Name: simultaneous focused ultrasound neuromodulation and fiber photometry recording in free-moving mouse
Uploaded: 2024-09-06T14:00:00.000+00:00
Duration: 8 min 38 s
Description: Focused ultrasound neuromodulation (FUN) represents a promising approach for non-invasive perturbation of neuronal circuits at deep brain regions. It is ...

This work is supported in part by the National Natural Science Foundation of China (32371151), Guangdong High Level Innovation Research Institute(2021B0909050004), the Hong Kong Research Grants Council Collaborative Research Fund (C5053-22GF), General Research Fund (15224323 and 15104520), Hong Kong Innovation Technology Fund (MHP/014/19), internal funding from the Hong Kong Polytechnic University (G-SACD and 1-CDJM), and the Natural Science Foundation of Liaoning Province- Joint Open Fund of State Key Laboratory of Robotics (2022-KF-22-03). The authors would like to thank the facility and technical support from the University Research Facility in Life Sciences (ULS) and University Research Facility in Behavioral and Systems Neuroscience (UBSN) of The Hong Kong Polytechnic University.

All procedures and animal handling complied with NSFC ethics guidelines and approved protocol requirements of the Institutional Animal Care and Use Committee of the Guangdong Institute of Intelligence Science and Technology.
1. Transducer preparation
<ol>
	<li>Prepare a piezoelectric plate with an inner diameter of 3 mm, an outer diameter of 7 mm, and a center frequency of 500 kHz. 
	NOTE: The outer diameter can be adjusted based on the specific brain region being targeted and should be maximized while maintaining stimulation accuracy and without exceeding the boundary of the mouse skull.</li>
	<li>Attach the wire to the two sides of the piezoelectric plate using epoxy silver paste (Figure 1). After the epoxy silver paste has solidified, use a multimeter to measure the resistance at both ends of the wire to ensure that it is approximately 0.</li>
	<li>Apply a layer of double-sided tape onto a clean glass sheet surface. Adhere to the piezoelectric plate and copper ring, with a height of 8 mm, an outer diameter of 8 mm, and an inner diameter of 7.6 mm, tightly to the glass sheet. 
	NOTE: The inner diameter of the copper ring is determined by the size of the piezoelectric plate to ensure the plate is covered by the copper ring.</li>
	<li>Securely insert the polypropylene pipe with an outer diameter of 3 mm into the center of the piezoelectric plate and firmly adhere it to the glass sheet (Figure 1).</li>
	<li>Prepare an appropriate amount of epoxy resin glue and vacuum it. Extract the epoxy using a disposable syringe and slowly inject it into the copper ring. Wait about 10 h until the epoxy has solidified (Figure 1).</li>
	<li>Solder the loose ends of two wires onto the bayonet nut connector using an electronic soldering iron. Remove the glass sheet. Clean the surface of the transducer with alcohol (Figure 1).</li>
</ol>
2. Reporting parameters for FUN
<ol>
	<li>Place the hydrophone and transducer in a water tank filled with deionized water (Figure 2A). Ensure that the central beam (Z-axis) of the positioning system is aligned with the transducer axis. This alignment can be achieved by firstly, discovering a field maximum in the focal plane through 2D scanning; secondly, identifying a field maximum in another plane with a clear maximum; thirdly, comparing the X and Y coordinates of the two maxima and then iteratively adjusting the position and/or orientation of the transducer if needed28.</li>
	<li>Adjust the tip of the hydrophone with the surface of the transducer at a distance of 1 mm, keeping this distance constant while positioning the hydrophone in the middle of the right edge of the transducer. Initiate the scanning program to capture the free acoustic field in the XZ plane (Figure 2B). 
	NOTE: The construction method of the hydrophone can be found at&#160;https://github.com/HQArrayLab/Hydrophone_system_control.</li>
	<li>Move the hydrophone along the Z-axis to determine the depths associated with the spatial peak pressure. In this experiment, the spatial peak pressure appears at a distance of 3.4 mm from the surface of the transducer; maintain this distance when moving the hydrophone to the bottom right corner of the transducer at the XY plane.Power up and initiate the scanning program to capture the free acoustic field in the XY plane (Figure 2B).</li>
	<li>Place the transducer on the skull of a mouse that underwent surgery as described in step 4. Acquire the transcranial acoustic field on the XZ plane and XY plane (Figure 2D) through hydrophone scanning as described in 2.1-2.3.</li>
