No Breach Needed: Ultrasound Improves Memory in Mice

The idea that sound waves knocking at the skull could boost memory continues to sound far-fetched to many Alzheimer's researchers, but researchers led by Jürgen Götz, University of Queensland, Brisbane, Australia, respectfully disagree. In the May 27 Molecular Psychiatry, they reported that scanning ultrasound improved synaptic signaling, increased neurogenesis, and sharpened spatial memory in old wild-type mice. Importantly, this worked without breaching the blood-brain barrier, a commonly used ultrasound trick to provoke a brain response. Whether this scanning ultrasound technique is appropriate for people remains to be seen, though early stage clinical trials in older adults indicate it may be safe.

  • Scanning ultrasound increased synaptic signaling, boosted neurogenesis in mice.
  • The treatment also improved memory.
  • No breach of the blood-brain barrier was required.

Older research has shown that using ultrasound to poke focal, temporary “holes” in the blood-brain barrier can benefit the brain. Tiny lipid sacs filled with an inert gas are injected into the blood, and then expand and contract as sound waves are pulsed into the brain, wedging open the blood-brain barrier. This allows medicinal molecule access to the brain that would otherwise be kept out; likewise, it allows toxic molecules, such as Aβ, to leave the brain that would otherwise be kept in. Götz previously found that ultrasound plus these microbubbles stirred microglia to clear plaques and rescue memory in the APP23 mouse model of amyloidosis (Mar 2015 news). 

Researchers led by Kullervo Hynynen, University of Toronto, found the same in TgCRND8 mice given ultrasound alone, without microbubbles (Burgess et al., 2014). How could ultrasound alone work?

To answer this, co-first authors Daniel Blackmore and Fabrice Turpin studied scanning ultrasound in 20-month-old C57Bl/6 wild-type mice. In the mice, memory falters by 18 months of age; they die around 26 months. The researchers tested three groups: 15 mice were intravenously injected with microbubbles plus treated with ultrasound, 20 were treated with ultrasound alone, and six stayed untreated. 

The scientists placed a scanning device over the mouse’s skull that emitted low-frequency ultrasound pulses in 1.5 mm increments over most of the mouse’s brain (see image below). Treated mice received half a dozen four-minute sessions over two weeks.

Scanning Helmet. An ultrasound scanner placed over the mouse’s head (left) moves in a grid pattern across the brain (right), pausing to deliver six-second sound pulses every 1.5 mm. [Courtesy of Blackmore et al., Molecular Psychiatry, 2021.]

To test the BBB’s integrity after treatment, the researchers intravenously injected Evans Blue. This dye binds to serum albumin and stays outside the barrier when it is intact. Only the brains of mice that were partially treated with scanning ultrasound (SUS) plus microbubbles turned blue, indicating BBB breach. The sound waves had opened the barrier over a narrow area, and had penetrated deeply into the brain (see image below).

After SUS, the mice were trained in an active place avoidance (APA) task, in which they associate certain locales with a foot shock. Ultrasound-treated mice better avoided shocks than untreated mice, indicating enhanced hippocampal-dependent spatial learning. The more ultrasound sessions they received, the better their memories became. Mice who received microbubbles along with the scanning ultrasound had no significant improvement.

Penetrating the Brain. Blue dye leaked into the brain after microbubbles pried open the BBB (left image, right); ultrasound alone left the barrier undisturbed (left image, middle). The barrier opened where the sound waves were aimed, penetrating deep into the treated area (right image). [Courtesy of Blackmore et al., Molecular Psychiatry, 2021.]

How could ultrasound alone boost memory? Previous work had suggested ultrasound somehow opens TRPA1 calcium channels in astrocytes, which then release glutamate to activate NMDA receptors on nearby neurons (Oh et al., 2019). Blackmore, Turpin, and colleagues looked for signs of astrocyte-mediated activation in mouse hippocampal tissue via Western blots, and found that tissue from mice exposed to ultrasound, regardless of microbubble use, contained more TRPA1 than tissue from control.

Evidence of NMDA activation came when the scientists separated hippocampal tissue into total and postsynaptic fractions. In the postsynaptic fraction, ultrasound had bumped up the amount of NR2B, a subunit of NMDA receptors that is needed for long-term potentiation, a form of synaptic plasticity. LTP is crucial for learning and memory and by 20 months of age, it has faded. However, the scanning ultrasound had restored LTP in aged mice, as judged by evoked potentials in hippocampal slices (see image below).

Sound Sans Bubbles. Scanning ultrasound alone (mode “A” in panels) evoked more robust changes in mouse brain than did SUS with microbubbles (mode “B”). After SUS alone, changes to the extracellular matrix, neurogenesis, membrane channels, and the proteome (top row) all culminated in improved LTP and spatial memory (bottom). Plus signs indicate magnitude of change; negative signs mean no change. [Courtesy of Blackmore et al., Molecular Psychiatry, 2021.]

