1–12 • The Journal of Neuroscience, March 13, 2024 • 44(11):e0784232024

Development/Plasticity/Repair

Transcranial Low-Intensity Focused Ultrasound Stimulation

of the Visual Thalamus Produces Long-Term Depression of
Thalamocortical Synapses in the Adult Visual Cortex
Lukas Mesik,1,2,3 Samuel Parkins,1,4 Daniel Severin,1 Bryce D. Grier,1,3 Gabrielle Ewall,1,3 Sumasri Kotha,1
Christian Wesselborg,1,4 Cristian Moreno,1 Yanis Jaoui,1 Adrianna Felder,1 Brian Huang,1 Marina B. Johnson,5
Timothy P. Harrigan,5 Anna E. Knight,5 Shane W. Lani,5 Théo Lemaire,6 Alfredo Kirkwood,1,3 Grace M. Hwang,2,5 and
Hey-Kyoung Lee1,2,3
1
Zanvyl-Krieger Mind/Brain Institute, Johns Hopkins University, Baltimore, Maryland 21218, 2Kavli Neuroscience Discovery Institute, Johns Hopkins
University, Baltimore, Maryland 21218, 3Solomon H. Snyder Department of Neuroscience, Johns Hopkins School of Medicine, Baltimore, Maryland
21205, 4Cell Molecular Developmental Biology and Biophysics Graduate Program, Johns Hopkins University, Baltimore, Maryland 21218, 5Johns Hopkins
Applied Physics Laboratory, Johns Hopkins University, Laurel, Maryland 20723, and 6Neuroscience Institute, New York University Langone Health,
New York, New York 10016

Transcranial focused ultrasound stimulation (tFUS) is a noninvasive neuromodulation technique, which can penetrate deeper and
modulate neural activity with a greater spatial resolution (on the order of millimeters) than currently available noninvasive brain
stimulation methods, such as transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS). While
there are several studies demonstrating the ability of tFUS to modulate neuronal activity, it is unclear whether it can be used for
producing long-term plasticity as needed to modify circuit function, especially in adult brain circuits with limited plasticity such
as the thalamocortical synapses. Here we demonstrate that transcranial low-intensity focused ultrasound (LIFU) stimulation of
the visual thalamus (dorsal lateral geniculate nucleus, dLGN), a deep brain structure, leads to NMDA receptor (NMDAR)-dependent
long-term depression of its synaptic transmission onto layer 4 neurons in the primary visual cortex (V1) of adult mice of both sexes.
This change is not accompanied by large increases in neuronal activity, as visualized using the cFos Targeted Recombination in
Active Populations (cFosTRAP2) mouse line, or activation of microglia, which was assessed with IBA-1 staining. Using a model
(SONIC) based on the neuronal intramembrane cavitation excitation (NICE) theory of ultrasound neuromodulation, we find that
the predicted activity pattern of dLGN neurons upon sonication is state-dependent with a range of activity that falls within the
parameter space conducive for inducing long-term synaptic depression. Our results suggest that noninvasive transcranial LIFU
stimulation has a potential for recovering long-term plasticity of thalamocortical synapses in the postcritical period adult brain.
Key words: adult plasticity; LIFU; long-term depression; noninvasive; synaptic plasticity; ultrasound

Significance Statement
Recovery of adult sensory cortical function is thought to be limited by the developmental decline in cortical plasticity mech-
anisms. In particular, thalamocortical (TC) synapses in the primary sensory cortices lose their ability to undergo experience-
dependent plasticity quite early in postnatal development. As such, recovery of adult sensory cortical function is often accom-
panied by restoration of thalamocortical plasticity. Here we used a noninvasive transcranial low-intensity focused ultrasound
(LIFU) stimulation, which can produce neuromodulation of localized structures deep in the brain, to elicit long-term depres-
sion of TC synapses in the primary visual cortex of adult mice. Our results suggest that transcranial LIFU stimulation could be
a useful therapeutic that can produce long-term plasticity of neural circuits to restore adult brain functions.

Received April 30, 2023; revised Dec. 13, 2023; accepted Jan. 30, 2024. The authors declare no competing financial interests.
Author contributions: L.M., S.W.L., G.M.H., and H-K.L. designed research; L.M., S.P., D.S., B.D.G., G.E., S.K., C.W., C.M., B.D.G.’s present address: Wu Tsai Neuroscience Institute, Stanford University, Stanford, California 94305.
Y.J., A.F., B.H., M.B.J., T.P.H., A.E.K., G.M.H., and H-K.L. performed research; L.M., S.P., D.S., B.D.G., G.E., S.K., C.W., G.M.H., G.M.H.’s present address: National Institute of Neurological Disorders and Stroke, National Institutes of Health,
and H-K.L. analyzed data; S.K. and T.L. contributed unpublished reagents/analytic tools; G.M.H. and H-K.L. wrote the paper. Rockville, Maryland 20852.
This work was supported by NIH Grant R21-EY031265 and Johns Hopkins Discovery Award to H-K.L. and GMH, Correspondence should be addressed to : Grace Hwang at [email protected] or Hey-Kyoung Lee at
Kavli Neuroscience Discovery Institute Distinguished Postdoctoral Fellowship to L.M., NIH predoctoral fellowship [email protected].
F31-EY031946 to S.P., and NIH Grant R01-EY012124 to A.K. The authors would like to thank Dr. Philip Feurtado for https://doi.org/10.1523/JNEUROSCI.0784-23.2024
helping with some of the LIFU stimulations. Copyright © 2024 the authors
2 • J. Neurosci., March 13, 2024 • 44(11):e0784232024 Mesik et al. • LIFU-Induced Thalamocortical LTD in Adult V1
Introduction
Traditional methods of brain stimulation have largely relied on (Guenthner et al., 2013), or a marker of microglia, which is upre-
electrical stimulation through implanted electrodes, which con- gulated following neuroinflammation (Muzio et al., 2021). Using
fer temporal precision and defined spatial localization. simulations based on the intramembrane cavitation model of
However, these methods require surgical implantation and ultrasound neuromodulation (Lemaire et al., 2019), we found
suffer from the potential damage of neural tissue from direct con- that the LIFU stimulation protocol is predicted to generate differ-
tact with the electrodes. To address these drawbacks, several non- ent firing patterns in dLGN neurons dependent on their state.
invasive methods for neural modulation have been developed, Our results suggest that transcranial LIFU stimulation can gener-
such as transcranial magnetic stimulation (TMS) (Thielscher ate long-term plasticity of TC synapses in the adult V1, which
and Kammer, 2002) and transcranial direct current stimulation could benefit the development of noninvasive therapeutics to
(tDCS) (Woods et al., 2016), but their utility is limited by low aid in functional recovery.
spatial resolutions (≥1 cm diameter) (Wagner et al., 2007) and
superficial cortical stimulations (Wagner et al., 2007; Siebner Materials and Methods
et al., 2009). Transcranial focused ultrasound stimulation Animals
(tFUS) is a rather unique noninvasive neuromodulation method Mice of both sexes were used in this study and reared in a normal
with a finer spatial resolution (in the range of millimeters) and 12-hour light/12-hour dark cycle. VGluT2-Cre (Slc17a6tm2(cre)Lowl/J,
penetration capacity for deep brain neuromodulation (Baek Jax mice stock# 016963; RRID:IMSR_JAX:016963) mice were
et al., 2017; Tyler et al., 2018; Dell’Italia et al., 2022). tFUS has used for targeting channelrhodopsin-2 (ChR2) expression in dLGN.
been used in different preparations to activate neurons and elicit cFosTRAP2 (Fostm2.1(icre/ERT2)Luo/J, Jax mice stock# 030323; RRID:
movement or behavioral changes (Tyler, 2011; Dell’Italia et al., IMSR_JAX:030323) mice were crossed with Ai14 (B6.Cg-Gt(ROSA)
26Sortm14(CAG-tdTomato)Hze/J, Jax mice stock# 007914; RRID:IMSR_
2022) by modulating neuronal activity through eliciting action
JAX:007914) to generate cFosTRAP2;Ai14 mice for histological analysis
potentials (Tufail et al., 2010; Tyler et al., 2018) or suppressing of neuronal activation. All animal procedures conform to the guidelines
activity (Yoo et al., 2011; Dell’Italia et al., 2022). However, a cur- of the US Department of Health and Human Services Office of
rent gap in knowledge is whether tFUS can modulate neuronal Laboratory Animal Welfare and were approved by the Institutional
activity to drive long-term plasticity, especially in adult circuits Animal Use and Care Committee at Johns Hopkins University.
with limited capacity. This understanding is much needed for
the development of tFUS-based therapeutics aimed at recovering In vivo stereotaxic injections to express channelrhodopsin-2 (ChR2) in
adult brain functions. We address this by using the thalamocor- dLGN
tical circuit in the primary visual cortex (V1) of adult mice as a VGluT2-Cre mice were used to specifically express ChR2 bilaterally in
dLGN using targeted injections. Adult mice were anesthetized using
model.
isoflurane (5% induction, 1–2% maintenance) and mounted on a stereo-
V1 has been used as a model of cortical plasticity in which the taxic instrument (Kopf). An incision was made along the midline and the
basic principles of experience-dependent plasticity have been skin was retracted to the sides to expose the skull. After stereotaxic lev-
elucidated. One of the key properties of cortical plasticity is eling, small craniotomies were made in both hemispheres, centered
that it has a defined critical period, which limits the ability of cor- 2 mm lateral and 2.3 mm posterior of Bregma. AAV9.Ef1alpha.
tical circuits to adapt to changes in sensory experience to an early dflox.hChR2(H134R) mCherry.WPRE.hGH (Addgene 20297-AAV9,
developmental period (Lee et al., 2005; Hooks and Chen, 2020). titer 1013 GC/ml; RRID:Addgene_20297) diluted 1:1 in sterile saline
At a cellular level, synaptic plasticity has been studied across all (∼200 nl) was then loaded into a glass pipette using a syringe pump
the cortical layers, and an emerging consensus is that thalamo- (Harvard Apparatus) and injected bilaterally into dLGN (coordinates
cortical (TC) plasticity terminates early in postnatal development relative to Bregma: 2 mm lateral, 2.3 mm posterior and 2.6 mm depth).
The skin was sutured, and mice were recovered on a heated pad (30°C)
(Crair and Malenka, 1995; Dudek and Friedlander, 1996; Desai
and returned to the animal colony housed with approximately 2–4
et al., 2002; Jiang et al., 2007; Barkat et al., 2011), while plasticity of same-sexed mice for about 2–4 weeks and prepared for LIFU stimula-
of other layers often persists through adulthood (Feldman et al., tion. Mice were monitored daily to ensure no postsurgery infection or
1998; Desai et al., 2002; Goel and Lee, 2007; Jiang et al., 2007). signs of distress.
These studies suggest that the loss of TC plasticity may be a pre-
cursor for the closure of the critical period. In support of this idea, Bilateral enucleation
recovery of cortical plasticity in adults is often accompanied by Mice were bilaterally enucleated at least the day before LIFU stimulation
restoration of TC plasticity (Montey and Quinlan, 2011; Yu of dLGN to prevent visually driven activity through the visual circuit.
Mice were put under isoflurane anesthesia (5% induction, 2% mainte-
et al., 2012; Petrus et al., 2014; Chung et al., 2017; Rodriguez
nance) and mounted on a stereotaxic apparatus (Kopf) without the use
et al., 2018). Such findings suggest that induction of TC plasticity of ear bars. The head was rotated sideways to facilitate easy eye access
may benefit the functional recovery of the adult sensory cortex. and the eye was popped out of the socket using curved tweezers. The
Here we investigated whether noninvasive tFUS of eye was then grabbed at the base by the optic nerve, twisted around at
dLGN would elicit TC synaptic plasticity in the adult V1. least three times to prevent bleeding, and cut off using surgical scissors.
Development of such a tool would have wide applications across Finally, eyelids were glued together using tissue adhesive (Vetbond).
different brain areas, as currently there is a paucity of methods
available for noninvasive stimulation of deep brain structures LIFU stimulation
like dLGN. We demonstrate that a pattern of low-intensity The LIFU stimulation device used was developed by the Johns Hopkins
Applied Physics Laboratory (JHU/APL) and utilized a single-element
focused ultrasound (LIFU) stimulation targeted to dLGN
focused ultrasound transducer with a center frequency of 0.5 MHz
through intact skin and skull of anesthetized adult mice produces (BII-7651H/500IMTS, Benthowave Instruments Inc.) fitted with an indi-
NMDAR-dependent long-term depression of TC synaptic trans- vidually tailored acoustic waveguide. While the design frequency for the
mission measured in V1 layer 4 (L4) neurons. This was not transducer is 0.5 MHz, acceptance testing showed that the optimal power
accompanied by changes in cFosTRAP2-mediated gene expres- efficiency occurred at 0.59 MHz. This frequency was found stable and
sion, which detects large abrupt increases in activity was then used for all procedures to minimize electrical impedance
Mesik et al. • LIFU-Induced Thalamocortical LTD in Adult V1 J. Neurosci., March 13, 2024 • 44(11):e0784232024 • 3

