We thank Shaili Jha and Heather Decot for assistance with filming.

Ethics Statement: This procedure is in accordance with the National Institutes of Health Guidelines for Animal Research (Guide for the Care and Use of Laboratory Animals) and is approved by the University of North Carolina Institutional Animal Care and Use Committee.
1. Electrode Implantation
The first step is electrode implantation. In this step, an electrode is unilaterally implanted into the subthalamic nucleus (STN), a small nucleus with translational significance for Parkinson&#8217;s disease treatment using the following methods:
<ol>
	<li>Sterilize all surgical equipment using an autoclave, or antiseptic solution where autoclaving is not possible (e.g.&#160;for electrode sterility). Note: this is a short-term survival surgery, and thus aseptic technique is essential. Following surgery, animals can be imaged after a brief recovery period (48 hr) or up to several weeks later.</li>
	<li>Anesthetize the rat (Adult Sprague-Dawley rats 250-400 g) using 2.5% isoflurane administered via endotracheal intubation and a small animal ventilator. Fix the rat to a stereotactic surgical frame and prepare the surgical site using aseptic techniques.</li>
	<li>Prepare and ensure that the electrode is sterile. A homemade 2-channel tungsten microwire electrode is used for this procedure, though many MRI-compatible electrode types will work. The electrode type used may affect the area of tissue mechanically damaged by the procedure, the area of tissue stimulated, and the accuracy of implantation, thus affecting the overall experimental result. If the electrode type is not able to be autoclaved, use povidone-iodine antiseptic to sterilize the electrode as far as is possible.</li>
	<li>Using scissors remove the scalp over the site of implantation with a diameter of approximately 1.5 cm, to uncover bregma and lambda on the skull. Remove the muscle and fascia overlying the skull and stop all bleeding using electrocautery.</li>
	<li>Scratch the skull surface in multiple directions with a scalpel to improve dental cement adhesion (step 1.8). Level Bregma and Lambda in the horizontal direction.</li>
	<li>To target STN, at 3.6 mm posterior to Bregma and 2.5 mm lateral to midline, use a small-tipped electric drill to create a burr hole measuring approximately 1.5 mm in diameter. Note: The exact location of the STN in reference to stereotaxic coordinates may vary by rat strain, weight, and gender. Adult rats of the same gender should be used to minimize any variation in location. If possible, pre-operational anatomical scans or intraoperative electrical recordings should be used to identify the STN location on an individual subject basis. Further, electrode termination sites should be histologically verified to ensure target accuracy.
	<ol>
		<li>Carefully make an incision in the dura, and use small blunt forceps to move the dura to the sides of the hole. Stop any bleeding using sterile cotton soaked in saline. Create holes for one&#160;or more MR-compatible screws) and insert them gently in the skull until they are stable. Screws may be placed in any location where they will not disrupt the placement of the external connector for the DBS electrode (e.g.&#160;not directly behind the STN ipsilateral to the electrode). We recommend placements at the lateral edges of the skull, ideally directly posterior to the lambda suture. At this point, the skull is relatively thick, reducing the likelihood that screws will damage the cortex.&#8221; Note: Brass screws cut to 4-5 mm in length are used in this protocol, although plastic screws are also suitable.</li>
	</ol>
	</li>
	<li>Place the electrode on the stereotactic arm, ensuring that it is straight and vertical. Touch the electrode to Bregma, then move the electrode exactly 3.6 mm posterior to Bregma and 2.5 mm lateral to midline and touch the cortical surface with the electrode. From the cortical surface, insert the electrode 7.8 mm ventrally. These coordinates are determined by reference to a neuroanatomical atlas1.</li>