	<li>Read the pressure amplitudes at the focal point, which is the spatial peak region in the free acoustic field and transcranial acoustic field. The pressure amplitude at the focal point in the free acoustic field is 730k Pa, and in the transcranial acoustic field is 580k Pa. Read the focal dimensions at -3 dB (Figure 2C,E), and the position on the XY and XZ planes within the transcranial acoustic field to assess whether the acoustic field of this transducer can cover the target brain areas.</li>
	<li>Compute the Mechanical Index (MI), which is constrained by the FDA guidance document to be below 1.9 in order to mitigate cavitation. The calculation of MI is given by the equation: 
	<img alt="Equation 1" src="/files/ftp_upload/67090/67090eq01.jpg" style="margin: auto;" />&#160; &#160; &#160;(1) 
	where pr,.3 represents the peak-rarefactional pressure in MPa adjusted by an attenuation coefficient of 0.3 dB cm-1 MHz-1, and f0 is the operating frequency in MHz. The transcranial-field peak rarefactional pressure measured is 580 kPa, , 3.4 mm from the transducer, the f0 is 500 kHz, so the derated pr,.3 is 576.6 kPa. The MI is 0.82.</li>
	<li>Calculate the spatial-peak pulse-average intensity (Isppa), which is required to be below 190 W/cm2 in the plane direction according to the FDA guidance document. The calculation of intensity is given by the equation: 
	<img alt="Equation 2" src="/files/ftp_upload/67090/67090eq02.jpg" style="margin: auto;" />&#160; &#160; &#160;(2) 
	where psp (t) is the time-varying acoustic pressure at the spatial-peak location, Z is the characteristic acoustic impedance of the medium (approximately 1.5 x 106 Rayls for soft tissue), and PD is pulse duration. In the case of the square envelope, this reduces to the equation: 
	<img alt="Equation 3" src="/files/ftp_upload/67090/67090eq03.jpg" style="margin: auto;" />&#160; &#160; &#160;(3) 
	where A is the spatial-peak pressure amplitude. The A measured at the ultrasound focused location is 580 kPa, and the Z of the brain is about 1.58 x 106 Rayls, so the Isppa of the square envelope is 10.65 W/cm2 and the Isppa of the sinusoidal envelope is 10.65 W/cm2.</li>
	<li>Calculate the spatial-peak time-average intensity (Ispta), which is constrained by the FDA guidance document to be below 430 mW/cm2 in the plane direction. The calculation of intensity is given by the equation: 
	<img alt="Equation 4" src="/files/ftp_upload/67090/67090eq04.jpg" style="margin: auto;" />&#160; &#160; &#160;(4) 
	where T&#160;is the time period over which the average is calculated. In the case of the square envelope, this reduces to the equation: 
	<img alt="Equation 5" src="/files/ftp_upload/67090/67090eq05.jpg" style="margin: auto;" />&#160; &#160; &#160;(5) 
	where DCpulse train is the duty cycle of pulse. Here, the DC pulse train is 1% because continuous waves were used, so the Ispta&#160;is equal to the spatial-peak pulse-average intensity, 106.5 mW/cm2 for a square envelope. The MI, Isppa, and Ispta can be calculated using the software (Figure 3A). A MATLAB-based code for easy use can be found at&#160;https://github.com/HQArrayLab/Ultrasound_Parameter_Caculation.</li>
	<li>Report the pulse timing parameters, including Amax, pulse duration, pulse repetition interval, pulse train duration, and envelope (Figure 3B).</li>
</ol>
3. Preparing the animal for surgery
<ol>
	<li>Weigh 8-week-old male GCaMP6s transgenic mice, with an approximate weight of about 20 g. Prepare a solution containing ketamine at 10 mg/mL and xylazine at 2 mg/mL in sterile saline. Administer the ketamine/xylazine solution by intraperitoneal injection at a dosage of 100 mg/kg ketamine and 20 mg/kg xylazine using a 26G needle and 1 mL disposable syringe. Commence surgical preparation once the animal is unresponsive to painful stimuli, such as toe pinch.</li>
	<li>Use a fader to trim the hair on the animal&#39;s head and disinfect the area with 70% ethanol and povidone-iodine prior to the surgical procedure.</li>
	<li>Place the mouse in a prone position on the stereotaxic frame and ensure that the skull is level. Place a protective ophthalmic ointment over the animal&#39;s eyes to maintain moisture.</li>
</ol>
4. Surgical procedure
<ol>
	<li>Make an incision along the sagittal suture, starting from the occipital bone to the beginning of the nasal bone. Use surgical scissors to remove the skin covering both hemispheres.</li>
	<li>Use sterile saline to cleanse the skull and eliminate any remaining periosteum.</li>
	<li>Apply 3% hydrogen peroxide to the exposed cranium using a cotton swab for approximately 2 s-3 s to create micropores. Rinse with sterile saline thoroughly and ensure the area is completely dry.</li>
	<li>Create a 0.6 mm diameter burr hole craniotomy using a sterile, autoclaved drill bit above the brain area location as determined by the stereotaxic atlas aligned to bregma and lambda. Wash away any debris with sterile saline and ensure thorough drying. Be careful not to damage any tissue.</li>
	<li>Insert the fiber optical ferrule (implant) into the probe holder and connect it to the stereotaxic arm.</li>
