Low-intensity focused ultrasound alters the latency and spatial patterns of sensory-evoked cortical responses in vivo

All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of New York Medical College. Adult C57BL/6J and C57BL/6J-Tg(Thy1-GCaMP6f) mice between two to five months were anesthetized with an initial dose of ketamine/xylazine (90/12 mg kg⁻¹) delivered interperitoneally, and were given maintenance doses of ketamine (30 mg kg⁻¹) every 45 min for the remainder of experimental sessions, which typically lasted 2–3 h, including periods of dye staining and the application of any pharmacological agents. Core body temperature was measured and maintained at 37 °C with a closed-loop temperature controlled heating pad (40-90-8D, FHC, Inc., Bowdoin, ME).

Following initial anesthesia induction, animals were positioned in a stereotaxic apparatus (Stoelting Co., Wood Dale, IL), and the eyes covered with petrolatum ophthalmic ointment (Puralube^®, Fera Pharmaceuticals, Locust Valley, NY). The scalp was infused with lidocaine delivered subcutaneously and a midline skin incision was performed to expose the skull. A ~$3\times 3$ mm square craniotomy was performed on the region overlying the forelimb's representation on primary somatosensory cortex (at the same point on the rostrocaudal axis as bregma and ~2.5 mm lateral), and the dura in the region was carefully retracted with a surgical dura hook.

In VSD imaging experiments, a fragment of Gelfoam dental sponge (Pfizer Inc., New York, NY) was saturated with an aqueous solution of di-4-AN(F)EPPTEA (184 µM, excitation/emission maxima at 444 nm/610 nm) (Yan et al 2012) and placed on the exposed region of the brain. The duration of the staining was 90 min, during which small volumes of dye solution were periodically added to the Gelfoam to prevent drying. The Gelfoam was subsequently removed and the brain was rinsed with saline solution to remove unbound dye. Following all staining and incubation periods, the brain surface was covered with 1.5% low-melting-point agarose and covered with a fragment of a glass coverslip; the glass window was sealed at its periphery with dental acrylic (Co-Oral-Ite Dental MFG. Co., Diamond Springs, CA). In experiments involving the use of tetrodotoxin (TTX), following the VSD staining and subsequent washout, another fragment of Gelfoam was saturated with a 10 µM TTX solution and placed on the brain; the gelfoam remained there for 30 min. For these experiments, the agarose solution underneath the glass window also contained 10 µM TTX.

After the glass window was sealed, an aluminum bar was fastened to the other side of the skull with cyanoacrylate glue (Vetbond, 3M Inc., Maplewood, MN) and dental acrylic. Subsequently, the stereotactic earbars and bite bar were removed, and the metal bar affixed to the skull was screwed into a custom articulating mount fastened to the same stereotactic base, which could be moved on and off of the microscope stage.

The experimental apparatus utilized a Nikon AZ100 multizoom macroscope as the main imaging device (figure 1). To accommodate the custom ultrasound transducer, which was in series with the optical epi-illumination path, a long working-distance objective was used (AZ-Plan Fluor; magnification: $5\times$; numerical aperture: 0.5; working distance: 15 mm). The image was de-magnified $0.18\times$ before being directed to the entry aperture of a CMOS camera (Zyla, Andor Technology Ltd., Belfast, UK). The diameter of the maximum field of view was ~4.5 mm.

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Illumination for VSD experiments was provided by a high power LED source (UHP-T-520-LN, Prizmatix Co., Givat-Shmuel, Israel) centered at 520 nm (±6 nm) with a low optical noise LED driver. The cooling fan for the LED source was temperature activated; to minimize mechanical artifacts, the LED source was turned on well before imaging sessions and the light was gated with an external mechanical shutter (Uniblitz VMM-DI, Vincent Associates, Rochester, NY). Excitation light was bandpass filtered at 520 nm  ±20 nm, and emission was long pass filtered  >575 nm (Chroma Technology Corp., Bellows Falls, VT). In Ca²⁺ imaging experiments involving Thy1-GCaMP6f animals, illumination was provided by a halogen lamp (Lambda LS, Sutter Instruments, Novato, CA). Excitation light was bandpass filtered at 470 nm  ±20 nm, and emitted fluorescence was bandpass filtered at 525 nm  ±25 nm. The imaging frame rate for VSD experiments was 537 Hz, and was 33 Hz for GCaMP6f experiments. Image processing was performed using custom Matlab (The Mathworks, Inc., Natick, MA) code. A 5-pixel Gaussian spatial filter was applied to raw images, and the average of frames prior to forelimb stimulation was used as a reference to obtain fractional fluorescence, i.e. ΔF/F. Z-scores represented the ratio of averaged fractional fluorescence to the standard deviation of pre-stimulus fluctuations. Some aspects of spatial data analysis were performed using ImageJ plugins (Abramoff et al 2004).