	<li>Place a layer of dental cement over the skull including the skull screws and electrode insertion point. Wait until the cement is completely hardened prior to removing the electrode from the stereotactic frame. Bend the electrode backward and use additional cement to cover the rest of the electrode tract and connector for durability.</li>
</ol>
2. fMRI Preparation
The second step is the setup for fMRI, including positioning of the coil and setup of physiological monitoring equipment.
<ol>
	<li>Secure the animal&#8217;s head to prevent motion during the scan. Note: A custom plastic intraauricular bar system is used here for head fixation. Place the bars into the ear canals and secure to the headpiece so that the head rotates smoothly in the vertical direction with no horizontal rotation. Secure the head position by fixing the upper teeth to the apparatus.</li>
	<li>Anesthetize the rat completely and monitor end tidal CO2 to ensure stability throughout all scans. To maintain anesthesia, ventilation and control end tidal CO2 levels during the scan, an MR-compatible small animal ventilation system combined with an isoflurane vaporizer is used here, though a variety of anesthetic and sedation agents may be used in a similar fashion. Set the ventilator to 45 breaths/min with a moderate volume, approximately 500 ml/min of air as a starting volume. Set the isoflurane to 2% and transfer the rat into the scan room. Attach the ventilator&#8217;s output to the rat&#8217;s endotracheal tube and press firmly to secure. Capnometry should be acquired using a tube connected as closely to the endotracheal tube connector as possible. Adjust ventilation volume to produce an end-tidal CO2 of 2.6% to 3.3%.</li>
	<li>Use an MR-compatible small animal holder to insert the rat into the scanner with a circulating hot water bath for temperature control. Tape the pad of the bath onto the holder and cover it with clean absorbent paper. Place the rat onto the hot water bed.</li>
	<li>Monitoring of temperature and carbon dioxide levels are essential to BOLD fMRI, while arterial oxygen saturation and heart rate are also useful physiological parameters. Insert an MR-compatible rectal temperature probe and tape it to the base of the tail, and then adjust the temperature of the water bath to maintain normal body temperature at 37&#160;&#176;C. Monitor arterial oxygen saturation and heart rate using a small animal pulse oximetry system, maintaining them at 95-98% and 250-350 bpm, respectively, which may vary depending on the type of anesthetics used. Oxygen saturation and heart rate are both influenced by depth of anesthesia, ventilation volume and ventilation rate. Ventilation volume and rate may need to be carefully balanced to maintain adequate end-tidal CO2 levels and adequate oxygen saturation.</li>
	<li>A surface coil is needed for BOLD fMRI acquisition. Place the surface coil as close to the surface of the head as possible. Once secured, place toothpaste on the surface of the head on the cement cap in order to reduce susceptibility artifacts near the brain surface. Note: We use a homemade transceiver surface coil with an internal diameter of approximately 1.6 cm, though larger surface coils may be used to optimize the BOLD response in deeper subcortical regions.</li>
	<li>Connect the stimulating electrode to a programmable electrical stimulator system. Note: We use a custom-made programmable TTL triggering system connected to a bipolar stimulator to deliver electrical pulses synchronized to the RF excitations from the MR scans.</li>
	<li>For sedation and paralysis during fMRI data acquisition, use a cocktail of dexmedetomidine (0.1 mg/kg/hr, i.p.) and pancuronium (1 mg/kg/hr, i.p.), combined with low dose isoflurane at 0.5% to prevent epileptic activity2. For drug infusion, an MR-compatible syringe pump must be used if the pump is to be placed in the magnetic environment. Alternatively, a noncompatible pump may be placed outside the magnetic environment provided that extended catheter tubing is used.</li>
</ol>
3. fMRI Data Acquistion
The third step is fMRI acquisition, including positioning, shimming, anatomical scans, and functional scans. A 9.4 Tesla system with a homemade surface coil is used here, though this technique may be adapted to other high-field systems and commercially made MRI coils.