	<li>Align the implant directly above the region of interest using the stereotaxic arm. When inserting the optical fiber into the brain tissue, advance the fiber slowly at a rate of approximately 2 mm/min.</li>
	<li>Mix the dental cement to achieve a viscosity that allows for easy application across the cranium. Use a sterile toothpick to spread a thin layer of dental cement over the cranium and onto the lower part of the implant. Allow it to dry completely.</li>
	<li>Detach the probe holder carefully. Prepare a polypropylene pipe with a height of 3 mm, an outer diameter of 3 mm, and an inner diameter of 2.6 mm, and then cut the pipe throughout its length.</li>
	<li>Attach the pipe to the bottom of the implant using tweezers. Pour the dental cement powder into the pipe ensuring sufficient length above the implant for recording the optical fiber signal. Add the required liquid and allow a few minutes for the dental cement to solidify.</li>
	<li>Locate the opening of the pipe and carefully clamp it to remove the pipe using tweezers. Prepare the dental cement mixture for application, ensuring an even and thin layer is spread across the cranium. Cover as much surface area on the cranium as possible with dental cement. Wait a few minutes for the dental cemental to solidify. 
	NOTE: Do not let the dental cement come into contact with the skin of the mouse.</li>
	<li>Drill three holes (1 mm diameter) into the 3D-printed ring, evenly divided on the horizon, with a height of 7 mm, an outer diameter of 10 mm, and an inner diameter of 8.4 mm. Secure the screws (1 mm length) into their respective holes.</li>
	<li>Insert the top of the implant into the hole of the pre-manufactured transducer. Make sure the inner wall of the 3D-printed ring is smooth, and then place it around the transducer positioned on the mouse skull. Ensure that the transducer is centered within the ring.</li>
	<li>Apply dental cement to the junction between the ring and the skull, then wait a few minutes for dental cement to solidify. Avoid placing the dental cement on the connection between the transducer and the skull.</li>
	<li>Carefully remove the transducer and securely tighten the screws.Transfer the mouse to a warm cage and ensure it is monitored until fully recovered before returning it to its original cage.</li>
	<li>After surgery, administer subcutaneous Carprofen (2 mg/kg) for analgesia and continue every 24 h for 3 days to manage inflammation and pain. Monitor the animals daily for any signs of distress, abnormal weight loss, pain, or infection. Normally, by the 3rd day post-surgery, all mice should be exhibiting normal behavior. If any signs of distress or illness are observed in a mouse after the 3rd day, follow institutional guidelines for euthanasia. 
	NOTE: In accordance with specific institutional guidelines and approvals, alternative analgesic regimens may be considered, such as the administration of a pre-procedural dose of analgesics.</li>
</ol>
5. Stimulation and signal recording
<ol>
	<li>At 7 days after surgery, turn on the oxygen supply to the gas anesthesia machine and adjust the oxygen flow regulator to set the gas flow to 300-500 mL/min.</li>
	<li>Place the mouse in the induction chamber and close the anesthetic gas delivery to the mask. Rotate the vaporizer dial to adjust the appropriate anesthetic concentration (2%- 2.5%).</li>
	<li>After the mouse is anesthetized, place it on the stereotaxic frame with an anesthetic mask. Close the induction line to let the anesthetic gas flow into the anesthetic mask. Adjust the appropriate maintenance anesthetic concentration (1%-1.5%).</li>
	<li>Clean the top surface of the implant with alcohol, and then insert the fiber optic patch cord into the center of the prepared transducer.</li>
	<li>Inject water into the space between the implant and the 3D-printed ring using a 26G needle and 1 mL disposable syringe to moisten the skull. Use paper towels to absorb excess water.</li>
	<li>Inject a coupling agent into the space between the implant and the 3D-printed ring using a 26G needle and 1 mL disposable syringe to facilitate the easy propagation of ultrasound from the transducer to the brain.</li>
	<li>Connect the implant to the fiber optic patch cord. Carefully insert the transducer into the area which is filled with a coupling agent and securely tighten the screws.</li>
	<li>Place the mouse in an open field and allow it to wake up. Attach the transducer to the ultrasonic excitation system and connect the fiber optic patch cord to the optical fiber recording system, enabling freedom of movement for the mouse. 
	NOTE: The fiber optic patch cord has a length of 2m and a diameter of 1.25 mm. The light intensity for the 405 channel is 20 &#181;W, and for the 470 channel is 40 &#181;W.</li>
	<li>Activate both the ultrasonic excitation device and the optical fiber recording system in order to synchronize ultrasonic neural modulation with optical fiber signal recording.</li>
</ol>

