Optimized ultrasound neuromodulation for non-invasive control of behavior and physiology

Abstract

Focused ultrasound can non-invasively modulate neural activity, but whether effective stimulation parameters generalize across brain regions and cell types remains unknown. We used focused ultrasound coupled with fiber photometry to identify optimal neuromodulation parameters for four different arousal centers of the brain in an effort to yield overt changes in behavior. Applying coordinate descent, we found that optimal parameters for excitation or inhibition are highly distinct, the effects of which are generally conserved across brain regions and cell types. Optimized stimulations induced clear, target-specific behavioral effects, whereas non-optimized protocols of equivalent energy resulted in substantially less or no change in behavior. These outcomes were independent of auditory confounds and, contrary to expectation, accompanied by a cyclooxygenase-dependent and prolonged reduction in local blood flow and temperature with brain-region-specific scaling. These findings demonstrate that carefully tuned and targeted ultrasound can exhibit powerful effects on complex behavior and physiology.

Keywords: fiber photometry; focused ultrasound; hypothalamus; optogenetics; thalamus.

Figure 1.. Stepwise parametric sweeps of ultrasound features reveal bidirectional manipulation of the midline thalamic nuclei

a) Illustration of parametric features examined at each step. b) Overlay of FUS intensity field on intended CMT target area accompanied by a fluorescence image overlay of CMT GCaMP6s expression. c) Overlay of FUS intensity fields and fluorescence images of the GCaMP6s expressing CAMKII+ CMT neurons d) CMT calcium responses during FUS stimulus as function of ultrasound PRF. The gray line (Quant) represents the period over which the response was quantified for all line plots (Bars represent mean ± S.E.M. and circles represent animals for all plots, n = 9 mice, repeated measures ANOVA p = 0.007, One sample t-test; * p<0.05, ** p<0.01). e) CMT calcium responses during post-FUS period (10–85s post stimulus onset) as function of ultrasound PRF (n = 9 mice, One sample t-test; * p=0.038). f) CMT calcium responses during stimulus (0–40s post stimulus onset) as function of 2.5 Hz temporal compression (n = 7 mice, repeated measures ANOVA p = 0.0098, One sample t-test; * p<0.05, ** p<0.01). g) CMT calcium responses during post-FUS period as function of ultrasound 20 Hz temporal compression (n = 8 mice, One sample t-test; * p< 0.05). h) CMT calcium responses during stimulus as function of 2.5 Hz, 5-s compressed ultrasound intensity (n = 6 mice, repeated measures ANOVA p = 0.0007, One sample t-test; * p<0.05, ** p<0.01, **** p<0.0001). i) CMT calcium responses during post-FUS period as function of ultrasound 20 Hz, 40-s compressed ultrasound intensity (n = 6 mice, One sample t-test; * p< 0.05).

Figure 2.. Stepwise parametric sweeps of variant cell types reveal conserved and variant waveform optima

a) Overlay of FUS intensity fields and fluorescence images of the GCaMP6s expressing DMH, LC, and BNST target areas. b) Time series and time-averaged calcium activity during FUS stimulus as function of ultrasound PRF for the DMH (left, n = 6 mice, Bars represent mean ± S.E.M. and circles represent animals for all plots, repeated measures ANOVA p< 0.0001, One sample t-test; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001), LC (middle, n = 6 mice, repeated measures ANOVA p= 0.026) and BNST (right, n = 7, One sample t-test; *; p= 0.012). c) Calcium responses during stimulus as function of 2.5 Hz temporal compressed FUS for the DMH (One sample t-test; * p<0.05, ** p<0.01) and LC. d) Calcium responses during stimulus as function of 2.5 Hz, 5-s compressed FUS for the DMH (repeated measures ANOVA p= 0.0074, One sample t-test; ** p<0.01, *** p<0.001) and LC (One sample t-test; * p<0.05). e) Spline fits of ΔF/F versus each feature examined across cell types examined.