mismatches and limit transducer heating. While this device is capable of (212.7 mM sucrose, 10 mM dextrose, 2.6 mM KCl, 1.23 mM
high-intensity focused ultrasound (HIFU), the stimulation parameters of NaH2PO4•H2O, 26 mM NaHCO3, 3 mM MgCl2, and 1 mM CaCl2)
LIFU were limited to time averaged intensity (ISPTA) < 720 mW/cm2, and and decapitated. A block of the brain containing V1 was dissected and
pulse average intensity (ISPPA) < 190 W/cm2 or mechanical index (MI) < sliced on a vibratome (model VT1200, Leica) in ice-cold dissection
1.9. These parameters are in line with current FDA requirements of buffer saturated with 95% O2/5% CO2 to obtain 300 µm thick coronal
acoustic output (FDA, 2023); hence it is considered LIFU stimulation. slices. Slices from each hemisphere were collected and placed separately
The APL group extensively characterized and demonstrated the abil- in a holding chamber containing artificial cerebrospinal fluid (ACSF:
ity of the LIFU system to focus ultrasound stimulation deep within the 124 mM NaCl, 5 mM KCl, 1.25 mM NaH2PO4•H2O, 10 mM dextrose,
tissue below the skull (Lani et al., 2017, 2018; Hwang et al., 2018; Tyler 1.5 mM MgCl2, and 2.5 mM CaCl2) continually bubbled with 95%
et al., 2018). The ultrasonic transducer generates an acoustic focus in O2/5% CO2 and recovered for ∼1 h at room temperature.
water 22 mm below the face of the transducer with a focal diameter of Brain slices were transferred to a submersion-type recording chamber
2.2 mm and focal length of 7.6 mm at the half power points (Fig. 1A). mounted on a fixed stage of an upright microscope with oblique infrared
To retain and not distort the properties of the focal spot during coupling illumination (model E600FN, Nikon) and were continually supplied with
to the subject, a rigid conical waveguide was fabricated with Conathane ACSF bubbled with 95% O2/5% CO2 at a flow rate of ∼2 ml/min.
EN-11 and EN-4 resin to match the acoustic specific impedance of water. Whole-cell patch-clamp recordings were performed on L4 neurons in V1
The resin waveguide was form fitted to the transducer face and coupled which were postsynaptic to ChR2-expressing dLGN axon terminals. To
using ultrasound gel (Medline). Additional details on the waveguide measure the strength of ChR2-evoked synaptic transmission, extracellular
design, fabrication, and testing can be found in (Lani et al., 2018). The Ca2+ was replaced with 4 mM Sr2+, and Mg2+ concentration was raised to
transducer with a custom fabricated waveguide is expected to place the 4 mM. To ensure that LED-evoked responses were monosynaptic, record-
focal point near dLGN (Fig. 1B). The delivered acoustic intensity was ings were performed in the presence of 1 µM TTX and 100 µM 4-AP. ChR2
measured and calibrated in a water tank using a calibrated needle hydro- activation was achieved by delivering a 5 msec duration pulse of LED
phone (Precision Acoustics model NH0500) with the waveguide present (455 nm, Thor Labs) light through the objective lens. Whole-cell recording
to ensure levels were within protocols. electrodes were filled with Cs-gluconate internal solution (130 mM
On the day of the LIFU stimulation, mice were put under isoflurane Cs-gluconate, 10 mM HEPES, 8 mM KCl, 1 mM EGTA, 4 mM diso-
anesthesia (5% induction, 1–1.5% maintenance) and mounted on a dium-ATP, 10 mM disodium-phosphocreatine, 0.5 mM sodium-GTP,
stereotaxic instrument (Kopf). Hair was removed from the head using and 5 mM lidocaine N-ethyl bromide; pH 7.4, 275–285 mOsm). L4 neu-
Nair hair removal lotion. A marker was used to draw a targeting cross rons were voltage-clamped at −80 mV holding potential. Recorded cells
centered approximately over the left or right dLGN (using the midline were routinely filled with biocytin (1 mg/ml; Sigma-Aldrich, catalog
and estimated position of lambdoid suture for guidance). For a group #B4261) to confirm their location post hoc. All recordings were amplified
of mice, NMDAR antagonist D-4-[(2E)-3-phosphono-2-propenyl]- using an amplifier (model 700B, Molecular Devices), digitized at 10 kHz by
2-piperazinecarboxylic acid (D-CPP; Tocris Biosciences catalog #1265) a data acquisition board (National Instruments), and acquired using a
was injected intraperitoneally (i.p., 10 mg/kg) 10–20 min prior to the custom-made IGOR program (WaveMetrics).
LIFU stimulation to test the role of NMDARs. The ultrasonic transducer
with the waveguide was mounted on a multi-axis positioning stage and Analysis of LED-evoked Sr2+-miniature excitatory postsynaptic currents
placed over the head of the mouse for precision sonication. The rigidity (Sr2+-mEPSCs)
and small contact point of the waveguide (8 mm diameter) allowed for Cells exhibiting series resistance (Rs) ≤ 25 MΩ, input resistance (Rin) ≥
visual alignment of the transducer over the anatomical markings of the 200 MΩ, and RMS noise ≤2 were used for final analysis. The detection
head. The skin was covered by ultrasound gel (Medline) and the contact threshold for selecting mEPSCs was set at three times the RMS noise
point of the waveguide was carefully centered over the targeting cross and analyzed following previous studies (Petrus et al., 2014, 2015;
and lowered until tight contact with the skin. LIFU stimulation was Rodriguez et al., 2018; Chokshi et al., 2019). There was no statistical
started after confirming good skin contact, absence of bubbles in the difference in the recording variables across the two hemispheres
ultrasound gel, and stable level of anesthesia. (Rs: CTL = 20.6 ± 1.26 MΩ, LIFU = 20.5 ± 0.64 MΩ, two-tailed unpaired
LIFU stimulation parameters were initially designed based on a prior t test, t = 0.0976, p = 0.9229; Rin: CTL = 576 ± 71.1 MΩ, LIFU = 548 ±
paper that reported transcranial ultrasound stimulation (Tufail et al., 43.7 MΩ, two-tailed unpaired t test, t = 0.3511, p = 0.7279; RMS noise:
2010). We subsequently used the SONIC model to determine the pre- CTL = 1.76 ± 0.064, LIFU = 1.71 ± 0.034, two-tailed Mann–Whitney
dicted neural activation patterns (see Computational modeling of thala- test, p = 0.2044). In brief, a 400 msec window before the LED stimulation
mocortical neural activity upon sonication section below). To generate was used for collecting spontaneous mEPSCs from each trace. Another
the wave train of tone bursts, two function generators were used in series. 400 msec window was set at 50 msec following the LED stimulation
The first function generator (Agilent model #33510B) set the pulse rep- to collect postLED events, which includes LED-evoked Sr2+-desynchor-
etition frequency of 500 Hz and triggered the second function generator onized mEPSCs. From each cell, 50–200 events were collected from
(Tektronix model #AFG3021B) to send a tone burst of 50 pulses or a tone pre-LED and postLED analysis windows. Analysis of mEPSCs was
burst duration of 0.0847 msec. The resulting wave train was passed to a done using a custom-made MATLAB script (https://github.com/
high voltage amplifier (MiniCircuits model LZY-22+). bdgrier). Cells with a <2 Hz difference in the frequency of events before
LIFU stimulation parameters used in this study are the following: and after LED stimulation were excluded from the final analysis because
waveform center frequency = 0.59 MHz, peak intensity = 2.4 W/cm2, they reflect cells with insufficient LED-evoked desynchronized events.
time average intensity (ISPTA) = 99–109.5 mW/cm2, pulse repetition fre- Spontaneous mEPSCs (pre-LED events) were mathematically subtracted
quency (PRF) = 500 Hz, cycles per pulse = 50 (at a duty cycle of 4.24%), from the postLED events as detailed in prior studies (Petrus et al., 2014,
peak pressure = 270 kPa, MI = 0.35–0.37, and volts out = 25–30 V. Each 2015; Rodriguez et al., 2018; Chokshi et al., 2019) to obtain the average
2+
mouse was subjected to unilateral LIFU stimulation targeting left or right amplitude of LED-evoked Sr -mEPSCs using the following equation:
dLGN using these parameters for a total duration of 1 h under isoflurane