Focused ultrasound was delivered by a custom transducer, shown in figure 2, that consisted of 16 elements aligned in a ring-shaped geometry, optimized at 510 kHz (modified version of H-205B, Sonic Concepts, Inc., Bothell, WA). The inner diameter was 18 mm and the radius of curvature was 11.5 mm. The transducer array's 16 elements were binned into four-channel quadrants which could operate independently, permitting the beam's focal profile to be shaped. Without knowing a priori the degree to which the spatial pattern of cortical responses are affected by FUS, we sought to perturb the full extent of the forelimb's receptive field on the primary somatosensory area. To achieve a focused beam despite an extremely short working distance (relative to the spacing between elements, which were separated by a wide diameter to accommodate optical imaging), we utilized the principle of a parametric acoustic array (Westervelt 1963). Operating the four quadrants at frequencies that differed by 4 kHz elicited a continuously changing pattern of constructive and destructive interference at the focus and enabled us to achieved a focal spot of lateral width 3.3 mm (full width at half-maximum). This effectively filled the entire field of view, which exceeded the spatial extent of the forelimb's representation. Stimulus waveforms were generated and amplified by a TPO-106 transducer drive system (Sonic Concepts, Inc., Bothell, WA). For neuromodulation experiments, sonication consisted of a 1 s burst of pulsed FUS of focal intensity I_sppa  =  0.69 W cm⁻² (peak pressure at the focus 0.17 MPa) wherein 500 µs pulses at center frequency 510 kHz were delivered at a repetition rate of 1 kHz. Beam properties were characterized using a RESON spherically directional hydrophone (characterization performed by Sonic Concepts, Inc.).

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The vertical profile of the transducer was sufficiently low as to permit optical imaging through the central aperture with a relatively high numerical aperture microscope objective. The acoustic focus of the transducer was co-localized with the microscope's optical focus. The interior of the ring transducer was filled with a volume of vacuum-degassed water that was bounded by a 25 mm optical window on the top side of the transducer (Edmund Optics, Barrington, NJ) and, at the bottom, a plastic membrane sealed to the transducer with an O-ring. The bottom volume effectively constituted a distensible 'cushion' which was necessary for coupling FUS from the large aperture to the mouse's brain at a short working distance. We verified that the acoustic and optical focal planes were co-localized by observing the emergence of thermally-induced holes in 90 µm thick live brain slices from a rat when sonicated at different focal depths (at 280 W cm⁻² I_sppa, constant-wave FUS). Holes in the tissue emerged at the center of the field of view only when the objective was within ~1 mm of the sharpest optical focus. Live slices were prepared and maintained following (Zhang et al 2006).

A layer of ultrasound transmission gel (Aquasonic 100, Parker Laboratories, Inc., Fairfield, NJ) was applied to the mouse's head before the transducer was lowered. Although the ultrasound gel and plastic membrane introduced slight warping of the optical plane, we were able to obtain relatively sharp images of cortical vasculature. Additionally, imaging through the transducer assembly's aperture revealed any remaining small air bubbles. If these were observed, the transducer assembly was removed and refilled carefully.

Imaging, sensory stimulation, and ultrasound components of the experimental apparatus were controlled by a single custom program in LabVIEW (National Instruments, Austin, TX). The general experimental protocol is depicted in figure 1(B). After data acquisition commenced, electrical stimuli were delivered at latencies sufficiently long as to obtain a period of pre-stimulus baseline data from which the average amplitude and standard deviation could be obtained. Somatosensory evoked cortical responses were elicited by biphasic pulses of current 0.2 ms in duration generated by a stimulus isolator (ISO-STIM 01M, NPI Electronic, Tamm, Germany) and delivered to the mouse's contralateral forelimb with a pair of 27-gauge stainless-steel needles inserted subcutaneously. In an effort to minimize the potential for nonspecific, antidromic stimulation of motor nerve fibers, the stimulus current was set to the lowest amplitude that still evoked a visible paw movement (typically ~1 mA). Sham experiments utilized the same settings except the stimulus isolator was switched off (analog input trigger remained, as did stimulating needles). In imaging trials that were preceded by ultrasound, frame acquisition began 200 ms after the end of FUS pulses in order to remove any possible mechanical artifacts due to sonication. Although we found such noise to be negligible at the low intensities of FUS used in our experiments, the magnitude of fractional fluorescence changes when using VSDs was typically on the order of 0.1%, so we took all precautions. For VSD experiments, the average of 25 single trials was used for data analysis, and for GCaMP6f data analysis the average of ten trials was used.

