such an effect. Scientists set out to find a protein that would make mammalian cells sensitive to ultrasound, and told about the search results in a new article.
TRP-4 is a pore—forming subunit of the channel that provides the transmission of a mechanical stimulus. At the first stage, the scientists screened a library of 191 channels and their homologues on HEK-293T cells, which normally do not respond to ultrasound, and investigated the change in the concentration of intracellular calcium during ultrasound stimulation. The strongest response to ultrasound was provided by the protein hsTRPA1, which in the human body reacts to toxic substances and activates the response to them. Cells expressing hsTRPA1 responded to ultrasound with a frequency from 1 to 7 MHz.
To understand the mechanism of sensitivity to ultrasound, scientists sequenced the human hsTRPA1 gene and nine of its homologues. It turned out that mammals have a highly conserved motif of 61 amino acids at the intracellular N-end of TRPA1. The loss of this motive leads to complete insensitivity to ultrasound. In addition, the response to ultrasound was inhibited when cells were treated with substances depolymerizing actin. Cholesterol turned out to be another important component of ultrasound signal transmission: cells expressing hsTRPA1 lost sensitivity to ultrasound during deletion of the transmembrane motif of the protein that recognizes cholesterol, as well as with cholesterol deficiency.
At the next stage, the scientists conducted experiments on mouse primary embryonic cortical neurons. hsTRPA1 expression was provided by infecting them with a construct based on an adeno-associated vector. As in the experiments with HEK-293T, hsTRPA1 stimulated an increase in the concentration of calcium inside the cell in response to ultrasound.
Finally, using an adeno-associated vector, the scientists expressed hsTRPA1 in the V layer of the cerebral cortex of mice. Normally, there is no TRPA1 in the brain. During ultrasound stimulation with a frequency of 7 MHz, neurons within a given area of the brain were activated in mice. Neither gene-therapeutic manipulations nor ultrasound stimulation resulted in a violation of the blood-brain barrier or a change in the behavior of mice.
Chalasani hopes that one day sonogenetics will replace invasive treatments for diseases such as Parkinson's disease, and will also be used to activate heart cells. Scientists are already working on ways to deliver therapeutic proteins across the blood-brain barrier. In addition, they plan to study in more detail the mechanism of response to ultrasound stimuli and find new proteins that are sensitive to them.
Article by Duque et al. Sonogenetic control of mammalian cells using exogenous Transient Receptor Potential A1 channels is published in the journal Nature Communications.
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