anesthesia. [(Apost · Fpost ) − (Apre · Fpre )]
,
(Fpost − Fpre )
Brain slice preparation and whole-cell recordings
After about 4 weeks following the unilateral LIFU stimulation, which is where, Apost is the average amplitude of postLED events, Fpost is the aver-
about 6–7 weeks after the AAV-dflox-ChR2-mCherry injection to allow age frequency of postLED events, Apre is the average amplitude of
sufficient expression of ChR2 at dLGN terminals in V1, mice were eutha- pre-LED events, and Fpre is the average frequency of pre-LED events.
nized for brain slice electrophysiology. Each mouse was deeply anesthe-
tized using isoflurane vapors until the absence of corneal and toe pinch cFosTRAP2 induction. For cFosTRAP2-mediated neural activity
reflexes, then transcardially perfused with ice-cold dissection buffer measurement, we used cFosTRAP2;Ai14 mice. The day before the
4 • J. Neurosci., March 13, 2024 • 44(11):e0784232024 Mesik et al. • LIFU-Induced Thalamocortical LTD in Adult V1
experiment, 4-hydroxytamoxifen (4OHT) was freshly prepared by dis- thalamocortical neuron with a Hodgkin-Huxley formalism, describing
solving 10 mg 4OHT (Sigma, catalog #H7904 or Hello Bio, catalog the evolution of the membrane charge density (Qm = Cm · Vm , where
#HB2508) in 1 ml of ethanol and then mixing the solution with corn Cm and Vm represent the membrane capacitance and potential, respec-
oil 1:1 and allowing ethanol to evaporate, resulting in 10 mg/ml 4OHT tively) under the action of a cell-type-specific set of ionic currents.
in corn oil. Specifically, this set included a sodium current (INa ), a delayed-rectifier
To validate the cFosTRAP2;Ai14 model, we examined tdTomato and leakage potassium currents (IKd and IKl , respectively), a low-
induction following monocular enucleation (ME). The enucleation threshold T-type calcium current (ICaT ), a mixed cationic current (IH )
method was the same as described above, except only performed unilat- and a nonspecific leakage current (ILeak ), as in (Destexhe et al., 1996).
erally. Mice were subsequently placed in a darkroom (dark exposure) It was complemented by a constant drive current term Idrive , representing
overnight to remove visually driven activity and brought out to light a simplified pre-synaptic input to the neuron. It results in the following
for 2 h with an injection of 4OHT (50 µg/g 4OHT, i.p.) as detailed below. membrane governing equation:
After the 2 h of light exposure, mice were returned to the darkroom to
prevent further visually driven activity through the open eye. After d(Cm · Vm )
1 week to allow for the sufficient expression of tdTomato, mice were pro- = −(INa + IKd + IKl + ICaT + IH + ILeak ) + Idrive . (1)
dt
cessed for histology as described below.
For LIFU stimulation experiments, cFosTRAP2;Ai14 mice were bilat- In this conductance-based model, passive currents are expressed as the
erally enucleated at least 1 d before the experiment and then underwent product of a constant conductance gx , and the difference between the
LIFU stimulation under isoflurane anesthesia as described above with the membrane potential and a specific reversal potential Ex :
transducer centered over left or right dLGN. Immediately following the
stimulation, the mice were weighed and injected intraperitoneally with IKl = gKl · (Vm − EKl )
5 µl/g of 10 mg/ml 4OHT (i.e., 50 µg/g 4OHT). The mice were then (2)
ILeak = gLeak · (Vm − ELeak ).
returned to their home cages for 1 week to allow sufficient expression
of tdTomato before being sacrificed for histology.
while active currents feature an additional gating term (represented by
one or multiple gating variables) to capture their voltage and time-
Immunohistochemical detection of cFosTRAP2;Ai14 and activated dependent conductance:
microglia. After 7 d following LIFU stimulation and cFosTRAP2 induc-
tion, mice were deeply anesthetized using isoflurane vapors. After the dis-
INa = gNa · m3 h · (Vm − ENa )
appearance of the corneal reflex, mice were kept under deep anesthesia via
a supply of isoflurane vapors through a nose-cone and transcardially per- IKd = gKd · n4 · (Vm − EKd )
(3)
fused with ∼5 ml of phosphate buffered saline (PBS, pH 7.4) followed by ICaT = gCaT · s2 u · (Vm − ECaT )
∼5 ml of 10% formalin solution (Sigma-Aldrich, catalog # HT5012). Mice IH = gH · (o + 2(1 − o − c)) · (Vm − EH ).
were then decapitation and the fixed brain was removed for postfixation
overnight in formalin solution at 4°C shielded from light. The postfixed Values of all conductances and reversal potentials are given in Table 1.
brain was coronally sectioned at 100 µm thickness on a vibratome The gating of sodium, delayed-rectifier potassium, and calcium
(Vibratome 1,000 plus, Ted Pella) and collected in PBS. We selected 6 sec- currents obey a simple two-state kinetic scheme, whereby gating
tions roughly spaced out (∼500 µm intervals) across the brain to cover transitions are regulated by voltage-dependent activation and inactiva-
anterior to posterior areas (Bregma coordinates: roughly between −0.5 tion rate constants (ax and bx , respectively), or by an equivalent
and −4 mm) and attempted to match the coronal sections across mice steady-state probability (x1 = ((ax )/(ax + bx ))) and time constant
to be consistent. Sections were counterstained with DAPI (1:1,000) fol- (tx = ((1)/(ax + bx ))):
lowed by two washes in PBS. Sections were then mounted on pre-cleaned
slides, coverslipped with Prolong Antifade mounting media dm
(ThermoFisher), nail polish sealed, and stored shielded away from light. = am (Vm ) · (1 − m) − bm (Vm ) · m
dt
We used IBA-1 as a marker for microglial activation. For LIFU stim-
ulated brains, we used the sections from cFosTRAP2;Ai14 mice prepared dh
= ah (Vm ) · (1 − h) − bh (Vm ) · h
as detailed above. IBA-1 staining was done on free-floating sections as dt
described previously (Hovens et al., 2014). Brain sections were pretreated dn
with 0.3% H2O2 in PBS for 20 min then washed twice in PBS (10 min = an (Vm ) · (1 − n) − bn (Vm ) · n (4)
dt
each) at room temperature. Sections were then incubated with rabbit
ds s1 (Vm ) − s
anti-IBA1 antibody (rabbit recombinant monoclonal IBA-1 antibody, =
Abcam, catalog # ab178846) diluted 1:2,500 in PBS with 2% bovine dt ts
serum albumin and 0.1% Triton X-100 for 3 d at 4°C. Sections were du u1 (Vm ) − u
= .
then washed twice in PBS (10 min each) at room temperature, then incu- dt tu
bated in Alexa Fluor 488 conjugated goat anti-rabbit IgG antibody
diluted 1:500 in PBS with 2% bovine serum albumin and 0.1% Triton
X-100 at room temperature for 1 h. Sections were then washed twice
in PBS (10 min each) at room temperature. Then counterstained with
DAPI (1:1,000 dilution) followed by washes in PBS (2× 10 min each). Table 1. Conductances and reversal potentials of the constituent ionic currents
Sections were then mounted on a pre-cleaned slide, coverslipped after of the thalamocortical neuron model
application of Prolong Antifade mounting media (ThermoFisher), nail
polish sealed, and stored shield away from light. Maximal conductance Reversal potential
Slides were imaged using a confocal microscope (LSM700, Zeiss) with (mS/cm2) (mV)
a 10× objective lens and stitched together using a tile function to acquire Current type Symbol Value Symbol Value
images of the whole coronal plane from LIFU stimulated brain sections.
Acquired images were quantified using FIJI (ImageJ) software. Cell depth Sodium (INa ) gNa 90 ENa +50
analysis was done using a custom-made MATLAB script (https://github. Delayed rectifier potassium (IKd ) gKd 10 EK −90
com/heykyounglee/Cell_Depth_Analysis). Leakage potassium (IKl ) gKl 0.0138
Low-threshold T-type calcium (ICaT ) gCaT 2 ECa +120
Mixed cationic (IH ) gH 0.0175 EH −40
Computational modeling of thalamocortical neural activity upon soni-
Nonspecific leakage (ILeak ) gLeak 0.01 ELeak −70
cation. We modeled the electrical membrane dynamics of a
Mesik et al. • LIFU-Induced Thalamocortical LTD in Adult V1 J. Neurosci., March 13, 2024 • 44(11):e0784232024 • 5