Integrated Protocol for FUN and Fiber Photometry in Free-Moving Mice

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The authors have no conflicts of interest to disclose.

This approach combines FUN with optical photometry recording, enabling the investigation of mouse brain function and in vivo FUN mechanism. The complete operational process, from transducer fabrication to surgical procedures, is outlined, allowing researchers to independently perform FUN from outside the field.
One crucial aspect of the protocol is ensuring that the optical implant is smoothly inserted into the transducer, the dental cement across the skull is thin enough for ultrasound penetration into the brain, the optical implant is securely connected to the skull to prevent dislodging during the experiment, and the energy output of the transducer is sufficient for effective neuromodulation. The thickness of dental cement surrounding the implant should be equal to or less than the diameter of the transducer hole. Therefore, it is advisable to use the same polypropylene pipe for both the transducer fabrication process and surgery. Since polypropylene pipe does not adhere to dental cement, it is selected to mold the dental cement around the implant, with a side cut, to facilitate easy removal of the polypropylene pipe.
Electrophysiological recording and optical photometry recording are commonly utilized technologies for monitoring brain activity in vivo, offering high temporal-spatial resolution. However, electrophysiological recording captures the firing activity signal from neurons attached to the electrodes directly. The ultrasound waves could directly vibrate the electrodes, inducing unnecessary confounding effects. Fortunately, the fiber photometry technology, which is less invasive, captures the activity of neurons beneath it, which could reduce the confounding effect of ultrasound vibration on the implant7,19,26. As a result, the technology of simultaneous focused ultrasound neuromodulation and fiber photometry recording in free-moving mice allows for the study of in vivo mechanisms of ultrasonic neuromodulation and enables the observation of the mice&#39;s behavioral responses without the interference of anesthesia.
However, fiber photometry&#39;s spatial resolution is restricted as it is unable to monitor the activity of subcellular and microcircuits24. Moreover, it provides an indirect representation of neuronal activity since it does not directly record the electrical signals produced by neuronal activity.

Focused ultrasound neuromodulation (FUN) has emerged as a promising and versatile neuromodulation tool, enabling the exploration of brain function and organization with great potential1. FUN is able to deliver acoustic energy noninvasively to any position within the brain tissue with pinpoint precision2. Its ability to transiently and reversibly modulate neuroactivity in deep brain structure, with high spatiotemporal specificity, in a safe and noninvasive manner, presents an appealing attribute that complements existing clinical neuromodulation technique3. Demonstration of effective FUN has been confirmed in both human subjects4,5,6 and in various animal models, encompassing small7,8,9,10 and large species11,12,13,14,15,16,17.
By observing the effect of FUN on specific neural types through neuroactivity monitoring during FUN, we can delve into the mechanism behind this process18,19. Fiber photometry based on genetically encoded calcium indicators (GECIs) has become widely utilized in the past decade as a versatile method for tracking cell-type-specific population activity in vivo20,21,22,23,24. Hence, the simultaneous application of FUN and fiber photometry can significantly enrich our comprehensive understanding of FUN. Nevertheless, the use of bulky single transducers necessitates fixation to a frame, while animals need to undergo anesthesia and be immobilized in a stereotaxic frame7,19,25,26. This approach may not be suitable for certain types of experiments related to perception, cognition, and behavior evaluation. It is crucial to establish a protocol that facilitates the amalgamation of FUN and fiber photometry without impeding the mice&#39;s mobilization7.
In this study, we present a refined protocol used in our previous studies to seamlessly and gracefully complement the method for crafting a single transducer and its temporary fixation on to the mice, as well as the secure fixation of a fiber optical implant to facilitate the smooth passage of the transducer7,19,26. It allows researchers to record the neuroactivity modulated by ultrasound in unrestrained mice. We opted for a smoother envelope, such as a sinusoidal envelope, to reduce the auditory confound27. This protocol&#39;s feasibility is confirmed through the simultaneous recording of neuroactivity in the anterior thalamic nucleus of free-moving mice during FUN. It demonstrates that the transducer&#39;s energy is sufficient to achieve neuromodulation, and the fixation methods for the fiber optical implant and the transducer can ensure their stability.