<ol>
	<li>Insert the rat into the scanner and position in the center of the magnet. Use a three-plane scout image to precisely center the rat within the magnet with respect to the brain regions of interest, and FASTMAP shimming to homogenize the magnetic field at the regions of interest.</li>
	<li>Use a sagittal T2-weighted RARE sequence (FOV, 2.56 x 2.56 cm2; matrix size, 256 x 256; slice thickness, 1.5 mm; TR/TE, 1500/11 ms; RARE Factor, 8; Flip Angle, 180&#176;) to find the location of the anterior commissure, and align the subsequent images to this location. Align eight-slice single-shot GE-EPI scans (FOV, 2.56 x 2.56 cm2; matrix size, 96 x 96, reconstructed to 128 x 128; slice thickness, 1mm; TR/TE, 1000/14 ms) to this point with coronal orientation.</li>
	<li>For functional scans, use 70 consecutive EPI scans with 1 second temporal resolution synchronized to the stimulation output, set to 20 sec rest, 10 sec stimulation, followed by 40 sec rest. Allow a minimum of 90 sec between scans to allow for neurovascular recovery. Acquire multiple repeated scans at each stimulation parameter to improve the signal-to-noise ratio by averaging. Use a series of dummy scans (typically 4-8) immediately prior to scanning for noise reduction. Confirm the BOLD response at the time of image acquisition to ensure success of the experiment using the method described in section 4, though averaging, coregistration and skull-stripping may be skipped in this setting.</li>
	<li>After functional scanning is complete, use a T2-weighted RARE spin-echo sequence (FOV, 2.56 x 2.56 cm2; matrix size, 256 x 256; slice thickness, 1 mm; TR/TE, 2500/33 ms; averages, 8) to measure the anatomical position of the electrode in vivo. Acquire multiple coronal and sagittal sections to measure the tip of the electrode artifact along anterior/posterior, medial/lateral and dorsal/ventral axes and confirm electrode placement. High resolution magnetic resonance microscopy (FOV, 1.8 x 1.28 cm; matrix size, 360 x 256; slice thickness, 0.5 mm; TR/TE, 2500/12.6 ms; RARE factor, 8; averages, 280) may be used to examine the precise location of the electrode tract after removal with respect to nearby neuroanatomical structures and confirm the accuracy of electrode placement3.</li>
</ol>
4. fMRI Data Processing and Analysis
The fourth step is processing and analysis of fMRI data, including generation of response maps and calculation of percent BOLD signal change. Custom programs running within a computing environment (e.g.&#160;MATLAB) or commercial fMRI software tools (e.g.&#160;SPM, FSL, or AFNI) may be employed.
<ol>
	<li>Begin with image coregistration and averaging of data first within-subject by frequency, followed by across-subject. Note: We accomplish this using SPM codes.</li>
	<li>Perform skull stripping to remove nonbrain tissue using manually defined region of interest (ROI) with signal thresholding. Automatic skull stripping algorithms may be employed.</li>
	<li>Compile response maps by calculating the correlation coefficient of the relationship between BOLD response over time and the stimulation paradigm for each voxel. Delaying the paradigm several seconds to account for delay in hemodynamic response may be necessary. Set significant level at P &#60; 0.05 after Bonferroni correction. Other statistical methods may be employed. Correcting for multiple comparisons using Random Field Theory or cluster-level correction based on Gaussian Random Field may be performed in place of the Bonferroni correction for more sensitive analysis4. Note: The hemodynamic delay may vary based on brain regions targeted, pharmacologic agents used, and physiological parameters. It is crucial to control these parameters in order to prevent variability within-subject and between-subjects.</li>
	<li>Quantify the BOLD response by defining a ROI to extract time-course data. Average the percent signal change across all voxels within the same anatomical structure. Voxel-wise analysis using the General Linear Model may also be used5.</li>
</ol>