The gating of the mixed cationic current obeys a more complex two group comparisons of normally distributed data sets, t tests were
kinetic scheme, depending on both voltage and intracellular calcium used for most experiments except for the cell depth analysis, which
concentration [Cai ], as described in detail in (Destexhe et al., 1996): used repeated measures two-way ANOVA. Data sets not normally dis-
tributed (assessed using the Shapiro–Wilk test with p < 0.05) were com-
do pared using a nonparametric Mann–Whitney test. Outliers were
= ao (Vm ) · c − bo · o − k3 · o · (1 − P0 ) + k4 · (1 − o − c ) removed from the final data set using the ROUT test with Q = 1%
dt
dc cutoff (only one data set, IBA-1 cell density in the cortex, had 2 outliers
= bo (Vm ) · o − ao (Vm ) · c (5) as noted in the figure legend). Data are presented as mean ± SEM.
dt
The specific tests used are specified and detailed in the figure legends.
dP0 P < 0.05 was taken as statistically significant. Final data sets are available
= k2 · (1 − P0 ) − k1 · P0 · [Cai ]2 .
dt on the Mendeley Data (DOI: 10.17632/zk33z4vc8k.1).

Here, 1 − P0 represents the fraction of a calcium regulating factor bound Code accessibility. Custom-made analysis codes and the SONIC
by intracellular calcium, while the constants k1 , . . . k4 [defined as in simulation codes are publicly available through GitHub (www.github.
(Destexhe et al., 1996)] govern transitions between the different gating com) with the links provided above.
states. The intracellular calcium concentration is regulated by the com-
bination of a first order decay and an influx due to the inward calcium
current ICaT : Results
LIFU stimulation of dLGN results in NMDAR-dependent
d[Cai ] [Cai ]0 − [Cai ] k long-term depression of dLGN synapses in V1 L4
= − · ICaT , (6)
dt tCa 2Fd To investigate whether tFUS can produce long-term synaptic
plasticity of TC inputs from dLGN to V1 L4, AAV carrying a
where the equilibrium intracellular calcium concentration [Cai ]0 is set to Cre-dependent ChR2 was injected bilaterally in dLGN of
50 nM, the intracellular calcium decay time constant tCa to 5 ms [as in VGluT2-Cre mice. VGluT2 is highly expressed in the thalamic
(Destexhe et al., 1996)], and the effective depth of the calcium influx shell
nuclei, including dLGN (Coleman et al., 2010). Hence the use
d to 100 nm [as in (Plaksin et al., 2016)], while F represents the Faraday
constant and k the conversion constant from molar units to current den-
of VGluT2-Cre allows the expression of ChR2 in dLGN and their
sity units. axon terminals in V1. After about 2–4 weeks, mice were prepared
The gating transition functions of sodium and potassium currents for LIFU stimulation using an ultrasound transducer fitted
(am , bm , ah , bh , an , and bn ) were taken from (Pospischil et al., 2008) with a waveguide to place the focal spot near dLGN (Fig. 1).
and those of the calcium current (s1 , ts , u1 and tu ) from (Plaksin Mice were bilaterally enucleated at least the day before to prevent
et al., 2016). The transition rate functions of the mixed cationic current visually driven activity in the dLGN to V1 circuit. The parameters
(ao and bo ) were derived from equivalent activation steady-state and used for the LIFU stimulation were modified from a prior study,
time constant functions found in (Huguenard and McCormick, 1992). which reported cFos and brain-derived neurotrophic factor
To simulate the impact of LIFU stimulation on thalamocortical activ- (BDNF) induction using a different ultrasound transducer
ity, we incorporated this point-neuron model into the SONIC paradigm
(Tufail et al., 2010). While the published study did not find
(Lemaire et al., 2019), a validated, open-source, and computationally
efficient implementation of the Neuronal Intramembrane Cavitation
sufficient penetration of ultrasound waves across the white mat-
Excitation (NICE) model (Plaksin et al., 2014). Under this paradigm, ter (Tufail et al., 2010), our transducer produces a focused beam
transmembrane ionic currents are re-expressed as a function of an effec- of ultrasound (Fig. 1A), which is predicted to penetrate deeper
tive membrane potential Vm∗ , which captures the cycle-averaged impact into the brain tissue (Fig. 1B). Mice were anesthetized and
of oscillatory membrane perturbations by the acoustic pressure: head-fixed, and the LIFU transducer was positioned above the
coordinates for dLGN using a multi-axis positioning stage. The
Ij = gx · (Vm∗ −Ex ). (7) fur on the head was removed and the transducer with the
attached waveguide was placed firmly on the exposed skin with
Analogously, gating transitions are re-expressed as a function of effective ultrasound gel (Fig. 2A). The skin of the mouse is thin, and we
rate constants a∗x and b∗x : were able to use the visible sagittal and lambdoid sutures on
the skull below as anatomical landmarks to position the tip of
dx
= a∗x · (1 − x) − b∗x · x. (8) the waveguide. LIFU stimulation (see Methods for details) was
dt
delivered to one hemisphere for 1 h. The mouse was returned
These effective variables were pre-tabulated using default geometrical to the home cage after recovery from anesthesia and kept for
and biomechanical parameters for the underlying “bilayer sonophore an additional 4 weeks before being used for ex vivo brain slice
model,” and assuming a sonophore coverage fraction set to 50%. electrophysiology (Fig. 2B). L4 principal neurons in V1 of both
Finally, to enable the simulation of neural dynamics upon sustained hemispheres (LIFU stimulated and nonstimulated) were
(i.e., neuroplasticity-inducing) LIFU exposure, we augmented the recorded under whole-cell voltage-clamp configuration. To com-
dimensionality of SONIC simulation protocols with an additional layer pare the strength of dLGN synapses on L4 neurons without the
of temporal granularity enabling the repetition of LIFU pulse trains at confounds of potential differences in the density of ChR2
a low (typically sub-Hz) frequency. The conductances and reversal
expressing dLGN terminals, the expression level of ChR2, or
potentials of the constituent ionic currents used for simulating thalamo-
cortical dLGN neuron is detailed in Table 1. Python code for the simula-
the intensity of light used for their activation, we measured
tion is publicly available at: https://github.com/tjjlemaire/PySONIC LED-evoked Sr2+-mEPSCs as in our prior studies (Petrus et al.,
2014, 2015; Rodriguez et al., 2018; Chokshi et al., 2019). We ver-
ified the expression of ChR2 by imaging the mCherry tag and
Experimental design and statistical analysis of data. The experimen-
tal design was done to allow within-subject comparisons (the unstimu- confirmed the location of the recorded neurons by processing
lated hemisphere served as a control for the LIFU stimulated for biocytin, which was added to the internal solution
hemisphere). Sample sizes were determined based on prior (Petrus (Fig. 2C). We found that the strength of dLGN inputs to V1 L4
et al., 2014, 2015; Chokshi et al., 2019) and preliminary studies. in the LIFU stimulated hemisphere, quantified as the average
Statistical analyses were done using Prism 9 (Graph Pad) software. For amplitude of LED-evoked Sr2+-mEPSCs, was significantly
6 • J. Neurosci., March 13, 2024 • 44(11):e0784232024 Mesik et al. • LIFU-Induced Thalamocortical LTD in Adult V1

