An implantable piezoelectric ultrasound stimulator (ImPULS) for deep brain activation

Precise neurostimulation can revolutionize therapies for neurological disorders. Electrode-based stimulation devices face challenges in achieving precise and consistent targeting due to the immune response and the limited penetration of electrical fields. Ultrasound can aid in energy propagation, but transcranial ultrasound stimulation in the deep brain has limited spatial resolution caused by bone and tissue scattering. Here, we report an implantable piezoelectric ultrasound stimulator (ImPULS) that generates an ultrasonic focal pressure of 100 kPa to modulate the activity of neurons. ImPULS is a fully-encapsulated, flexible piezoelectric micromachined ultrasound transducer that incorporates a biocompatible piezoceramic, potassium sodium niobate [(K,Na)NbO3]. The absence of electrochemically active elements poses a new strategy for achieving long-term stability. We demonstrated that ImPULS can i) excite neurons in a mouse hippocampal slice ex vivo, ii) activate cells in the hippocampus of an anesthetized mouse to induce expression of activity-dependent gene c-Fos, and iii) stimulate dopaminergic neurons in the substantia nigra pars compacta to elicit time-locked modulation of nigrostriatal dopamine release. This work introduces a non-genetic ultrasound platform for spatially-localized neural stimulation and exploration of basic functions in the deep brain.

From the comparison, it is clear that KNN has comparable piezoelectric performance to the most widely used piezoelectric material, PZT.KNN also possesses superior biocompatibility with regard to the cytotoxicity of its breakdown byproducts compared to PZT (SI ref 19).Furthermore, its high Curie temperature enables advanced fabrication techniques to create device architectures such as the piezoelectric micromachined ultrasound transducer (pMUT), which greatly enhances the piezoelectric properties of the device without poling or chemical modification.Indeed, piezoelectric devices with KNN have been fabricated, implanted, and evaluated for biocompatibility (SI ref 20 or the diaphragm radius must be increased significantly, which decreases the spatial resolution of the device.
The precedent literature serves as the basis to make an informed decision of fabricating flexible pMUT using SU-8 instead of using rigid Si.We performed a COMSOL multiphysics simulation for resonance

Supplementary Note 3: Potential mechanisms of action of Ultrasound Neuromodulation
The In our study, the calcium indicator GCaMP7F labels primary excitatory neurons, and we demonstrated activation of excitatory cells when the transducer is placed adjacent to the neuron bodies of granule cells in the dentate gyrus (supplementary fig.18).It is important to note, however, that its expression does not imply the lack of activation of astrocytes/glial cells.We achieved robust stimulation using an ultrasound driving protocol that is known to excite neurons (refs 23, 60 in manuscript), but the mechanism of action can be partially driven by indirect excitation via astrocytes 'gliotransmitters release.
In the scope of this work, we have demonstrated the ability of a new implantable and spatially precise device to cause neuron excitation, and we envision that further studies can help elucidate the mechanism of action of this activation in the various regions we tested, whether via direct or indirect stimulation.

Supplementary Note 4: Rationale for Ultrasound parameters
The driving frequency of 500 kHz in water is well described in the literature as an appropriate frequency for transcranial neuromodulation in mice brains (refs 15, 22, 23 and 24 in manuscript), as it presents a good tradeoff between skull transmission (more efficient in ranges below 1 MHz) and spatial selectivity (focus size decreases with frequency increase).Although our proposed implantable device bypasses the skull and is focused in a volume <100 μm 3 , we chose to design a device with 500 kHz resonant frequency as it could reproduce or explore similar established protocols for neuron activation.Ye et al. (2016) investigated the frequency dependence of ultrasound neuromodulation in the mouse brain, and in the range of 0.3 -2.9 MHz, demonstrated that the activation success rate was nearly flat at lower frequencies but, at higher frequencies, higher spatial peak intensities were necessary to attain comparable success rates when contrasted with lower ones (ref 16 in manuscript).
The Pulse Repetition Frequency dependency on ultrasound neuromodulation was explored by Manuel et al. (2020) , who demonstrated that pulsed stimulation was more efficient than continuous wave stimulation (ref 62 in the manuscript).Among pulsed US, the parameter space that led to the biggest activation of neurons (quantified by calcium imaging) had PRF of 1500 Hz, center frequency of 500 kHz, acoustic pressure of 100 kPa, and burst duty cycle of 60%, which are very similar to our stimulation parameters.protocol.Often the choice of parameters is limited by the availability of commercial ultrasound probes or the manufacturing capabilities of research facilities.One advantage of our chosen method of pMUT fabrication is the possibility of creating an ultrasound element or an array of elements in many sizes and center frequencies, a flexibility that will allow for the continuation of parameter investigations in various ultrasound modulation applications.Therefore, for the current work investigating neuron excitation in mice, our ultrasound driving parameters of 500 kHz, 10-50% duty cycle, 1500 Hz pulse repetition frequency are consistent with literature and corroborated our findings of ImPULS's ability to elicit a modulatory response.

Supplementary Note 5: COMSOL simulation parameters
The geometric parameters used in the COMSOL simulation model: fig.4), however, to reduce the resonance frequency of Si-based pMUT to ~500 kHz needs significant increase in cavity size thereby overall device size (SI ref25).Considering all this, we have decided to fabricate flexible SU-8 based pMUT instead of rigid Si-based pMUT.
ability of ultrasound waves to activate neural cells of various types has been demonstrated in several past works with explorations into mechanisms encompassing the activation of mechano-sensitive PIEZO and TRP channels, demonstrated in vitro (refs 11, 14 in manuscript) and in vivo with transcranial focused ultrasound (ref 14 in manuscript and SI ref 26).These channels exist in both neurons and astrocytes, but studies have shown that different neural cell types might respond differently to US stimulation.Zhu et al.(2023) demonstrated that knocking out the highly mechano-sensitive PIEZO channels in neurons resulted in the loss of US modulation sensitivity while knocking out PIEZO channels in astrocytes did not (SI ref likely employing different mechanisms.Indeed, the ultrasound frequency and stimulation parameters can potentially be tuned to achieve a degree of cell selectivity as has been demonstrated by other groups (ref 61 in the manuscript).Genetic or pharmacological studies that disable mechanosensitive ion channels simultaneously affect other physiological processes that maintain cell or 13).Leeet al. (2023) and Oh et al. (2019) demonstrated that TRPA-channels from astrocytes can be ultrasonically activated and are sufficient to indirectly excite neurons via glutamate release (Ref 14 in manuscript and SI ref 27).Therefore, we hypothesize that ultrasound can activate both neurons and astrocytes, although Pressures close to 100 kPa have been shown to activate neural circuitry, as exemplified by Tufail et al. (2010) (ref 22 in the manuscript), who tested pulsed driving frequencies in the 0.25-0.5 MHz range with max pressure of 97 kPa and published a separate protocols paper (ref 21 in the manuscript).Notably, they found that their pulsed protocol with PRFs in the 2.5 kHz range was sufficient to activate robust stimulation in hippocampal circuits.In other studies by Yoo et al. (2022) (ref 11 in the manuscript) in cortical neurons, pressures exceeding 150 kPa were needed to reliably drive neural activation but subthreshold pressures still had modulatory effects.Taken together, we can see there are a number of parameter investigations for the ultrasonic modulation of neuronal tissue, but no consensus on which optimal parameters should be used.Recommendations for successful parameters of ultrasound neuromodulation encompass a range, and our chosen parameters are contained in the recommendations of both Blackmore et al.'s (2019) review and Tufail et al.'s (2011)