ASIC1a is required for neuronal activation via low-intensity ultrasound stimulation in mouse brain
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Summary: This is an interesting manuscript suggesting that ultrasound stimuli induce movements of the extracellular matrix and the cytoskeleton to cause mechanical activation of ASIC1a in cortical neurons. This is a novel finding.
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Abstract
Accumulating evidence has shown transcranial low-intensity ultrasound can be potentially a non-invasive neural modulation tool to treat brain diseases. However, the underlying mechanism remains elusive and the majority of studies on animal models applying rather high-intensity ultrasound that cannot be safely used in humans. Here, we showed low-intensity ultrasound was able to activate neurons in the mouse brain and repeated ultrasound stimulation resulted in adult neurogenesis in specific brain regions. In vitro calcium imaging studies showed that a specific ultrasound stimulation mode, which combined with both ultrasound-induced pressure and acoustic streaming mechanotransduction, is required to activate cultured cortical neurons. ASIC1a and cytoskeletal proteins were involved in the low-intensity ultrasound-mediated mechanotransduction and cultured neuron activation, which was inhibited by ASIC1a blockade and cytoskeleton-modified agents. In contrast, the inhibition of mechanical-sensitive channels involved in bilayer-model mechanotransduction like Piezo or TRP proteins did not repress the ultrasound-mediated neuronal activation as efficiently. The ASIC1a-mediated ultrasound effects in mouse brain such as immediate response of ERK phosphorylation and DCX marked neurogenesis were statistically significantly compromised by ASIC1a gene deletion. Collated data suggest that ASIC1a is the molecular determinant involved in the mechano-signaling of low-intensity ultrasound that modulates neural activation in mouse brain.
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Reviewer #2:
In this study the authors claim that short lasting low intensity ultrasound stimulation activates many neurons in the whole brain. They further claim that the activation mechanism is via the ASIC1a channel. There are some intriguing results in this paper, but there are also many open questions and methodological issues that should be addressed. The authors use pERK as a surrogate for neuronal activation by a global ultrasound stimulus. Some but not all neurons in cortex seem to show activation (it seems only large pyramidal cells, why not interneurons? More analysis needed here.
This experiment is followed by an in vitro experiment with cultured cortical neurons from neonates (no ages given for the animals used in this experiment as far as I can see). These are also not equivalent to the adult cells tested in the in vivo …
Reviewer #2:
In this study the authors claim that short lasting low intensity ultrasound stimulation activates many neurons in the whole brain. They further claim that the activation mechanism is via the ASIC1a channel. There are some intriguing results in this paper, but there are also many open questions and methodological issues that should be addressed. The authors use pERK as a surrogate for neuronal activation by a global ultrasound stimulus. Some but not all neurons in cortex seem to show activation (it seems only large pyramidal cells, why not interneurons? More analysis needed here.
This experiment is followed by an in vitro experiment with cultured cortical neurons from neonates (no ages given for the animals used in this experiment as far as I can see). These are also not equivalent to the adult cells tested in the in vivo experiment. In the bulk of the experiments calcium imaging is used as a surrogate to measure neuronal activation. Unfortunately, in none of the graphs displayed of the Delta F/Fo is there any indication of the number of cells selected and measured. This is critical to evaluate the robustness of the results. In addition, it is normal at the end of the experiment to permeabilize the neurons to calcium by using an ionophore. This allows the assessment of the maximum fluorescence signal when calcium outside concentration equilibrates with the intracellular concentration. This was not done which means the experiments have no internal calibration.
It is for me impossible to assess the robustness of the calcium imaging experiment when I do not know what each data point corresponds to, take Figure 2I as an example. Are these individual cells or means values from many cells from individual cultures? Many critical methodological details are indeed missing from the paper.
The idea that ASIC1a is THE critical mediator of this effect is quite surprising and the more dramatic and implausible the conclusion may seem, the more solid the evidence needed. The authors should use ASIC1a mutant mice both in vivo and in vitro to prove that ASIC1a really is critical. The same applies to the apparent effect on neurogenesis.