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The authors have nothing to disclose.

Simultaneous DBS and fMRI represents a promising experimental toolkit for the identification and characterization of global downstream responses to neural circuit stimulation, in vivo.&#160;The major advantage of this technique over other available tools, such as electrophysiological recordings, lies in the relatively unbiased nature of fMRI, whereby a large and diverse area of brain tissue can be examined for responsiveness to DBS at any target. Although the described protocol is specific for DBS-fMRI in the ra...

Determining the global downstream effects of neural circuit activity represents a major challenge and goal for many areas of systems neuroscience. A paucity of tools are currently available that meet this need, and thus there is a demand for increased accessibility of the appropriate experimental setups.&#160;One such method for evaluating the global consequence of neural circuit activation relies on the simultaneous application of deep brain electrical stimulation (DBS) and functional MRI (fMRI). DBS-fMRI allows for the detection of downstream responses to circuit activation on a large spatial scale, and can be applied at virtually any stimulation target. This toolset is highly suitable for translational preclinical studies, including the characterization of responses to therapeutic high frequency stimulation.
In addition to access to a suitable MRI scanner, successful DBS-fMRI experiments require consideration of a number of variables, including electrode type, sedation method, and maintenance of physiological parameters.&#160;For example, electrode choice should be based on factors relating to stimulation efficacy (e.g.&#160;lead size and conductance, mono- vs. bipolar), as well as MR compatibility and electrode artifact size. Electrode artifacts vary according to electrode material and size, as well as the scan sequence used; thorough pre-experimental testing should be employed to determine the appropriate electrode type for each study. In general, tungsten microwire electrodes are recommended for this protocol. Choice of paralytic and sedative should be made to effectively immobilize the animal and reduce the suppressive effects of certain sedatives on blood-oxygen-level-dependent (BOLD) signal.&#160;Lastly, it is critical to maintain the animal at optimal physiological parameters, including body temperature and oxygen saturation.
The protocol that we have developed for DBS-fMRI overcomes many of these potential obstacles, and in our hands, provides robust and consistent results.&#160;Additionally, these experimental procedures may be readily adopted for the combination of fMRI with alternative stimulation methods, including optogenetic stimulation.&#160;

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Isoflurane (Forane)</td>
			<td style="width:71px;">Baxter</td>
			<td style="width:119px;">1001936060</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Dexmedetomidine (Dexdomitor)</td>
			<td style="width:71px;">Pfizer</td>
			<td style="width:119px;">145108-58-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Pancuronium Bromide</td>
			<td style="width:71px;">Selleckchem</td>
			<td style="width:119px;">S2497</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">9.4T Small Animal MRI</td>
			<td style="width:71px;">Bruker</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">BioSpec System with BGA-9S gradient</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sterotactic Frame</td>
			<td style="width:71px;">Kopf</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model 962</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Small Animal Ventilator</td>
			<td style="width:71px;">CWE, Inc.&#160;</td>
			<td style="width:119px;">12-02100</td>
			<td style="width:167px;">Model SAR-830</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Dental Cement</td>
			<td style="width:71px;">A-M Systems</td>
			<td style="width:119px;">525000</td>
			<td style="width:167px;">Teets Cold Curing</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">MouseOx Plus System</td>
			<td style="width:71px;">STARR Life Science Corp.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Capnometer</td>
			<td style="width:71px;">Surgivet, Smith Medical</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">V9004 Series</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Stimulus Isolator</td>
			<td style="width:71px;">World Precision Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model A365</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">MR-compatible Brass Screws</td>
			<td style="width:71px;">McMaster Carr</td>
			<td>94070A031</td>
			<td style="width:167px;">0-80 thread size, 1/4 inch. Can be cut to desired length.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Tungsten Wire</td>
			<td style="width:71px;">California Fine Wire Company</td>
			<td>100211</td>
			<td style="width:167px;">Used to construct MR-compatible stimulating microelectrode</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Syringe Pump</td>
			<td style="width:71px;">Harvard Appartus</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model PHD 2000 (not MRI-compatible)</td>
		</tr>
	</tbody>
</table>

deep brain stimulation with simultaneous fmri in rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

Representative functional data were acquired according to the above protocol in a single rat with a stimulating electrode implanted to the subthalamic nucleus on the right side. An illustration of essential setup for DBS fMRI image acquisition is provided in Figure 1. Stimulation was applied consistent with the above protocol, with an amplitude of 0.3 mA, frequency of 130 Hz and pulse width of 0.09 msec. Robust activation of ipsilateral motor cortex has been consistently visualized using this protocol wi...