Indications of neural injury were assessed based on the expression of glial fibrillary acidic protein (GFAP) 24 h after sonication. This tissue analysis was performed on mice that were not involved in VSD or Ca²⁺ imaging experiments. Mice were anesthetized with a single dose of ketamine/xylazine (90/12 mg kg⁻¹). To ensure that the transcranial sonication was performed at the same site as the imaging experiments, the skull was exposed to reveal cranial landmarks. The scalp was shaved and disinfected with Betadine (Purdue Pharma L.P., Stamford, CT). A region of skin was then infused with lidocaine (0.5% solution) and a 1 cm incision was made, exposing the surface of the skull. The skull location overlying the forelimb's representation on the somatosensory cortex, roughly 2.5 mm lateral of the midline at the level of bregma (see Paxinos and Franklin (2004)), was marked with a fine-tipped surgical marking pen, and ultrasound transmission gel was applied to the head. The FUS transducer was lowered onto the head and the microscope was used to center the field of view at the marked spot. In preliminary experiments, we verified that the acoustic focal location was at the center of the field of view by sonicating with high intensity and observing the removal of marker ink at the center of the spot. After the brains were postfixed, slices were taken from a span of ~5 mm along the anteroposterior axis, centered approximately at bregma. Slices were subsequently determined to be localized at bregma based on morphological comparison with a mouse stereotaxic brain atlas (Paxinos and Franklin 2004). After sonication, the incision was closed and sealed with cyanoacrylate tissue adhesive and the animal was removed from the apparatus. To prevent dehydration, 1 ml of warmed lactated Ringer's solution was injected subcutaneously and topical antibiotic ointment was applied to the closed incision. Animals that appeared to be in pain following recovery received subcutaneous injections of Buprenorphine (0.1 mg kg⁻¹) twice daily. Animals were individually housed with food and water available ad libitum. 24 h after sonication, mice were overdosed with ketamine/xylazine and perfused through the left cardiac ventricle with heparinized physiological saline followed by 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). Brains were dissected out and postfixed in 4% PFA for 24 h at 4 °C, after which they were washed in PBS for one hour and equilibrated in a 30% sucrose solution. Brains were then embedded in OCT compound (Tissue-Tek, Sakura Finetechnical Co., Tokyo, Japan) and sectioned into 40 µm thick coronal sections on a cryotome. Sections were washed and blocked by incubation with 1% bovine serum albumin (BSA) in PBS supplemented with 0.4% Triton X-100 for one hour at room temperature. They were then incubated with rabbit anti-GFAP polyclonal antibody (1:400 dilution, ThermoFisher No. 18-0063) in 1% BSA and 0.4% Triton X-100 at room temperature overnight. Sections were then incubated for one hour with Alexa-488 labeled secondary antibody (1:500 dilution, donkey anti-rabbit, A21206, Life Technologies, Carlsbad, CA), after which sections were mounted on slides, dehydrated, cleared in xylene, and coverslipped with non-fluorescent mounting medium (Krystalon, 64969-95, EMD, Darmstadt, Germany).

BBB disruptions were assessed based on the leakage of intravenously-administered Evans blue dye into the brain's parenchyma. 30 min prior to FUS treatment, 0.1 ml of a 2% solution of Evans blue dye was injected intravenously through the tail vein. FUS was administered as described above for exploring changes in GFAP expression. Two hours following recovery, mice were overdosed with ketamine/xylazine and perfused through the left cardiac ventricle with saline solution. The brains were then dissected out and postfixed, embedded in OCT compound, sliced on a cryotome, and mounted on slides as described above for GFAP analysis. Subsequently, the Evans blue dye distribution was observed by means of fluorescence imaging (560  ±  28 nm excitation, 645  ±  38 nm emission) with a Nikon Eclipse 90i upright microscope. We performed this experiment at low FUS intensity in two animals and at high intensity in two other animals.