<table><tbody><tr><td>1ml disposable syringe</td><td>DOUBLE-DOVE</td><td>1ml</td><td>Injection needles</td></tr><tr><td>26-gauge needle</td><td>Jin mao</td><td>JM-J02</td><td>Preparation needles</td></tr><tr><td>70% ethanol</td><td>Dong de alcohol&amp;nbsp;</td><td>0.7</td><td>Disinfect</td></tr><tr><td>alcohol</td><td>Dong de alcohol&amp;nbsp;</td><td>0.75</td><td>Clean the transducer surface</td></tr><tr><td>Bayonet Nut Connector</td><td>Risym</td><td>75-5</td><td>The other end of the connecting wire is connected to the ultrasonic excitation device</td></tr><tr><td>copper ring</td><td>Guowei Metal Materials</td><td>Outer diameter, wall thickness, height (8mm, 0.2mm, 8mm)</td><td>The outer protective case of the transducer</td></tr><tr><td>disposable syringe</td><td>DOUBLE-DOVE</td><td>1ml</td><td>The inhalation of epoxy resin allows precise small amounts to be injected into the copper pipe</td></tr><tr><td>double-sided tape</td><td>3M</td><td>3M55236</td><td>It is used to fix the transducer and the wire to ensure that the epoxy silver glue does not move before drying</td></tr><tr><td>electronic soldering iron</td><td>Victor</td><td>868A&lt;sup&gt;+&lt;/sup&gt;</td><td>The soldered wires are connected to the BNC</td></tr><tr><td>epoxy resin glue</td><td>Kraft</td><td>K 9741</td><td>Seal the rear of the transducer</td></tr><tr><td>epoxy silver paste</td><td>Vonroll</td><td>CB-052</td><td>The wire is attached to the positive and negative poles of the piezoelectric ceramic sheet and the resistance is kept low</td></tr><tr><td>fader&amp;nbsp;</td><td>JOQO</td><td>YP-7021</td><td>Remove the head hair of the mouse</td></tr><tr><td>gas anesthesia machine</td><td>RWD</td><td>R500</td><td>It is used for anesthesia in mice</td></tr><tr><td>glass sheet</td><td>Square glass</td><td>80mm*80mm</td><td>A temporary operating surface for placing piezoelectric ceramics and wires can be used to coat the surface of the glass plate with double-sided tape</td></tr><tr><td>ketamine/xylazine&amp;nbsp;</td><td>Shutai/shengxin</td><td>Zoletil 50/2ml*10</td><td>Anesthetize the mouse</td></tr><tr><td>medical coupling agent</td><td>Bestman</td><td>120g</td><td>The couplant acts as a medium to conduct the ultrasound signal</td></tr><tr><td>mouse</td><td>Bai shi tong</td><td>GCaMp6</td><td>Test subject</td></tr><tr><td>ophthalmic ointment</td><td>Yun Zhi</td><td>0.5% x 2.5 g x1</td><td>Moistens the eye area to prevent blindness</td></tr><tr><td>&amp;nbsp;piezoelectric plate</td><td>Jiaming Electronics Factory</td><td>Diameter, pore, thickness (7mm, 3mm, 3.56mm)</td><td>The electrical energy is emitted in the form of ultrasound</td></tr><tr><td>polypropylene pipe</td><td>Baihao Pipe Factory</td><td>Outer diameter, inner diameter, length (3mm, 2mm, 500mm)</td><td>Prevent the epoxy resin from plugging the holes and leaving the holes</td></tr><tr><td>povidone-iodine</td><td>lefeke</td><td>500ml</td><td>Disinfect</td></tr><tr><td>signal record of fiber</td><td>Thinker Tech Nanjing Biotech</td><td>Three-color single-channel fiber optic recording system</td><td>Record fiber photometry signals</td></tr><tr><td>stereotaxic frame</td><td>RWD</td><td>68805</td><td>Fix the head of the mouse and localize the brain region</td></tr><tr><td>sterile saline</td><td>Shijiazhuang si yao</td><td>500ML,4.5g</td><td>As a solvent, dissolves the drug</td></tr><tr><td>stimulation of ultrasound&amp;nbsp;</td><td>Deep Brain Technology</td><td>DB-USNM</td><td>Provides stable input to the transducer</td></tr><tr><td>weighing machine</td><td>Qin bo shi</td><td>1718</td><td>Weigh the mouse</td></tr><tr><td>wire</td><td>Jinpeng Cable Factory</td><td>0.3mm&lt;sup&gt;2&lt;/sup&gt;</td><td>Voltage is supplied to the transducer</td></tr></tbody></table>

simultaneous focused ultrasound neuromodulation and fiber photometry recording in free-moving mouse