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Deep Brain Stimulation with Simultaneous fMRI in Rodents

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

deep-brain-stimulation-with-simultaneous-fmri-in-rodents

Research

JoVE Journal

Neuroscience

14.0K Görüntüleme.  University of North Carolina.  Bu protokol kemirgen eşzamanlı fonksiyonel manyetik rezonans görüntüleme ve derin beyin stimülasyonu için standart bir yöntem açıklanır. Bu deneysel araçların kombine kullanımı hemen hemen herhangi bir beyin hedefe elektriksel uyarıya tepki olarak küresel alt aktivitesinin araştırılması için izin verir. 

Video: Deep Brain Stimulation with Simultaneous fMRI in Rodents

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

A General Method for Evaluating Deep Brain Stimulation Effects on Intravenous Methamphetamine Self-Administration

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people. There is a need for novel, effective and practical treatment strategies that are validated in animal models. Neuromodulation, including deep brain stimulation (DBS) therapy, refers to the use of electricity to influence pathological neuronal activity and has shown promise for psychiatric disorders, including drug dependence. DBS in clinical practice involves the continuous delivery of stimulation into brain structures using an implantable pacemaker-like system that is programmed externally by a physician to alleviate symptoms. This treatment will be limited in methamphetamine users due to challenging psychosocial situations. Electrical treatments that can be delivered intermittently, non-invasively and remotely from the drug-use setting will be more realistic. This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence. Methamphetamine dependence is rapidly developed in rodents using an operant paradigm of intravenous (IV) self-administration that incorporates a period of extended access to drug and demonstrates both escalation of use and high motivation to obtain drug.

This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence.

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people.  There is a need ...

Behavior

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, which is unlike the protocols used for human subjects. We report a successful application of rTMS to the unilateral hemisphere of rat brain. The rTMS was delivered with a low-frequency (1 Hz), high-frequency (20 Hz), or sham stimulation protocol to one side of the brain by using a small 25-mm figure-8 coil. We placed the center of the coil 1 cm lateral to the vertex on the biauricular line and angulated the coil 45&#176; to the ground to minimize a potential direct effect of rTMS on the contralateral cortex. We also used an in-house water cooling system to enable repetitive magnetic stimulation for more than 20 min, even at a 20-Hz stimulation frequency. Increases in the transcriptions of immediate early genes (Arc, Junb, and Egr2) were greater after rTMS than after sham stimulation. After 5 consecutive days of 20-min 1-Hz rTMS, bdnf mRNA expression was significantly higher in stimulated cortex than in contralateral side. The model presented herein will elucidate the molecular mechanisms of rTMS by allowing analysis of the inter-hemispheric difference in its effect.

We applied repetitive transcranial magnetic stimulation (rTMS) to the unilateral hemisphere of rat brain, by placing a 25-mm figure-8 coil 1 cm lateral to the vertex on the biauricular line and angulating the coil by 45&#176;. An in-house water cooling system was used for rTMS for more than 20 min.

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, ...

Simultaneous Transcranial Alternating Current Stimulation and Functional Magnetic Resonance Imaging

Transcranial alternating current stimulation (tACS) is a promising tool for noninvasive investigation of brain oscillations. TACS employs frequency-specific stimulation of the human brain through current applied to the scalp with surface electrodes. Most current knowledge of the technique is based on behavioral studies; thus, combining the method with brain imaging holds potential to better understand the mechanisms of tACS. Because of electrical and susceptibility artifacts, combining tACS with brain imaging can be challenging, however, one brain imaging technique that is well suited to be applied simultaneously with tACS is functional magnetic resonance imaging (fMRI). In our lab, we have successfully combined tACS with simultaneous fMRI measurements to show that tACS effects are state, current, and frequency dependent, and that modulation of brain activity is not limited to the area directly below the electrodes. This article describes a safe and reliable setup for applying tACS simultaneously with visual task fMRI studies, which can lend to understanding oscillatory brain function as well as the effects of tACS on the brain.