In VSD experiments, it was possible to discern prominent somatosensory evoked cortical responses through the transducer aperture (figure 3). The average peak fractional fluorescence change (ΔF/F) observed with di-4-AN(F)EPPTEA was ~0.2%  ±  0.08% (mean  ±  SEM). Consistent with previous in vivo findings with forelimb stimuli (e.g. Brown et al (2009)), onset latency was 15.3  ±  0.9 ms (mean  ±  SEM) following electrical stimuli (figure 4). We defined the onset of the response as the time point at which the response ΔF/F exceeded two times the standard deviation observed during baseline periods (i.e. z-score  >  2). The spatial region on which temporal analysis was performed was selected based on the pixels' Pearson's correlation coefficient (cc) when compared with a step function describing the somatosensory stimulus; pixels were included in the temporal analysis if their cc was greater than one standard deviation above the mean cc, which was assessed over all pixels in the image.

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As depicted in figure 4, when trials were preceded by 1 s of low-intensity FUS, the onset of optical responses began 3.0  ±  0.7 ms earlier (P  <  0.05, obtained through Wilcoxon rank sum test, n  =  7). We did not observe a significant reduction in response latency at a modestly higher FUS intensity (I_sppa  =  3.5 W cm⁻², versus 0.69 W cm⁻²), although the sample size was smaller (n  =  4). Subsequently, in the 'recovery' period, consisting of averaged responses obtained in the same animals 20 min after the FUS-preceded trials, the onset latency did not differ significantly from baseline (non-FUS) experiments. Inhibiting action potentials by applying tetrodotoxin (TTX) abolished all evoked voltage responses, whether preceded by FUS or not. Although the peak amplitude of the optical responses preceded by FUS generally increased, when averaged over all animals, the responses varied significantly among experiments and the change was not statistically significant for our sample size.

In addition to alterations in temporal kinetics, pre-treatment with FUS altered the spatial morphology of evoked cortical responses. The peak fractional fluorescence signal was low in VSD experiments (on the order of 0.1%), and the spatial patterns of electrophysiological responses were therefore typically obscured by photon shot noise. In contrast, it was easier to observe spatially focal responses in Ca²⁺ imaging experiments using Thy1-GCaMP6f mice. In these experiments, stimulating the forelimb evoked clear fluorescence responses that peaked ~180 ms following stimulus. Mirroring VSD experiments, TTX blocked all calcium responses. Although we did not observe a significant change in response temporal kinetics, administering FUS just before sensory stimulation increased the spatial solidity of the Ca²⁺ response at the forelimb region of the primary somatosensory cortex (figure 5). Using z  >2 as a criterion for inclusion in spatial analysis, we quantified spatial solidity as the ratio of the area within the convex hull—the area enclosed within an encapsulating perimeter of minimum length—to the area of pixels with z  >  2. This emergent, co-varying property of form-factor and density has been previously used to describe morphologies in biological structures that change yet maintain a constant volume, such as the development of fibrils or processes emerging from a soma (e.g. Sołtys et al (2001)). FUS-pretreatment caused a 13.2  ±  3.2% (mean  ±  SEM) increase in response solidity. Additionally, FUS-pretreatment caused cortical responses to become more radially symmetric. The spatial 'circularity' of the evoked Ca²⁺ response, defined as $4\pi \frac{{\rm area}}{{\rm perimete}{{{\rm r}}^{2}}}$, where area is the area spanned by the pixels with z  >2 and perimeter is the linear pathway conforming to the exact spatial extent of the area, increased by 40.5  ±  16.4%. In subsequent trials that were not preceded by FUS, these morphological aspects did not differ significantly from baseline properties. It should be noted that the morphometric properties of solidity and circularity are not necessarily related to neural mechanisms, but served to quantify our empirical findings.

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Although the average and peak intensity parameters used in this study were within the range of those used in previous neuromodulation studies, we sought to assess any potential tissue-level damage or modification given that our transducer design and measurement configuration was relatively unique. We assessed the expression of GFAP 24 h after mice were exposed to FUS. Alterations in the expression level and morphology of labeled cells (primarily astrocytes) are often used as an indicator of brain injury (Chen and Swanson 2003). At the FUS stimulation parameters used in our imaging experiments, there did not appear to be any differential expression of GFAP between the sonicated and control hemispheres (figure 6). At the neurovascular level, to assess whether the observed alterations in electrical activity were associated with BBB compromise in our implementation, we explored the degree to which intravenously injected Evans blue dye permeated into the parenchyma (figure 7). Evans blue binds to serum albumin and does not leave the vasculature unless the BBB is permeated. At the neuromodulatory intensities used in this study, FUS did not induce observable alterations in the distribution of Evans blue dye fluorescence.

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