Figure 1. LIFU transducer acoustic intensity profile. A, Normalized acoustic intensity profile (normalized to the peak intensity) of the LIFU transducer measured in a water tank using a
calibrated needle hydrophone. The tip of the transducer and the tip of the custom-fabricated waveguide (23 mm length) are shown. The intensity profile was measured without the waveguide
present; however, the focal pattern remains the same with part of the focal pattern occurring within the waveguide as depicted in the figure because the waveguide matches the acoustic specific
impedance of water. Long dashed gray line outlines −3 dB of maximum acoustic intensity. Short dashed gray line outlines 90% of maximum acoustic intensity. B, The tip of the waveguide is
placed flat on the head of a mouse after removing the fur to expose the skin. The acoustic intensity profile in (A) is overlaid with a traced outline of a mouse brain taken from a mouse brain atlas
[Plate 50, Bregma −2.3 mm in (Paxinos and Franklin, 2001)] to show the expected location of the peak acoustic energy inside the skull. The boundaries of dLGN are outlined in pink. The center of
the LIFU transducer was placed approximately ∼2 mm lateral to the midline and the lambdoid suture, which is visible through the exposed skin, was used as a guide for estimating the
anterior-posterior target location.

depressed compared to that recorded in the control hemisphere under the activity-dependent cFos promoter; hence, it allows
(CTL: 12.7 ± 0.65 pA, n = 11 cells; LIFU: 10.7 ± 0.51 pA, n = 21 temporal control of Cre-dependent recombination by the timing
cells; 4 mice; two-tailed unpaired t test with Welch’s correction: of intraperitoneal (i. p.) injection of 4OHT, a tamoxifen analog.
t = 2.372, p = 0.0268; Fig. 2D). This suggests that LIFU stimula- To visualize the activated neurons, we crossed the cFosTRAP2
tion produces long-term synaptic depression, which can be mea- with a tdTomato reporter line (Ai14). We first verified the
sured at least 4 weeks after induction. The observed synaptic cFosTRAP2;Ai14 mouse line by examining tdTomato induction
depression was not due to a global depression of synaptic trans- in the visual circuit following monocular enucleation (ME). ME
mission on V1 L4 neurons because we did not observe statisti- mice were dark exposed overnight to remove visually driven
cally significant difference in the average amplitude of activity and brought out to light for 2 h with 4OHT injection
spontaneous mEPSCs, which were acquired during a time win- to induce cFosTRAP2-mediated tdTomato expression. The
dow prior to the LED stimulation (CTL: 12.0 ± 0.63 pA, n = 11 mice were then subsequently placed back in the dark room for
cells; LIFU: 11.6 ± 0.41 pA, n = 21 cells; 4 mice; two-tailed 7 d to prevent further visually driven activity and allow for
unpaired t test with Welch’s correction: t = 0.5585, p = 0.5833). sufficient expression of tdTomato (Guenthner et al., 2013).
Next, we tested whether the observed long-term synaptic Fixed brain sections were counterstained with DAPI, which
depression was dependent on NMDARs. To do this, we injected was used to outline different brain areas based on the anatomical
the NMDAR antagonist CPP (10 mg/kg, i.p.) 10 to 20 min prior landmarks that match those seen in the mouse brain atlas
to the 1 h LIFU stimulation in one hemisphere. V1 slices were (Paxinos and Franklin, 2001) (Fig. 3A). We found significant
obtained approximately 4 weeks after the LIFU stimulation increases in tdTomato-labeled cells in visual cortices, both V1
with CPP. We found no statistically significant difference in and V2, ipsilateral to the enucleated eye, which predominantly
the strength of dLGN inputs to V1 L4, as quantified by the aver- receive the open eye input (Fig. 3A–C). This result suggests
age amplitude of LED-evoked Sr2+-mEPSCs (CTL + CPP: 13.0 ± that cFosTRAP2;Ai14 can indeed report the 2 h of visually driven
0.56 pA, n = 20 cells; LIFU + CPP: 13.1 ± 0.44 pA, n = 21 cells; 4 activity. There was no significant difference in tdTomato-labeled
mice; two-tailed Mann–Whitney test: p = 0.6891; Fig. 2E). This cell density across the two hemispheres in other cortical areas
suggests that LIFU stimulation-induced long-term synaptic examined suggesting that the cFosTRAP2;Ai14 is a good model
depression is dependent on activation of NMDARs. to detect activated cells. Unfortunately, we found that the
cFosTRAP2 did not drive tdTomato expression much in the
LIFU stimulation does not induce neuronal activity needed to dLGN. Thus, the density of labeled cells in the ipsilateral
activate cFosTRAP2-mediated gene expression dLGN, which should have received visual activity from the
Next, we attempted to measure neural activation resulting from open eye, was much lower than that seen in the visual cortices
LIFU stimulation. Because of the large size of the transducer, such that we could not detect a significant difference from that
which occupies the space above the head of the mouse seen in the deprived contralateral dLGN. We further verified
(Fig. 2A), we were unable to find a way to directly measure neural that the cFosTRAP2;Ai14 does not have baseline leakage of the
responses using in vivo recordings. Thus, we turned to an immu- cFos promoter in the absence of 4OHT. To do this we compared
nohistological method by utilizing the cFosTRAP2 mouse line the tdTomato signal in normal sighted mice with and without
(Guenthner et al., 2013). This mouse line expresses Cre-ERT2 4OHT injection (Fig. 3D). We found a near absence of
Mesik et al. • LIFU-Induced Thalamocortical LTD in Adult V1 J. Neurosci., March 13, 2024 • 44(11):e0784232024 • 7