Focused ultrasound neuromodulation (FUN) represents a promising approach for non-invasive perturbation of neuronal circuits at deep brain regions. It is compatible with most of the existing modalities for monitoring brain functions in vivo. Integration with brain function recording modalities not only enables us to address orders and disorders of specific brain functions with closed-loop feedback but also provides us with mechanistic insights about FUN itself. Here, we provide a modified, simple, dependable, and robust protocol for the simultaneous application of FUN and fiber photometry GCaMP6s fluorescence recording in free-moving mice. This involves the fabrication of a well-sized single transducer and its temporary placement on the mice, along with the secure fixation of a fiber optical implant to facilitate the smooth passage of the transducer. The combination of FUN and fiber photometry provides for the optical recording of neural circuitry responses upon FUN in real-time in deep brain regions. To demonstrate the efficiency of this protocol, Thy1-GCaMP6s mice were used as an example to record the neuroactivity in the anterior thalamic nucleus during FUN while the mice are freely moving. We believe that this protocol can promote the widespread use of FUN in both the neuroscience field and the biomedical ultrasound field.

The acoustic pressure distribution in the free acoustic field on the XY plane and XZ plane located 3.4 mm away from the transducer surface, corresponding to the position of the mouse&#39;s anterior thalamic nucleus, is shown in Figure 2B,C. These measurements were acquired through hydrophone scanning in the XY domain and XZ domain. The acoustic pressure distribution in the transcranial acoustic field on the XY plane and XZ plane located 3.4 mm away from the transducer surface is shown in Figure 2D,E. The free acoustic pressure measured is 730 kPa, and the transcranial acoustic pressure measured is 580 kPa for 500 kHz center frequency. The thickness of the skull measured is about 0.2 mm, on average. We assume that the dispersion relationship is approximately linear, so the skull has an attenuation coefficient of 19.98 dB/cmMHz. The lightweight transducer, weighing around 1.66 g, allows the mouse to move easily, facilitating the observation of the response behavior of the mouse under FUN and the motion trail.
The optical fiber signals were recorded under FUN (Figure 4B,D), with the envelope being square and sinusoidal, respectively. Five male mice were used in the experiment. The square lasted for 300 ms, while the continuous sinusoidal lasted for 471 ms, which can ensure the total energy is the same in two different FUNs (Figure 4A,C). An enhancement in optical fiber signal indicates an increase in neural activity. The neural response is rapid under the FUN, suggesting that the transducer has sufficient energy and excellent focusing capabilities.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67090/67090fig01.jpg" /> 
Figure 1: Production process of the transducer. This involves, in turn, connecting a piezoelectric sheet to a wire and then packaging it. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67090/67090fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67090/67090fig02v2.jpg" /> 
Figure 2: Ultrasound field measurement setup and characterization for ultrasonic transducer.&#160;(A) The setup for ultrasound field measurement includes a hydrophone, motor system, control software, signal generator, and oscilloscope. (B, D) Schematic diagram of ultrasonic transducer measurements in free and transcranial acoustic fields and the results of transverse and longitudinal sound field measurements. (C, E) Diagram of the transverse sound field at the transducer focal position, with the red line indicating the sound field at the -3 dB position. (F, G) Waveform diagram of the output measured by the hydrophone for the transducer. The area within the red dashed box and the area within the blue dashed box represent the periods before the waveform reaches a stable amplitude and the ringing period of the transducer at the end, respectively. The area within the orange dashed box represents the stable part of the waveform, which is used for calculating the pressure amplitude, noted as p. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67090/67090fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67090/67090fig03.jpg" /> 
Figure 3: Calculation software and ultrasound parameter. (A) A homemade ultrasound parameter calculation interface. MI, Isppa, and Ispta were calculated. The interface could be obtained from https://github.com/HQArrayLab/Ultrasound_Parameter_Caculation.&#160;(B) Schematics of ultrasound pressure waveforms. A sinusoidal Pulse Envelope and a rectangular Pulse Envelope are used. The period (T) represents the duration of a single cycle of the operating frequency. A pulse, known as a single continuous sonication, lasts for a specified duration called the pulse duration (PD). Typically, pulses are repeated in a sequence known as a pulse train. The time interval between two consecutive pulses in a pulse train is referred to as the pulse repetition interval (PRI), calculated as the reciprocal of the pulse repetition frequency (PRF). The entire sequence of pulses, known as the pulse train, has a specific duration known as the pulse train duration. The interval time means the duration of a single trial. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67090/67090fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67090/67090fig04v2.jpg" /> 
Figure 4: Signal of fiber photometry during FUN. (A, C) Ultrasound parameters enveloped by square (B) and sinusoidal (D). (B, D) The fiber photometry signal respectively during FUN of (A) and (C). The green shadow is the duration of FUN. The solid line is the mean, and the shades of blue and red are the mean and standard deviation of recorded signals. Five male mice were used in the experiment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67090/67090fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Integration of Fiber Photometry and Focused Ultrasound Neuromodulation for Investigating Neural Modulation in Freely Moving Mice

The protocol includes transducer manufacturing, parameters reporting, surgical procedure, and signal recording for the entire operational workflow of concurrent focused ultrasound neuromodulation and fiber photometry recording in free-moving mice.