The videos show quite large physical effects of the ultrasound on the cultures (cells moving around). This is problematic as it may be that what the calcium signals are purely indicative of cell damage. Controls should be provided to ensure this was not the case.
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Reviewer #1:
In the manuscript entitled “ASIC1a is required for neuronal activation via low-intensity ultrasound stimulation in mouse brain", Lim et al. investigate the mechanism underlying the activation of brain neurons by transcranial low-intensity ultrasound stimulation. The authors propose that ultrasound stimuli-induced movements of the extracellular matrix and the cytoskeleton cause mechanical activation of ASIC1a in cortical neurons, which leads to Ca2+ influx and subsequent expression of pERK, which the authors used as a surrogate marker for neuronal activation.
While I agree that the finding that ultrasound activates neurons via activation of a mechanosensitive ion channel is per se very interesting, I have to say that in my opinion most of the conclusions and claims are not supported by the actual data.
The entire study is …
Reviewer #1:
In the manuscript entitled “ASIC1a is required for neuronal activation via low-intensity ultrasound stimulation in mouse brain", Lim et al. investigate the mechanism underlying the activation of brain neurons by transcranial low-intensity ultrasound stimulation. The authors propose that ultrasound stimuli-induced movements of the extracellular matrix and the cytoskeleton cause mechanical activation of ASIC1a in cortical neurons, which leads to Ca2+ influx and subsequent expression of pERK, which the authors used as a surrogate marker for neuronal activation.
While I agree that the finding that ultrasound activates neurons via activation of a mechanosensitive ion channel is per se very interesting, I have to say that in my opinion most of the conclusions and claims are not supported by the actual data.
The entire study is purely correlative. Thus, the authors made two independent experiments; on the one hand they show that in-vivo transcranial ultrasound stimulation induces pERK in various brain regions and on the other hand they shown that ultrasound-evoked Ca2+ influx in cultures of cortical neurons is probably mediated by ASIC1a. From this data they conclude that pERK activation is also mediated by ASIC1a activation. This is, however, pure speculation. The authors must provide additional evidence to support their claim. In my opinion the sole use of PcTx1 is not sufficient to prove that the Ca2+ signals are mediated by ASIC1a. Hence, firstly the authors should demonstrate that ASIC1a is indeed activated by ultrasound. This is a very simple experiment. All they would have to do is express ASIC1a in a cell line (e.g. HEK293, CHO, etc) and show that this expression renders the cells sensitive to ultrasound. Second, I would appreciate it if the authors would show that cortical neurons, especially those that show pERK activation, express ASIC1a in the first place. This would also be quite simple - just co-stain the brain sections with an anti-ASIC1a antibody. Third, if the authors want to keep up their claim (see title) that ASIC1a is required for ultrasound activation of brain neurons they should examine ultrasound-induced pERK activation in ASIC1a-knockout mice.
It is difficult to evaluate the Ca2+ imaging experiments, because the method - especially the ultrasound stimulation - is not very well described. Hence it is unclear to me how close to the cell the ultrasound stimulator was placed. Moreover, the N-numbers of the Ca2+ imaging experiments are rather small (by the way, it would make reading much easier if the N-numbers were indicated in the figure). Most importantly, it is unclear if the inhibitors (Gadolinium, GsMTx4 etc - Figure 2B-H) were applied to the control cells from the same panel or to different cells. In this context it would be important to know how many control cells actually responded to the ultrasound stimulation. Considering the low N-number, I was wondering if the authors may have had a hard time finding cells that responded and that this is the reason why the N-numbers are so small? I suggest examining many more control neurons and provide information about the proportion of cells that respond. If only for the controls as well as for the cells treated the various channel inhibitors.
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Summary: This is an interesting manuscript suggesting that ultrasound stimuli induce movements of the extracellular matrix and the cytoskeleton to cause mechanical activation of ASIC1a in cortical neurons. This is a novel finding.
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