Transcranial alternating current stimulation (tACS) is a promising tool for noninvasive investigation of brain oscillations, though its effects are not completely understood. This article describes a safe and reliable setup for applying tACS simultaneously with functional magnetic resonance imaging, which can increase understanding oscillatory brain function and effects of tACS.

Transcranial alternating current stimulation (tACS) is a promising tool for noninvasive investigation of brain oscillations. TACS employs ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for various neurological and psychiatric disorders. HFS in the deep region of the brain, also named deep-brain stimulation (DBS), is becoming increasingly important in clinical trials. Recent progress in the field of high-frequency DBS (HF-DBS) surgery has begun to spread the possibility of utilizing this invasive technique to other situations, such as treatment for major depression disorder (MDD), obsessive-compulsive disorder (OCD), and so on. 
Despite these expanding indications, the underlying mechanisms of the beneficial effects of HF-DBS remain enigmatic. To address this question, one approach is to use implanted electrodes that sparsely activate distributed subpopulations of neurons by HFS. It has been reported that HFS in the anterior nucleus of the thalamus could be used for the treatment of refractory epilepsy in the clinic. The underlying mechanisms might be related to the increased neurogenesis and altered neuronal activity. Therefore, we are interested in exploring the physiological alterations by the detection of neuronal activity as well as neurogenesis in the mouse dentate gyrus (DG) before and after HFS treatment. 
In this manuscript, we describe methodologies for HFS to target the activation of the DG in mice, directly or indirectly and in an acute or chronic manner. In addition, we describe a detailed protocol for the preparation of brain slices for c-fos and Notch1 immunofluorescent staining to monitor the neuronal activity and signaling activation and for bromodeoxyuridine (BrdU) labeling to determine the neurogenesis after the HF-DBS induction. The activation of the neuronal activity and neurogenesis after the HF-DBS treatment provides direct neurobiological evidence and potential therapeutic benefits. Particularly, this methodology can be modified and applied to target other interested brain regions such as the basal ganglia and subthalamic regions for specific brain disorders in the clinic.

This protocol shows how to set up a reliable HFS method in mice. Neurons throughout the hippocampal dentate gyrus are electrically stimulated by HFS directly and indirectly in vivo. Neuronal activity and molecular signaling are examined by c-fos and Notch1 immunofluorescent staining, respectively; neurogenesis is quantified by bromodeoxyuridine labeling assay.

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for ...

Acquisition of Resting-State Functional Magnetic Resonance Imaging Data in the Rat

Resting-state functional magnetic resonance imaging (rs-fMRI) has become an increasingly popular method to study brain function in a resting, non-task state. This protocol describes a preclinical survival method for obtaining rs-fMRI data. Combining low dose isoflurane with continuous infusion of the &#945;2 adrenergic receptor agonist dexmedetomidine provides a robust option for stable, high-quality data acquisition while preserving brain network function. Furthermore, this procedure allows for spontaneous breathing and near-normal physiology in the rat. Additional imaging sequences can be combined with resting-state acquisition creating experimental protocols with anesthetic stability of up to 5 h using this method. This protocol describes the setup of equipment, monitoring of rat physiology during four distinct phases of anesthesia, acquisition of resting-state scans, quality assessment of data, recovery of the animal, and a brief discussion of post-processing data analysis. This protocol can be used across a wide variety of preclinical rodent models to help reveal the resulting brain network changes that occur at rest.

This protocol describes a method for obtaining stable resting-state functional magnetic resonance imaging (rs-fMRI) data from a rat using low dose isoflurane in combination with low dose dexmedetomidine.

Resting-state functional magnetic resonance imaging (rs-fMRI) has become an increasingly popular method to study brain function in a resting, non-task ...