Sonogenetics - A non-invasive method to manipulate neurons
During the AM poster sessions, one that caught my eye was from the Chalasani Lab at the Salk Institute in La Jolla, California. Several years ago, they described a method by which they could control neural activity in the nematode worm C. elegans using focused ultrasound. This paper demonstrated that ectopic expression of the mechanosensitive channel TRP-4 in neurons rendered them sensitive to ultrasound stimulation. This is a big deal because other so called ‘non-invasive’ neural manipulation techniques like optogenetics require a fiber optic probe to be placed near the cells of interest, making the manipulation of deep brain structures with high temporal precision tedious.
Sonogenetics allows for non-invasive control of neural activity. Here, in C. elegans with PVD neurons expressing the ultrasound-sensitive protein (TRP-4) and the calcium indicator GCaMP3, we can see that ultrasound exposure drastically increases calcium activity in these neurons, indicating ultrasound mediated neural activation. Warmer colors indicate more GCaMP3 fluorescence = more activity (Credit: Ibsen et al., 2015; Nature Communications)
This was to be just the first step in a long process of isolating different mechanosensitive proteins and screening them in mammalian cells to find one just right for use in more complicated organisms. During the poster session this morning, Corinne Lee-Kubli, a post-doc in the Chalasani lab, provided an update on the progress in sonogenetics to date.
Using an in vitro screening method to identify ultrasound-sensitive mechanoreceptors, Corinne expressed a large variety of putative channels in cells in a dish. These cells were co-transfected with the calcium indicator GCaMP6f, a powerful and fast reporter of cell activity. The fluorescent signal was then monitored before, during, and after ultrasound stimulation in a high-throughput manner.
A subset of the putative mechanoreceptors were packaged into cre-dependent AAV-viral vectors and delivered to AgRP neurons deep in the brain (arcuate nucleus) of AgRP-cre mice. Validation of the excitatory actions of the ultrasound sensitive protein was done using a feeding assay, as AgRP neurons strongly promote feeding. Upon ultrasound (10 MHz) stimulation of the head (through the skull and entire brain), a few of the channels strongly promoted feeding responses, a trait not observed in mice expressing the control virus (encoding GFP). An important note is that ultrasound stimulation alone had no effect on feeding responses, indicating a specific effect of the putative mechanoreceptor in AgRP neurons.
AgRP neurons in the arcuate nucleus expressing the calcium indicator GCaMP6. These cells are powerful regulators of feeding behavior and metabolism (Credit: Srisai et al., 2017; Nature Communications)
This proof-of-principle application represents a significant advancement for the nascent field of sonogenetics. Much more research needs to be done to discover the most potent and specific ultrasound sensitive protein, the kinetics of said protein, and additional tools for cell inhibition. In the future, we can expect to see multiple channels expressed in different cellular populations, each sensitive to different ultrasound frequencies. Then, ‘nested’ delivery of different ultrasound waveforms could putatively activate and/or inhibit discrete cell populations across the entire brain, simultaneously or with tight temporal control.
The power of this technique is impressive, as ultrasound can easily reach through the entire mouse brain at 10 MHz, and can go much deeper (e.g., in rat or primate brain) using lower frequencies. I look forward to what’s to come!
Jeremy C. Borniger, PhD
Department of Psychiatry & Behavioral Sciences
Stanford University SoM