Simultaneous Focused Ultrasound Neuromodulation and Fiber Photometry Recording in Free-Moving Mouse

Focused ultrasound neuromodulation (FUN) represents a promising approach for non-invasive perturbation of neuronal circuits at deep brain regions. It is ...

simultaneous-focused-ultrasound-neuromodulation-fiber-photometry

Nanyang Technological University

Research

JoVE Journal

Neuroscience

820 Views.  Guangdong Institute of Intelligence Science and Technology. Ons laboratorium is gewijd aan de toepassing van op echografie gebaseerde technologieën om de hersenfunctie te bestuderen en hersenaandoeningen aan te pakken. Ons huidige onderzoek draait voornamelijk om twee belangrijke fouten: functionele echografie en gefocusseerde ultrasone neuromodulatie. Gericht op echografie wordt neuromodulatie beschouwd als een mogelijke benadering voor herseninterventie bij mensen, maar de mechanismen zijn onduidelijk.Onlang...

Video: Auteur in de schijnwerpers: integratie van vezelfotometrie en gefocusseerde ultrasone neuromodulatie voor het onderzoeken van neurale modulatie bij vrij bewegende 

Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and human studies to investigate the cortical processing of nociceptive information. These laser-evoked brain potentials (LEPs) consist of several transient responses that are time-locked to the onset of laser stimuli. However, the functional properties of the LEP responses are still largely unknown, due to the lack of a sampling technique that can simultaneously record neural activities at the surface of the cortex (i.e., electrocorticogram [ECoG] and scalp electroencephalogram [scalp EEG]) and inside the brain (i.e., local field potential [LFP]). To address this issue, we present here an animal protocol using freely moving rats. This protocol is composed of three main procedures: (1) animal preparation and surgical procedures, (2) a simultaneous recording of ECoG and LFP in response to nociceptive laser stimuli, and (3) data analysis and feature extraction. Specifically, with the help of a 3D-printed protective shell, both ECoG and LFP electrodes implanted on the rat&#39;s skull were securely held together. During data collection, laser pulses were delivered on the rat&#39;s forepaws through gaps in the bottom of the chamber when the animal was in spontaneous stillness. Ongoing white noise was played to avoid the activation of the auditory system by the laser-generated ultrasounds. As a consequence, only nociceptive responses were selectively recorded. Using the standard analytical procedures (e.g., band-pass filtering, epoch extraction, and baseline correction) to extract stimulus-related brain responses, we obtained results showing that LEPs with a high signal-to-noise ratio were simultaneously recorded from ECoG and LFP electrodes. This methodology makes the simultaneous recording of ECoG and LFP activities possible, which provides a bridge of electrocortical signals at the mesoscopic and macroscopic levels, thereby facilitating the investigation of nociceptive information processing in the brain.

We developed a technique that simultaneously records both electrocorticography and local field potentials in response to nociceptive laser stimuli from freely moving rats. This technique helps establish a direct relationship of electrocortical signals at the mesoscopic and macroscopic levels, which facilitates the investigation of nociceptive information processing in the brain.

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and ...

Behavior

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely manipulate neural firing, hormonal secretion and genetically-reprogrammed cells. However, the translation of this technology for medical applications is currently hampered by a lack of biophysical mechanisms by which targeted tissues sense and respond to LIPUS. A suitable approach to identify these mechanisms would be to use optical biosensors in combination with LIPUS to determine underlying signaling pathways. However, implementing LIPUS to a fluorescence microscope may introduce undesired mechanical artefacts due to the presence of physical interfaces that reflect, absorb and refract acoustic waves. This article presents a step-by-step procedure to incorporate LIPUS to commercially-available upright epi-fluorescence microscopes while minimizing the influence of physical interfaces along the acoustic path. A simple procedure is described to operate a single-element ultrasound transducer and to bring the focal zone of the transducer into the objective focal point. The use of LIPUS is illustrated with an example of LIPUS-induced calcium transients in cultured human glioblastoma cells measured using calcium imaging.