tdTomato-positive cells in normal sighted controls without

4OHT injection when compared to those injected with 4OHT
suggesting minimal baseline leakage of Cre activity in the absence
of 4OHT.
We proceeded to use this mouse line for LIFU stimulation,
despite very weak induction in the dLGN, based on the fact
that the estimated activation spot (∼2 mm in diameter and
∼5 mm in length) includes the overlaying cortex and hippocam-
pus (Fig. 1B), both of which show good cFosTRAP2 induction
(Fig. 3) (Guenthner et al., 2013). cFosTRAP2;Ai14 mice were
bilaterally enucleated the day before the LIFU stimulation to
remove the visually driven activity. LIFU stimulation was done
unilaterally to one hemisphere targeting the dLGN using the
same parameters as used for ex vivo experiments. After 1 h of
LIFU stimulation, mice received an injection of 4OHT and
were kept under anesthesia for an additional 1 h to suppress neu-
ronal activity during the time window in which cFosTRAP2
could be induced. After that, the mice recovered from anesthesia
and returned to their home cage for 1 week to allow for sufficient
expression of tdTomato. We analyzed the confocal images of the
brain sections counterstained for DAPI, which were used to out-
line the anatomical boundaries (Fig. 4A). We first analyzed brain
sections containing dLGN and outlined a 1-mm-wide rectangle
(the estimated LIFU acoustic activation path) in both hemi-
spheres, which was centered ∼2 mm lateral to the midline to
pass through the dLGN. We also outlined the cortex, hippocam-
pus, and dLGN within this rectangle as additional regions of
interest (ROIs) for analysis, as well as the areas corresponding
to V1 in more posterior sections. Images were thresholded in
the tdTomato channel to select positively labeled cells. We first
plotted the cells within the rectangle ROI and quantified their
depth relative to pia (Fig. 4B). We did not see any difference in
the distribution of tdTomato-positive cells in the unstimulated
(CTL) and LIFU stimulated hemispheres (Fig. 4C). This suggests
Figure 2. LIFU stimulation of dLGN results in NMDAR-dependent long-term depression of
that LIFU stimulation does not produce sufficient activity to
dLGN synaptic strength measured in V1 L4. A, Illustration of LIFU stimulation of a mouse. The
fur on the top of the head was removed to expose the skin of an anesthetized mouse, which induce cFosTRAP2 which could localize the activation spot.
was head-fixed on a stereotaxic frame, and the LIFU waveguide tip was firmly coupled using We also did not find any significant difference in the density of
ultrasound gel. Mouse was enucleated at least the day before to remove any potential visually tdTomato-positive cells in the dLGN or the overlaying cortex
evoked responses. LIFU stimulation was delivered under isoflurane anesthesia. B, Schematics and hippocampus in the two hemispheres (Fig. 4D). Neither
of the experiment. ChR2-mCherry was expressed in dLGN of VGluT2-Cre mice at least 2 weeks was there a change in the tdTomato positive cell density in V1
before the LIFU stimulation and 6 weeks before the ex vivo brain slice electrophysiology. across the two hemispheres (Fig. 4D).
Whole-cell voltage-clamp recordings were done in V1 L4 principal neurons and dLGN axon
terminals expressing ChR2-mCherry were activated by LED (455 nm, 5 ms pulse width) deliv-
ered through the objective lens. V1 of both hemispheres (LIFU stimulated and unstimulated LIFU stimulation does not produce activation of microglia
CTL) were used for recording LED-evoked Sr2+-mEPSCs. C, An example of recorded V1 L4 neu- While we did not observe tissue damage due to LIFU stimulation
rons visualized by processing for biocytin, which was present in the internal solution. After the from visual inspection of the tissue integrity and DAPI-stained
recording, the slice was fixed and counterstained with DAPI (blue). Three L4 neurons were images, we proceeded to examine this further by staining for
recorded from this particular slice. Left, A tiled image using a 10× objective lens showing IBA-1 (Fig. 5A), which is a marker for activated microglia asso-
the location of the recorded cells (green). Right, A higher magnification (63×) confocal image ciated with neuroinflammation (Hovens et al., 2014; Muzio et al.,
showing the same recorded cells (green) counterstained with DAPI (blue) and overlaid with
2021). Previous studies have shown changes in the density of
mCherry (magenta) signals showing dLGN axons expressing ChR2-mCherry. D, Left,
Comparison of the average amplitude of LED-evoked Sr2+-mEPSCs recorded from CTL and IBA-1 positive microglia with sensory deprivation and denerva-
LIFU stimulated hemispheres. The average amplitude from each V1 L4 neuron is shown as tion paradigms (Fuentes-Santamaria et al., 2012; Grier et al.,
open circles and overlaid on the bars showing the group averages (mean ± SEM).
*p < 0.05 (two-tailed unpaired t test with Welch’s correction, t = 2.372, p = 0.0268).

Middle, Average traces of calculated LED-evoked Sr2+-mEPSCs (see Methods for details).
Right, Example recording traces (top, from a neuron in CTL hemisphere; bottom, from a neu- depression of LED-evoked Sr2+-mEPSCs. Left, Comparison of the average amplitude of
ron in LIFU stimulated hemisphere). The Gray dotted line represents the time window to mea- LED-evoked Sr2+-mEPSCs recorded from CTL + CPP and LIFU + CPP hemispheres. The average
sure spontaneous mEPSCs. The blue arrow denotes the time of LED stimulation (5 ms pulse amplitude from each V1 L4 neuron is shown as open circles and overlaid on the bars showing
duration), which is followed by an initial synchronized release mediated synaptic responses. the group averages (mean ± SEM). CTL + CPP data set did not pass the normality test
The blue line represents the time window used to measure LED post events (started 50 ms (Shapiro–Wilk test, p = 0.0110); hence, statistical comparison was done using a nonparamet-
after the LED to remove the initial synchronized release event), which include LED-evoked ric test (two-tailed Mann–Whitney test, p = 0.6891). n.s., not statistically significant. Middle,
Sr2+-mEPSCs in the background of spontaneous mEPSCs (see Methods for details on math- Average traces of calculated LED-evoked Sr2+-mEPSCs (see Methods for details). Right,
ematical subtraction of spontaneous mEPSCs to quantify LED-evoked Sr2+-mEPSCs). E, Example recording traces (top, from a neuron in CTL + CPP hemisphere; bottom, from a neu-
Systemic application of an NMDAR antagonist (CPP, 10 mg/kg, i.p.) prevents LIFU-induced ron in LIFU + CPP hemisphere). Annotations are the same as in panel D.
8 • J. Neurosci., March 13, 2024 • 44(11):e0784232024 Mesik et al. • LIFU-Induced Thalamocortical LTD in Adult V1