To find out whether ultrasound altered other molecular pathways, the scientists turned to proteomics. Mass spectrometry suggested SUS altered the amount of 51 proteins, whereas SUS with microbubbles altered 39. Eight were in common between these two groups. They included proteins involved in synaptic regulation and memory, such the metabotropic glutamate receptor mGluR1, leucine-rich repeat transmembrane neuronal protein 4 (Lrrtm4), and others that associate with neurogenesis.

Could the latter account for the memory effect? Based on dentate gyrus expression of doublecortin, a marker of new neurons, the authors concluded that SUS upped neurogenesis 13-fold, while SUS with microbubbles coaxed a sevenfold increase in new neurons. Taken together, the researchers believe ultrasound enhanced synaptic plasticity and neurogenesis, which could explain how the mice performed better on the APA task.

The scientists did not track how long the memory changes lasted. “Because there are changes at the NDMA receptor level, my gut feeling is that ultrasound leads to long-lasting changes,” he said, noting that his group is studying the short- and long-term cognitive effects of ultrasound in APP23 mice.

How about people? Clinical trials using ultrasound to pry open the BBB are underway in people with Alzheimer’s disease or mild cognitive impairment. Researchers led by Hynynen and Sandra Black, Sunnybrook Research Institute, Toronto, reported that in five people with mild to moderate AD, sound pulses paired with microbubbles opened the barrier, which closed within a day and caused no adverse events (Aug 2018 conference news). 

A helmet-shaped ultrasound device used in that study, called Exablate, is being tested in two larger AD safety studies. One in the U.S. will enroll 20 people, and one in Canada 30 people. Early results from three women in the U.S. trial showed the barrier opening, closing within one day, without ill effects (Mehta et al., 2021). Likewise, Elisa Konofagou, Columbia University, New York, is leading a safety study of focused ultrasound with microbubble treatment in six people with MCI or early AD.

Taking a different approach, the French company CarThera is testing a device in 10 people with mild AD in a recently completed Phase 1/2 trial. Called Sonocloud, its device is surgically implanted into the brain. It can open the BBB with low-intensity ultrasound since it avoids the dulling of sound waves by the skull. Results are not publicly available yet.—Chelsea Weidman Burke

Comments

  1. Blackmore et al. is another exciting paper from the Götz group, building on their success with low-intensity ultrasound. They show powerful activation of the hippocampal circuitry, manifested by enhanced plasticity and memory restoration. This treatment increased hippocampal neurogenesis, upregulated synaptic proteins, improved synaptic receptor function, and restored LTP induction in senescent mice.

    The scanning ultrasound (SUS) is an intriguing approach that seems to have a profound effect on neural function. Blackmore et al. speculate that SUS may cause the opening of TRPA1 channels in astrocytes, resulting in the entry of Ca2+ ions. It would be fascinating to learn more about the underlying mechanisms and the concerted effect on astrocytes and neurons that lead to increased plasticity.

    This approach is noninvasive, focal, and capable of targeting subcortical areas. The low energy minimizes the chance of peripheral damage. These qualities should translate well into human therapy. Since it induces transient plasticity, it would be necessary to determine treatment frequency.

    View all comments by Orly Lazarov

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References

Research Models Citations

  1. APP23
  2. TgCRND8

News Citations

  1. Stop, Hey, What’s That Sound? ... Amyloid Is Going Down?
  2. Focused Ultrasound Breaches Blood-Brain Barrier in People with Alzheimer’s

Paper Citations

  1. Burgess A, Dubey S, Yeung S, Hough O, Eterman N, Aubert I, Hynynen K. Alzheimer disease in a mouse model: MR imaging-guided focused ultrasound targeted to the hippocampus opens the blood-brain barrier and improves pathologic abnormalities and behavior. Radiology. 2014 Dec;273(3):736-45. Epub 2014 Sep 15 PubMed.
  2. Oh SJ, Lee JM, Kim HB, Lee J, Han S, Bae JY, Hong GS, Koh W, Kwon J, Hwang ES, Woo DH, Youn I, Cho IJ, Bae YC, Lee S, Shim JW, Park JH, Lee CJ. Ultrasonic Neuromodulation via Astrocytic TRPA1. Curr Biol. 2019 Oct 21;29(20):3386-3401.e8. Epub 2019 Oct 3 PubMed.
  3. Mehta RI, Carpenter JS, Mehta RI, Haut MW, Ranjan M, Najib U, Lockman P, Wang P, D'haese PF, Rezai AR. Blood-Brain Barrier Opening with MRI-guided Focused Ultrasound Elicits Meningeal Venous Permeability in Humans with Early Alzheimer Disease. Radiology. 2021 Mar;298(3):654-662. Epub 2021 Jan 5 PubMed.

External Citations

  1. U.S.
  2. Canada
  3. safety study
  4. Phase 1/2 trial

Further Reading

Primary Papers

  1. Blackmore DG, Turpin F, Palliyaguru T, Evans HT, Chicoteau A, Lee W, Pelekanos M, Nguyen N, Song J, Sullivan RK, Sah P, Bartlett PF, Götz J. Low-intensity ultrasound restores long-term potentiation and memory in senescent mice through pleiotropic mechanisms including NMDAR signaling. Mol Psychiatry. 2021 May 27; PubMed.

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