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts.

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely ...

Engineering

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a ...

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ultrafast doppler angiography. As brain perfusion is strongly linked to local neuronal activity, this technique allows the whole-brain 3D mapping of task-induced regional activation as well as resting-state functional connectivity, non-invasively, with unmatched spatio-temporal resolution and operational simplicity. In comparison with fMRI (functional magnetic resonance imaging), a main advantage of fUS imaging consists in enabling a complete compatibility with awake and behaving animal experiments. Moreover, fMRI brain mapping in mice, the most used preclinical model in Neuroscience, remains technically challenging due to the small size of the brain and the difficulty to maintain stable physiological conditions. Here we present a simple, reliable and robust protocol for whole-brain fUS imaging in anesthetized and awake mice using an off-the-shelf commercial fUS system with a motorized linear transducer, yielding significant cortical activation following sensory stimulation as well as reproducible 3D functional connectivity pattern for network identification.

This protocol describes the quantification of volumetric cerebral hemodynamic variations in the mouse brain using functional ultrasound (fUS). Procedures for 3D functional activation map following sensory stimulation as well as resting-state functional connectivity are provided as illustrative examples, in anesthetized and awake mice.

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy (pCLE) of Hippocampal Capillaries in Awake Mice

The goal of this protocol is to describe fiber-optic-bundle-coupled pre-clinical confocal laser-scanning endomicroscopy (pCLE) in its specific application to elucidate capillary blood flow effects during seizures, driven by mural cells. In vitro and in vivo cortical imaging have shown that capillary constrictions driven by pericytes can result from functional local neural activity, as well as from drug application, in healthy animals. Here, a protocol is presented on how to use pCLE to determine the role of microvascular dynamics in neural degeneration in epilepsy, at any tissue depth (specifically in the hippocampus). We describe a head restraint technique that has been adapted to record pCLE in awake animals, to address potential side-effects of anesthetics on neural activity. Using these methods, electrophysiological and imaging recordings can be conducted over several hours in deep neural structures of the brain.

In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy pCLE of Hippocampal Capillaries in Awake Mice

Whereas multiphoton imaging is only effective at limited depths from the tissue&#8217;s surface, it is possible to achieve 3 &#181;m resolution imaging at any depth via pCLE. Here, we present a protocol to conduct pCLE imaging to measure microvascular dynamics in the hippocampus of ictal and wild-type mice.

The goal of this protocol is to describe fiber-optic-bundle-coupled pre-clinical confocal laser-scanning endomicroscopy (pCLE) in its specific application ...

Focused Ultrasound Neuromodulation of Human In Vitro Neural Cultures in Multi-Well Microelectrode Arrays

The neuromodulatory effects of focused ultrasound (FUS) have been demonstrated in animal models, and FUS has been used successfully to treat movement and psychiatric disorders in humans. However, despite the success of FUS, the mechanism underlying its effects&#160;on neurons remains poorly understood, making treatment optimization by tuning FUS parameters difficult. To address this gap in knowledge, we studied human neurons in vitro using neurons cultured from human-induced pluripotent stem cells (HiPSCs). Using HiPSCs allows for the study of human-specific neuronal behaviors in both physiologic and pathologic states. This report presents a protocol for using a high-throughput system that enables the monitoring and quantification of the neuromodulatory effects of FUS on HiPSC neurons. By varying the FUS parameters and manipulating the HiPSC neurons through pharmaceutical and genetic modifications, researchers can evaluate the neural responses and elucidate the neuro-modulatory effects of FUS on HiPSC neurons. This research could have significant implications for the development of safe and effective FUS-based therapies for a range of neurological and psychiatric disorders.

Focused Ultrasound Neuromodulation of Human In Vitro Neural Cultures in Multi-Well Microelectrode Arrays

Here, we present a protocol for using a high-throughput system that enables the monitoring and quantification of the neuromodulatory effects of focused ultrasound on human-induced pluripotent stem cell (HiPSC) neurons.

The neuromodulatory effects of focused ultrasound (FUS) have been demonstrated in animal models, and FUS has been used successfully to treat movement and ...

Recording Neural Activity in Freely Moving Mice Using Fiber Photometry

Source: Martianova, E., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60278">Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals.</a> J. Vis. Exp. (2019).
This video demonstrates a method for recording neural activity in a freely moving mouse using fiber photometry. In this method, neurons in a specific brain region express a genetically encoded calcium indicator. A stimulus is applied to this brain region, and the stimulus-dependent change in fluorescence is measured to identify neural activity.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Simultaneous Focused Ultrasound Neuromodulation and Fiber Photometry Recording in Free-Moving Mouse

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