Transcranial Pulsed Ultrasound Stimulates Intact Brain Circuits
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1 Neuron, Volume 66 Supplemental Information Transcranial Pulsed Ultrasound Stimulates Intact Brain Circuits Yusuf Tufail, Alexei Matyushov, Nathan Baldwin, Monica L. Tauchmann, Joseph Georges, Anna Yoshihiro, Stephen I. Helms Tillery, and William J. Tyler SUPPLEMENTAL FIGURE LEGENDS Figure S1 related to Figures 1-7. A general experimental configuration for conducting intact brain circuit stimulation using transcranial pulsed ultrasound is shown. The illustration is meant as a guide to facilitate the set-up of same or similar approaches in other laboratories.
2
3 Figure S2 related to Figure 1. To further outline the strategy for constructing stimulus waveforms a typical low-intensity US waveforms is illustrated. The example US stimulus waveform was constructed using single US pulses (A) containing cycles of.5 MHz US. (B) Fifty US pulses were repeated at a PRF of 2. KHz to produce final stimulus waveforms lasting 25 msec. (C) Multi-dimensional pressure output profiles obtained using a 25 mm planar US transducer alone (top) and with a 5 mm acoustic collimator tapered to a 2 mm output diameter (right) used to differentially target US stimulus waveforms to intact brain regions are illustrated. (D) Normalized pressure profiles obtained with a 25 mm planar transducer (black) and with a tapered 2 mm output diameter (red) collimator are illustrated for X (left) and Y (right) planes. The full-width half-maximum (FWHM) values are illustrated for each plane.
4 Single US Pulse A Hydrophone Voltage Trace (mv) Cycle Pulse; f =.5 MHz Time (msec) ISPTA = 13 mw/cm Time (msec) (m m) ce (mm ).8 FWHM mm 1.89 mm X-axis Distance (mm).8 FWHM mm 1.61 mm Y-axis Distance (mm) ce Dis tan -. Y-a xis Distan 7.5 Dis tan X-axis ) Y-a x ce (mm -. Normalized Pressure Normalized Pressure D Distan X-axis.4 is -7.5 ce (m m ) Normalized Pressure Normalized Pressure ISPPA =.225 W/cm2 5 5 US Pulses; PRF = 2. KHz C PII =.65 mj/cm2 1 ulus Waveform. Hydrophone Voltage (mv) B
5 Figure S3 related to Figure 5. (A) Representative images from coronal sections immunolabeled with antibodies against c-fos. Images are depicted for dorsal cortex of slices rostral and caudal to the stimulus zone, as well as within the stimulus zone (indicated by red). The 2 mm diameter stimulus zone is marked by red arrows for two coronal sections within the stimulus region along the rostral-caudal axis. C-fos + cells are best viewed when using a "magnifying glass" option in the document reader to zoom in on regions of cortex. Within stimulated zones, one can clearly observe a greater density of c-fos + cells. In these regions a rough medial-lateral demarcation zone of positive cells can also be observed, which helps one visualize the path of pulsed US through the tissue. Mean c-fos+ cell densities are plotted for the stimulated hemispheres and zones, as well as the contralateral control hemispheres for the medial-lateral (B) and dorsal-ventral (C) brain axes.
6 A 1. mm Bregma -1. mm Stim Zone Bregma -1.7 mm Stim Zone Bregma -2.2 mm Bregma -4.1 mm 4 *P <.5 C Stim Zone * * * ** * 3 c-fos+ cells/6.25 x 1-2 mm2 Contralateral Control Dorsal ** * 1 2 *P < midline 4 Pial Surface Cortex ** * 1 Depth (mm) c-fos+ cells/6.25 x 1-2 mm2 B Stimulated Hemisphere Corpus Callosum Hippocampus 2 Geniculate Nuclei 3 Thalamic Nuclei VPL VPM 4 Cerebral Peduncle 4 lateral midline 1 distance (mm) 2 3 Subthalamic Nucleus 5 4 lateral 6 Ventral * Stimulated Hemisphere Contralateral Control
7 Figure S4 related to Figure 6. (A) Representative EMG recordings obtained from the left triceps brachii of the same mouse across multiple stimulation trial days in response to stimulation of motor cortex with transcranial pulsed US. The histogram shows the mean EMG amplitudes obtained from mice undergoing multiple repeated trials across days as illustrated above. (B) Electron micrographs illustrate neuropil from the motor cortex of an unstimulated control (left) compared to a US stimulated cortical region (right). Cellular membranes, synapses, dendrites, mitochondria, endoplasmic reticulum, and myelination are observed in these images and do not appear to be different across treatment groups indicating low-intensity transcranial US stimulation produces no gross ultrastructural damage to brain tissue. (C) Histograms illustrating mean rotorod running times (left and middle) and mean wire hang times (right) obtained 24 hr prior to, and 24 hr and 7 d following sham-treatment or US stimulation of motor cortex.
8 A Transcranial US Brain Circuit Stimulation of Right M1 Repeated Across Weeks Control B 1. um 2 µv Left Tricep EMG Response 3 sec Day 7 2 µv Left Tricep EMG Response 3 sec 19,5X Magnification Day EMG Amplitude (µv) d 7d 14d US stim day 17 RPM Control pre 24h 7d post stim 26 RPM pre 24h 7d post stim Hang Time (sec) Figure 3 Rotorod Run Time (sec) 2 µv 3 sec Left Tricep EMG Response Day 14 Performance C Motor pre 24h 7d post stim
9 Figure S5 related to Figure 7. Individual extracellular recording traces obtained in the CA1 s.p. region of intact hippocampus in response to transcranial pulsed US. Traces (black) are illustrated for US-evoked LFP (1-12 Hz; left) and corresponding US-evoked SWP (16-2 Hz; middle) frequency-bands. A 25 msec region of the US-evoked SWP response is expanded (red) to illustrate SWP "ripples" (right) in response to transcranial stimulation of the intact mouse hippocampus with pulsed US.
10 LFP (1-12 Hz) SWP (16-2 Hz) SWP ripples 1 mv 5 µv 1 µv 25 msec region expanded 5 msec 5 msec 25 msec
11 Table S1. Low-intensity transcranial US waveform properties used to stimulate intact mouse motor cortex. PD PRF p r P II I SPTA f (MHz) c/p (msec) (Hz) Np length (sec) (MPa) (mj/cm 2 ) (mw/cm 2 ) MI * * Relatively high-intensity US stimulus waveform used to assess safety
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