Figure 4. LIFU stimulation does not induce cFosTRAP2-driven gene expression. A, Left,
Unilateral LIFU stimulated cFosTRAP2;Ai14 mouse brain sections were counterstained with
DAPI to identify landmarks for outlining the ROIs in the tile scanned confocal image. In sec-
tions corresponding to the LIFU target region, we outlined a 1-mm-wide rectangle that is
Figure 3. Validation of cFosTRAP2;Ai14 mouse. A, Coronal sections of monocularly enucle- within the predicted LIFU acoustic beam path (∼2-mm diameter based on acoustic intensity
ated cFosTRAP2;Ai14 mice were counterstained with DAPI and confocal tiled images were measurements, see Figure 1). Within this rectangle, the overlaying cortex (Ctx, labeled 1),
taken. Using the DAPI channel, brain areas of interest were outlined by comparing the land- hippocampus (HC, labeled 2), and dLGN (labeled 3) were outlined. Right, Using the outlined
marks with corresponding plates in a mouse brain atlas (Paxinos and Franklin, 2001). Left, ROIs from the DAPI channel, tdTomato positive cells were quantified in the tdTomato (tdT)
DAPI imaging with ROIs outlined. 1, retrospleneal cortex (RSP); 2, V2; 3, V1; 4, somatosensory channel. Hemispheres were identified by a nick to the bottom of one hemisphere as shown in
cortex (S1); 5, auditory cortex (A1); 6, dLGN. Right, ROIs selected in the DAPI channel were these images. B, Left, A plot showing the depth profile of identified tdT positive cells (orange
used to quantify tdTomato (tdT) positive cells in each brain area. Contra, contralateral hemi- circles) within the outlined rectangles in panel (A) for CTL and LIFU stimulated hemispheres.
sphere from the enucleated eye (i.e., visually deprived hemisphere). Ipsi, ipsilateral hemi- Blue line, pia. Right. A graph showing quantification of the depth of each tdT positive cell in
sphere from the enucleated eye (i.e., dominantly driven by the open eye). Hemispheres relation to the outlined pia from the plot shown in the left panel. C, Quantification of the
were identified by a nick made to the bottom of one hemisphere as is visible in this image. depth profile of tdT positive cells across five mice with unilateral LIFU stimulation. Only
B, Zoomed-in images of each brain area from the example brain shown in (A). Left panels, the brain sections corresponding to the similar coronal plane as shown in panel (A) were
DAPI. Right panels, tdTomato (cFosTRAP2;Ai14). Images from contra- and ipsilateral hemi- analyzed. There was no statistical significance in the fraction of tdT positive cells across
spheres are shown for V1, V2, RSP, and S1. C, Comparison of quantified density of the depth between CTL and LIFU hemispheres, while there is a significant effect on the depth
tdTomato positive cells in contra (C) and ipsi (I) hemispheres for V1, V2, dLGN, RSP, S1, [two-way repeated measures ANOVA, depth × group (CTL/LIFU), F (35, 70) = 4.159, p =
and A1. Note a significant increase in tdTomatoe labeling of active cells in V1 and V2 of ipsi- 0.7851; depth, F (35, 70) = 4.159, p < 0.0001]. D, Quantification of the density of
lateral hemispheres to the enucleated eye. dLGN only showed minimal labeling in both hemi- tdT-positive cells in dLGN, Ctx, HC, and V1. Open circles, Data from each brain section con-
spheres. cFosTRAP2-induced tdTomato cell density (mean ± SEM), Contra V1 = 8.4 ± taining the specific ROI. Bars, Average tdT positive cell density (mean ± SEM, dLGN, CTL =
3.21 mm−2, Ipsi V1 = 71.8 ± 13.06 mm−2, n = 4 sections; Contra V2 = 15.5 ± 3.08 mm−2, 18.2 ± 7.83 mm−2, LIFU = 14.8 ± 4.47 mm−2, n = 8 sections; Ctx, CTL = 34.7 ± 9.46 mm−2,
Ipsi V2 = 32.4 ± 4.90 mm−2, n = 10 sections; Contra dLGN = 4.4 ± 1.59 mm−2, Ipsi dLGN LIFU = 34.5 ± 9.54 mm−2, n = 8 sections; HC, CTL = 8.7 ± 1.96 mm−2, LIFU = 11.5 ±
= 8.7 ± 2.32 mm−2, n = 9 sections; Contra RSP = 31.4 ± 6.54 mm−2, Ipsi RSP = 35.7 ± 3.20 mm−2, n = 8 sections; V1, CTL = 19.1 ± 5.10 mm−2, LIFU = 18.8 ± 5.21 mm−2,
7.56 mm−2, n = 10 sections; Contra S1 = 9.4 ± 2.30 mm−2, Ipsi S1 = 8.5 ± 1.68 mm−2, n = 11 sections) quantified from five mice. n.s., not significant (two-tailed paired t tests,
n = 11 sections; Contra A1 = 13.1 ± 3.34 mm−2, Ipsi A1 = 26.3 ± 1.00 mm−2, n = 3 sec- Ctx, t = 0.2227, p = 0.8347; HC, t = 1.131, p = 0.3213; V1, t = 0.243, p = 0.8200). dLGN
tions; 2 mice. *p < 0.05, **p < 0.01, n.s., not significant (two-tailed paired t tests, dLGN, data sets did not pass the normality test (Shapiro–Wilk test, CTL, p = 0.0131; LIFU,
t = 1.747, p = 0.1187; RSP, t = 1.937, p = 0.0848; A1, t = 3.058, p = 0.0924). Ipsi V1, Ipsi p = 0.0123); hence, the statistical comparison of dLGN cell density was done using a nonpara-
V2, and both S1 data sets did not pass the normality test (Shapiro–Wilk test, Ipsi V1 metric test (two-tailed Mann–Whitney test, p = 0.7024).
p = 0.0108; Ipsi V2 p = 0.0455; Contra S1 p = 0.246; Ipsi S1 p = 0.0080); hence, the statistical
comparisons of V1, V2, and S1 tdT-positive cell densities were done using a nonparametric
test (two-tailed Mann–Whitney test; V1, p = 0.0286; V2, p = 0.0232; S1, p = 0.7969). D, Left, 2016). We performed an analysis similar to that used for quanti-
Image of a negative control brain section of cFosTRAP2;Ai14, which did not receive an injec- fying cFosTRAP2;Ai14 signals by outlining a rectangular ROI of
tion of 4-OHT. There is very little background cFosTRAP2-driven tdTomato (tdT) expression. 1 mm width that spans the dorsoventral thickness of the brain
Right, Image of a control brain of a normal-sighted cFosTRAP2;Ai14, which received an injec- and is centered around 2 mm lateral to the midline to pass
tion of 4-OHT. Note induction of tdTomato across both hemispheres. through the dLGN. Only brain sections that contained dLGN
Mesik et al. • LIFU-Induced Thalamocortical LTD in Adult V1 J. Neurosci., March 13, 2024 • 44(11):e0784232024 • 9

were analyzed for comparison of the distribution of IBA-1 posi-

tive microglia across the depth from pia (Fig. 5B,C). We did not
observe a significant difference in their distribution profile across
the depth between the unstimulated and LIFU stimulated hemi-
spheres (Fig. 5C). We further analyzed the density of IBA-1 pos-
itive microglia in each brain region of interest, as well as the
IBA-1 intensity, and did not find any significant difference
between the two hemispheres (Fig. 5D,E). We did notice a ten-
dency of an increase in IBA-1 positive cell density in dLGN of
LIFU stimulated hemisphere, which did not reach statistical
significance.