Figure 3.. Optimized FUS protocols induction of distinct walking and head motion behavior

a) Illustration of behaviors examined using overhead infrared image tracking. b) Time series of change in head speed with varying target and FUS waveform stimulation (Δ relative to pre-stimuli baseline, shaded grey area represents stimuli period, bars represent mean ± S.E.M., 10-s bins). Associated GCaMP6s photometry recordings are shown for all behavioral time series (trace represents 10-trial average). Individual animal means during stimuli (“Quant” period, grey line) are collated to the right of the time series (n = 6–7 mice per group, Bars represent mean ± S.E.M. and circles represent animals for all plots, One sample t-test; **, p<0.01. Groups compared using a one-way ANOVA with Bonferroni correction; # p < 0.05, ## p < 0.01). c) Time series of change in body speed, d) stretch attend posture, and e) head speed with varying target and FUS waveform stimulation (Δ relative to pre-stimuli baseline, shaded grey area represents stimuli period, bars represent mean ± S.E.M., 10-s bins) Individual animal means during FUS stimulation are collated to the right of the time series (n = 6–7 mice per group, One sample t-test; * p<0.05, ** p<0.01. Groups compared using a one-way ANOVA with Bonferroni correction; # p < 0.05, ## p < 0.01).

Figure 4.. Hypothalamic sub-regions differentially respond to FUS stimulation

a) Horizontal plane view of the mouse brain with dorsomedial and lateral hypothalamic sub regions. The circle approximates the lateral full-width half-maximum boundaries when targeting either region (Green, lateral hypothalamus; Blue, dorsomedial hypothalamus). b) Overlaid time series of change in walking speed and stretch attend posture when stimulating either of the hypothalamic sub regions (n = 6–7 mice, DMH data repeated from Fig. 5b, c, Bars represent mean ± S.E.M. for all plots, One sample t-test; * p<0.05, ** p<0.01, Q1; 0–50 seconds post stimulation). c) Illustration of rotarod task where time is measured between FUS onset and task failure. d) Change in time to failure on the rotarod task with and without FUS stimulation applied to either of the hypothalamic sub regions, or the locus coeruleus (circles represent individual animals for all plots, paired two-tailed t-test; ** p<0.01). e) Linear correlation of freely behaving motion changes during the stimulus (Quant, panel b) and change in time to failure with and without FUS stimulation (panel d) (Pearson correlation, DMH; two-tailed p=0.0066, LH; two-tailed p=0.5275). The quantified period was chosen to be inclusive of all rotarod failure times (0–50 sec post stimulus onset).

Figure 5.. FUS induces cyclooxygenase dependent vasoconstriction and deep brain cooling

a) Illustration of a thermocouple probe implanted in place of the optical fiber within the PhoCUS apparatus. b) Time series of change in focal brain temperature relative to CMT targeted FUS stimulation (Line and shaded area represent mean ± S.E.M.) and c) comparison of the maximum decrease in temperature within animals (n = 5 animals, Bars represent mean ± S.E.M. and circles represent animals for all plots, One sample t-test; # p<0.05, paired two-tailed t-test; ** p<0.01). d) Average traces of thermal recording from skull probe. e) Illustration of rhodamine B dextran fluorescence monitored in blood with spectrally separate measurement of GCaMP6s with a time series average of recording from the CMT. f) Time series average averages of neural activity (gray) and blood fluorescence (red) relative to FUS stimulus. g) Time series average averages of neural activity and h) blood volume at the CMT with and without COX inhibitor or saline vehicle administration prior to FUS stimulation with individual change across post stimulus period (paired two-tailed t-test; * p=0.019, One sample t-test; ## p<0.01).

Figure 6.. FUS induced vasodynamics are brain region dependent

a) Illustration of a PhoCUS apparatus for simultaneous recording of brain GCaMP and blood volume b) Time series average averages of neural activity for arousal regions during blood fluorescence monitoring c) Time series averages of blood fluorescence for all arousal regions during FUS stimulation with quantification during post FUS period (15–180 sec) (Bars represent mean ± S.E.M. and circles represent animals for all plots, n = 5–6 mice, One sample t-test; # p<0.05, paired two-tailed t-test; ** p<0.01). d) Illustration of system for simultaneous functional ultrasound imaging and stimulation with an intensity overlay. e) Representative time series of changes in cerebral blood volume (ΔCBV) relative to a 5 second FUS stimulation (2.5 Hz, 5 seconds, 20% D.C, 18.9 W/cm²). f) Example 2D brain segmentation for quantification (hypothalamus visible in 3/5 experiments) g) time series averages of ΔCBV across different brain areas during FUS stimulation with quantification during post FUS period (30–180 sec) (Bars represent mean ± S.E.M. and circles represent animals for all plots, n = 3–5 mice, One sample t-test; # p<0.05, ## p<0.01).