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

Abstract

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.

Fig. 1. An implantable piezoelectric ultrasound stimulator: ImPULS.

a Schematic illustration of an implantable piezoelectric ultrasound stimulator (ImPULS) implanted in a subcortical brain region of a wild-type mouse. A magnified view showing the activated neurons with ultrasound application. Schematic created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. b Schematic of a peeled view of the ImPULS, revealing each layer. The ImPULS is a piezoelectric micromachined ultrasound transducer (pMUT) structure where biocompatible potassium sodium niobate (KNN) is sandwiched between two thin SU-8 layers, and an air-filled cavity and a backing layer is formed underneath the piezoelectric thin-film membrane. c Optical image of the ImPULS assembled with flexible ACF cable and a custom printed circuit board (PCB) with a magnified view of the ImPULS probe (right, top inset) and further zoomed version of the tip of the probe (ultrasound unit) under a microscope (bottom, left inset). Scale bars, 5 mm, 2 mm, and 100 µm, respectively. d Colorized cross-sectional scanning electron microscope (SEM) image of the ImPULS. The device consists of (1) SU-8 encapsulation layer, (2) Top Pt electrode, (3) KNN thin film, (4) Bottom Au electrode, (5) SU-8 membrane layer, (6) Air-cavity, and (7) SU-8 backing layer. Scale bars, 20 µm and 500 nm, respectively.

Fig. 2. Characterization of implantable piezoelectric ultrasound stimulator (ImPULS).

a The impedance and phase angle spectra of the ImPULS in air and water, showing the resonance frequency in both mediums. b Displacement of ImPULS in air and water mediums measured using laser Doppler vibrometer (LDV) at 4 V (p-p) when the inputs are a periodic chirp (bottom) and a sinusoidal signal (top). c Displacement of ImPULS as a function of input voltage (p-p) with inset showing two-dimensional (2-D) point scan of displacement indicating the lateral resolution of the beam of the device. Error bar represents the standard deviation in measurement, N = 3 devices. Data represents mean values ± SD. d Simulated acoustic pressure profile of the ImPULS showing a spherical pressure distribution. e Comparison of simulated and experimentally measured pressure using a fiber-optic hydrophone at different distances. Error bar represents the standard deviation in measurement, N = 3 devices. Data represents mean values ± SD in the x and y directions. f 2-D mapping of pressure generated by the ImPULS measured at z = 15 μm. Scale bar, 25 µm. g Microscopic image of the ImPULS taken each 24 h apart during the aging test (left), and normalized displacement of ImPULS before the start of the test and after 7 days. Scale bar, 100 µm. Error bar represents a standard deviation in measurement, N = 3 devices. Data is normalized to the displacement measured on day 0 per device. Day 8 data represents the mean displacement ± SD. (a.u. arbitrary units). h Temperature change in water medium when a continuous sinusoidal signal of 500 kHz at 20 V (p-p) applied to the ImPULS. Ultrasound was ‘off’ for 10 min, ‘on’ for 10 min and ‘off’ for 10 min. i 2-D mapping of temperature generated by the ImPULS measured at z = 15 μm. Scale bar, 25 µm.

Fig. 3. Robust stimulation of the dCA1 in anesthetized mice.

a Experimental design and schematic diagram of surgical procedure. Schematic created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. b Representative images of the hippocampus across experimental conditions: No-stim (top), and 500 kHz, 500 kHz, continuous wave for 60 s (middle), and 10% duty factor for 60 s (bottom). Yellow dashed boxed area approximates the dorsal CA1 (dCA1) area used for cell counts. Scale bar, 450 µm. Color-coded dashed box indicates the area shown in the magnified section. Scale bar, 100 µm. c cFos+ cells in dCA1 normalized by area across experimental conditions (One-way ANOVA; N = 4 mice control implant no-stim; N = 3 mice for 500 kHz condition; N = 3 mice for 500 kHz, 10% duty factor; No-stim vs. 500 kHz: p = 0.0506; No-stim vs. 500 kHz, 10% duty factor: p = 0.0184).

Fig. 4. Stimulation of nigrostriatal dopamine release in anesthetized mice.

a Schematic diagram of the experimental approach to stimulate substantia nigra pars compacta (SNc) dopaminergic (DA) neurons, including post-hoc histological validation of on-target implantation and DA2m sensor expression. Scale bar, 500 µm. Schematic created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. b Averaged DA2m fluorescence responses for control (top right) and SNc (bottom right) stimulation trials. Average heatmaps of fluorescence across trials for control (top left, 3 mice) and SNc (bottom left, 3 mice) stimulation trials. Solid line data represents mean data and bands represent ± SD. c, Full recording trace of Z-score DA2m fluorescence across stimulation trials, with onset and offset of stimulation (5 s,1500 Hz, 50% duty factor) indicated by solid and dashed red lines, respectively. d, Area under the curve (AUC) analysis for 5 s pre-stimulation versus 5 s during stimulation for average control and SNc stimulation trials (3 mice/group). Repeated-measures 2-way ANOVA with Sidak’s multiple comparisons test (***p = 0.0009, ****p < 0.0001).