Simulation of predicted neuronal activity in dLGN with LIFU

stimulation
Since we were unable to detect neuronal activation with LIFU
stimulation using the cFosTRAP2-mediated gene expression sys-
tem, we turned to simulations to see what type of neuronal activ-
ity is predicted from our stimulation parameters. There are many
models and hypotheses on how ultrasound could activate neu-
rons (Dell’Italia et al., 2022), but we decided to utilize a
biophysics-based model that can produce clear predictions on
physiological neuronal output. One such model is multi-scale
optimized neuronal intramembrane cavitation (SONIC)
(Lemaire et al., 2019), which is based on the neuronal intramem-
brane cavitation excitation (NICE) model of ultrasound activa-
tion (Plaksin et al., 2014, 2016). According to the NICE model,
ultrasound-induced neuronal activation is due to oscillations of
small areas (submicron-sized) of membranes, which are called
bilayer sonophores (Krasovitski et al., 2011). It is hypothesized
that these sonophores provide mechanoelectrical coupling that
induces membrane currents that can lead to the depolarization
of neurons to generate action potentials (Plaksin et al., 2014).
Figure 5. LIFU stimulation does not produce changes in activated microglia. A, Brain The predictions of the NICE model are consistent with the basic
sections from unilateral LIFU stimulated cFosTRAP2;Ai14 mice were stained for activated observations of ultrasound stimulation studies showing that
microglial marker IBA-1 and counterstained with DAPI. Left, DAPI images (10× objective effective stimulation of cortical neurons requires long durations
lens, tiled confocal image) were used to outline the ROIs as described above. Right, The of continuous wave stimulation over pulse stimulation (Tufail
ROIs were then used for quantifying the IBA-1 images. In this particular example, images
et al., 2010, 2011; King et al., 2013). The SONIC model is a
from each hemisphere were saved as different image files, hence displayed as separate pan-
els. Hemispheres were identified by a nick to the bottom of one side as shown. B, Left, A plot
revised version of the NICE model that is optimized to be com-
showing the depth profile of identified IBA-1 positive cells (orange circles) within the outlined putationally more efficient (Lemaire et al., 2019).
rectangles in panel (A) for CTL and LIFU stimulated hemispheres. Blue line, pia. Right, A graph We used the SONIC model to simulate the activity of dLGN
showing quantification of the depth of each IBA-1 positive cell in relation to the outlined pia neurons under the LIFU stimulation parameters utilized for our
from the plot shown in the left panel. C, Quantification of the depth profile of IBA-1 positive study. Importantly, the SONIC model contains built-in parame-
cells across 5 mice with unilateral LIFU stimulation. Only the brain sections corresponding to ters for simulating thalamocortical neurons, which include
the similar coronal plane as shown in panel (A) were analyzed. There was no statistical sign- T-type Ca2+ channels (see Methods for details of the model).
ificance in the fraction of IBA-1 positive cells across the depth between CTL and LIFU hemi- Because thalamocortical neurons exhibit different firing states
spheres, while there was a significant effect of the depth [two-way repeated measures (tonic and burst modes) due to their T-type Ca2+ channels, we
ANOVA, depth × group (CTL/LIFU), F (35, 560) = 0.5992, p = 0.9682; depth, F (5.277,
ran our simulations using two distinct values of resting mem-
84.43) = 5.027, p = 0.0003]. D, Quantification of the density of IBA-1 positive cells in
dLGN, Ctx, HC, and V1. Open circles, Data from each brain section containing the specific
brane potential (each artificially constrained by a specific driving
ROI. Bars, Average IBA-1 positive cell density (mean ± SEM, dLGN, CTL = 8.8 ± current), −55 mV for tonic mode and −70 mV for burst mode, to
18.19 mm−2, LIFU = 18.2 ± 6.48 mm−2, n = 8 sections; Ctx, CTL = 7.2 ± 2.48 mm−2, LIFU allow predictions of neuronal activity under the two different
= 8.6 ± 2.63 mm−2, n = 6 sections; HC, CTL = 7.7 ± 1.92 mm−2, LIFU = 6.2 ± 1.93 mm−2, states. We found that in the different modes, the same LIFU stim-
n = 6 sections; V1, CTL = 18.1 ± 5.74 mm−2, LIFU = 14.8 ± 3.64 mm−2, n = 7 sections) ulation parameters result in different predicted firing patterns
quantified from five mice. n.s., not significant (two-tailed paired t tests, Ctx, t = 1.161, and intracellular Ca2+ transients (Fig. 6). Under the tonic
p = 0.2980; HC, t = 0.9803, p = 0.3824; V1, t = 0.5349, p = 0.6298). dLGN LIFU data set
did not pass the normality test (Shapiro–Wilk test, p = 0.0121); hence, the statistical com-
parison of dLGN cell density was done using a nonparametric test (two-tailed Mann–Whitney
test, p = 0.1605). Two data points for Ctx were removed based on an outlier test (ROUT, Q = 
1%), but the inclusion of these data points does not alter the conclusion (two-tailed paired CTL = 165 ± 7.0 a.u., LIFU = 164 ± 6.6 a.u., n = 6 sections; HC, CTL = 164 ± 6.4 a.u., LIFU =
t test, t = 0.7689, p = 0.4671). E, Quantification of the intensity (a.u., arbitrary unit of fluor- 164 ± 5.8 a.u., n = 6 sections; V1, CTL = 165 ± 4.1 a.u., LIFU = 167 ± 3.8 a.u., n = 7 sections)
escence intensity) of IBA-1 positive cells in dLGN, Ctx, HC, and V1. Open circles, Data from quantified from five mice. n.s., not significant (two-tailed paired t tests, dLGN, t = 0.3307, p
each brain section containing the specific ROI. Bars, Average IBA-1 positive cell intensity = 0.7574; Ctx, t = 0.6487, p = 0.5519; HC, t = 0.2322, p = 0.8278; V1, t = 0.3281,
(mean ± SEM, dLGN, CTL = 169 ± 6.5 a.u., LIFU = 164 ± 6.6 a.u., n = 8 sections; Ctx, p = 0.7644).
10 • J. Neurosci., March 13, 2024 • 44(11):e0784232024 Mesik et al. • LIFU-Induced Thalamocortical LTD in Adult V1
around 25 Hz under tonic firing mode, while a series of short
bursts repeated around 4 Hz under burst firing mode (Fig. 6).
Observation of synaptic plasticity after tFUS is not unique to
our system. A recent study demonstrated that tFUS produces
synaptic depression in the hippocampal CA1 of adult rats, which
was rather transient and lasted about 20–60 min (Niu et al.,
2022). There is also a report where tFUS of the motor cortex
(M1) in human subjects shows potentiation of TMS-evoked
motor evoked potentials (MEPs) for at least an hour (Zhang
et al., 2021), but whether such plasticity was due to synaptic plas-
ticity or changes in neuronal excitability was not determined. The
novelty of our study is that we observed long-lasting synaptic
depression of TC inputs to V1 in adult mice, which was measured
at least 2 weeks after the LIFU stimulation. Unlike the CA1 or
M1, which exhibits robust synaptic plasticity throughout the life-
span of rodents (Hess and Donoghue, 1996; Kumar, 2011), TC
synapses in V1 display an early critical period which closes
around the third postnatal week (Jiang et al., 2007) and is absent
in adults (Rodriguez et al., 2018). The percentage of synaptic
depression observed in our study with LIFU stimulation
(∼15%) is comparable to the magnitude of LTD observed at
TC synapses measured ex vivo in V1 L4 of young juvenile mice
(P18–23) (Jiang et al., 2007). The developmental loss of long-
term synaptic plasticity in the dLGN to V1 synapses has been
suggested to be correlated with the diminished experience-
Figure 6. Simulation of predicted neuronal activity in dLGN with LIFU stimulation param- dependent plasticity in the adult V1 (Lee and Whitt, 2015). As
eters used experimentally. A, Results from simulation in the SONIC model using the param- such, recovery of TC plasticity in the adult V1 has been consid-
eters for dLGN when in a depolarized tonic firing mode. Top, Predicted membrane potential
ered critical for recovering juvenile-like ocular dominance plas-
(Vm) changes show a train of single action potentials at ∼25 Hz. Bottom, Predicted intracel-
lular Ca2+ concentration. B, Results from simulation in the SONIC model using the parameters ticity (Montey and Quinlan, 2011) or accelerating the adult
for dLGN when in a hyperpolarized burst firing mode. Top, Predicted membrane potential form of it (Rodriguez et al., 2018) in rodents. Therefore, our
(Vm) changes show bursts of action potentials repeated at ∼250 msec inter-burst interval. observation that a noninvasive transcranial LIFU stimulation
Bottom, Predicted intracellular Ca2+ concentration. See Table 1 for the details on ionic can result in LTD of TC synapses in the adult V1 paves a way
conductances used for the simulation. for developing new noninvasive therapies that could potentially
help recover adult V1 functionality.
One of the remaining questions is how LIFU stimulation leads
to the long-term synaptic plasticity we observe here. Due to the
mode, the model predicted a train of action potentials at around size of the LIFU transducer used in this study, we were unable
25 Hz with corresponding small increases in intracellular Ca2+ to directly measure neuronal activity in vivo using conventional
transients (∼3 µM peak increases in concentration) (Fig. 6A). electrophysiological recording techniques or in vivo two-photon
With the burst mode simulation, it predicted the generation of Ca2+ imaging. Instead, we utilized an immunohistochemical
bursts repeated at roughly 250 msec inter-burst intervals with method of visualizing activated neurons using the cFosTRAP2
corresponding higher increases in intracellular Ca2+ concentra- mouse line (Guenthner et al., 2013). While we were able to visu-
tion (∼35 µM peak) (Fig. 6B). The simulation results suggest alize activated neurons in visual cortices following monocular
that the neuromodulation of dLGN activity by LIFU is state visual stimulation (Fig. 3), we were unable to see differences in
dependent but has a range of single spiking activity under tonic labeled active neurons between the LIFU stimulated and unsti-
mode that could in principle drive LTD in the postsynaptic V1 mulated control hemispheres (Fig. 4). This could suggest the fol-
neurons. lowing several possibilities: (1) our LIFU stimulation protocol
does not activate neurons; (2) it produces neuronal activity, but
it falls below the activity threshold for cFos promoter activation;
Discussion or (3) it only provides subthreshold neuromodulation. To clearly
Here, we report that transcranial LIFU stimulation of a deep distinguish between these possibilities, further studies will need
brain structure, dLGN, produces long-lasting weakening of to be done to directly measure neuronal activity ideally using
dLGN synaptic strength onto V1 L4 neurons in adult mice, which intracellular recording techniques to discern even subthreshold
was dependent on NMDAR activation (Fig. 2). LIFU stimulation or hyperpolarization events that may accompany tFUS.
did not produce neural activity levels sufficient to drive However, our observation that the LTD of dLGN inputs V1 L4
cFosTRAP2-induce gene expression (Fig. 4) or activation of is dependent on NMDAR activity (Fig. 2E) suggests that LIFU
microglia as visualized by IBA-1 staining (Fig. 5). Using the stimulation would have caused some level of neural activity
SONIC model (Lemaire et al., 2019), which is based on the neu- that can activate NMDARs in the range for LTD induction.
ronal intramembrane cavitation theory of ultrasound neuronal While the parameter space for inducing LTD of TC synapses
activation (Plaksin et al., 2014, 2016), we found that the particu- even in juvenile V1 has not been fully characterized, it is expected
lar LIFU stimulation parameters used in our study are predicted to be similar to the range (1–10 Hz) seen in L4 to L2/3 synapses
to generate different patterns of activity dependent on the state of ex vivo (Kirkwood et al., 1996). It is known that cFos induction
the dLGN neuron. The model predicts a train of action potentials requires a threshold of higher activity, in particular, repeated
Mesik et al. • LIFU-Induced Thalamocortical LTD in Adult V1 J. Neurosci., March 13, 2024 • 44(11):e0784232024 • 11

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Disclaimer Huguenard JR, McCormick DA (1992) Simulation of the currents involved in
This material is based on work while one of the authors (G.M.H.) rhythmic oscillations in thalamic relay neurons. J Neurophysiol 68:1373–
was supported by (while serving at) the National Science 1383.
Foundation. Any opinions, findings, conclusions, or recommen- Hwang GM, Lani SW, Rosenberg AP, Congedo MB, Tyler WJ (2018)
dations expressed in this material are those of the authors and do Forward-looking engineering concepts for ultrasonic modulation of neu-
not necessarily reflect the views of the National Science ral circuit activity in humans. Proc. SPIE, 10639:106391J.
Jiang B, Trevino M, Kirkwood A (2007) Sequential development of long-term
Foundation. This article was prepared while Dr. Grace Hwang potentiation and depression in different layers of the mouse visual cortex.
was employed at Johns Hopkins University. The opinions J Neurosci 27:9648–9652.
expressed in this article are the author's own and do not reflect Jo Y, et al. (2022) General-purpose ultrasound neuromodulation system for
the view of the National Institutes of Health, the Department chronic. Closed-loop preclinical studies in freely behaving rodents. Adv
of Health and Human Services, or the United States Government. Sci (Weinh) 9:e2202345.
Kaczmarek L (1992) Expression of c-fos and other genes encoding tran-
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