Supported by the National Natural Science Foundation of China Grants 31522029, 31770929 and 31371149 (to Haitao Wu), Program 973 (2014CB542203) from the State Key Development Program for Basic Research of China (to Haitao Wu), and Grant Z161100000216154 from the Beijing Municipal Science and Technology Commission (to Haitao Wu). The authors thank all the members of the Haitao Wu laboratory for their encouragement and discussions. The authors are extremely grateful to Zhenwei Liu for his help with debugging the apparatus.

Animal experimental procedures followed the institutional guidelines of the Beijing Institute of Basic Medical Sciences (Beijing, China) and the Chinese governmental regulations for the Care and Use of Laboratory Animals. The mice (adult male, 26 ~ 30 g) were housed and kept at a constant temperature of 23 &#176;C, with water and food ad libitum, under a 12-h light/12-h dark cycle (lights on at 7:00 a.m.). All experimental procedures were performed during the light cycle.
1. Surgical Preparation
NOTE: A custom electrode was homemade using the method modified from Halpern&#39;s report23.
<ol>
	<li>Use 2 copper micro-wires (with an outer diameter of 200 &#181;m) as the parallel bipolar electrode. Wrap the electrodes around each other to form cylindrical electrodes for the stimulation. 
	NOTE: Here, the electrode length within either the PP or DG was varied according to the anatomical depth of the nucleus.</li>
	<li>Sterilize the electrodes using an Ethylene oxide disinfection cabinet. Handle and store the homemade 2-channel micro-wire electrodes without any contamination. The electrodes should be strictly sterilized before any surgical implantation.</li>
	<li>Autoclave the surgical instruments.</li>
	<li>Before the surgery, inject the mouse with pentobarbital (20 - 50 mg/kg) intraperitoneally, to achieve a surgical plane of anesthesia.</li>
	<li>Inject the mouse with a sterile penicillin G procaine suspension (75,000 U, I.M.) and an analgesic agent (carprofen, 0.05 - 1&#160;mg/kg, SC) to decrease the risk of infections and pain, respectively24.</li>
	<li>After the animal anesthesia, apply an eye lubricant to both eyes to prevent eye dryness. Ensure the induction of the sedation and the depth of the anesthesia by the animal&#39;s reflexes in response to toe pinching and proceed with the operation only when no response occurs. 
	NOTE: Vet ointment is another option to prevent eye dryness.</li>
	<li>Shave most of the fur on the top of the mouse head with electrical hair clippers or a razor blade, sterilize the shaved area with three alternating scrubs of 70% alcohol and betadine solution and proceed with the operation after the betadine has dried on the skin.</li>
</ol>
2. High-Frequency Stimulation Surgery25,26
NOTE: In this step, an electrode is unilaterally implanted into the dorsal DG or medial PP area, a part of the hippocampus with critical roles in episodic learning and memory.
<ol>
	<li>Place the anesthetized mouse onto the stereotactic frame. To keep the mouse&#39;s head immobile, align the ear bars properly and install them quickly. Gently tighten the ear bars so that they support the centered head and secure the mouse slightly.</li>
	<li>Use a thermostatic pad to maintain the animal&#39;s body temperature at 37 &#177; 0.5 &#176;C.</li>
	<li>Use forceps to grasp the skin anterior to the mouse ears and cut it with sterilized scissors to remove about 1-cm2 area of skin on the top of the mouse&#39;s skull. 
	NOTE: A small amount of bleeding could be observed along the edges of the cut skin.</li>
	<li>Remove the muscle and other attachment overlying the skull with a cotton swab to expose the skull surface. Use a cotton swab and dissecting chisel to clean all periosteum, tendons and connective tissue from the skull&#8217;s surface completely to identify the sagittal and lambda sutures lines.</li>
	<li>Identify the bregma and lambda point. The intersection of the coronal suture and the sagittal suture on the superior middle portion of the calvaria is where the bregma is located. The intersection of the lambdoid suture by the sagittal suture is where the lambda is visible. Use the bregma as the original point to set the other nucleus position.</li>
	<li>After the identification of the bregma and lambda position on the skull, adjust these two points in the horizontal level. Make sure that the height of these two points is almost the same in the dorsal-ventral direction (any errors should be &#60; 0.05 mm). Also, make sure that the skull is level in the medial-lateral direction in the same way26.</li>
	<li>For a unilateral surgery, mount a homemade 2-channel copper micro-wire electrode into the rotating electrode holder on the stereotactic surgical frame.</li>
	<li>Place the electrode vertically above the bregma point at first, and set it as original site. Use the digital displayed stereotactic surgical frame to indicate the anterior-posterior (AP), medial-lateral (ML), and dorso-ventral (DV) positions. 
	NOTE: To prevent the electrode from getting bent, do not touch the electrode tip onto the skull during the operation.</li>
	<li>Move the electrode gently to the correct position (Figure 1A, AP-2.1/ML &#177; 1.0 for DG and AP-3.8/ML &#177; 3.0 for PP, respectively)27 above the skull without any touch. When the desired site was identified for electrode insertion, use the dorsoventral stereotactic adjustments to higher the electrodes holder, and mark the position with a black marker. 
	NOTE: The exact location in reference to the stereotaxic coordinates may vary by mouse strain, weight, and sex. Adult mice of the same sex should be used to minimize any variation in the location. If possible, pre-operational anatomical scans should be used to identify the location on an individual subject basis27.</li>
	<li>Use a hand-held micromanipulator-assisted drill (burr size = 0.5 mm) to make burr holes at the marked position precisely. Sterilize the tip of the burr with 70% ethanol before drilling. Move up the mini drill bit through the skull along the Z-axis 0.8 - 1.0 mm without changing its X-Y position. Use a sterile cotton to stop any bleeding during the operation. 
	NOTE: To avoid over-drilling and excessive heat generation during the drilling, ensure the drilling speed is 15,000 - 18,000 revolutions per minute (RPM), and frequently withdraw the drill bit from burr hole every 8 - 10 s.</li>
	<li>Keep drilling until the dura is exposed. Use a bent needle to remove any bone scraps that are generated during the drilling and make a pinhole on the dura without damaging the underlying soft brain tissue. 
	NOTE: To avoid damaging the brain tissue, the bent blunt needle can also be replaced with a fine blunt forceps.</li>
	<li>To confirm that the burr holes have been made properly at the desired position, reduce the electrode height by an adjustment of the stereotactic surgery frame and ensure that the electrode can be inserted smoothly through the burr hole without touching any obstacles. If so, slowly insert the electrodes to a depth of 1.8 mm for the DG (or 3.0 mm for the PP) from the surface of the skull located at the medial PP to the DG (PP-DG). 
	NOTE: Wipe off any cerebrospinal fluid or blood outflows from the burr holes with sterile cotton during the process of the electrode implantation.</li>
	<li>After inserting the micro-wire electrode into the skull hole, hold it in place with adequate dental cement using a small spatula. As the cement thickens, mold it around the inserted electrode and screws to form a nice smooth cap. Debride and disinfect the wound edges.</li>
	<li>Disengage the electrode from the electrode holder. Remove the mouse from the stereotactic frame and return it to a warm cage. 
	NOTE: After surgery, wait with returning the animals to their cages until they have fully recovered from the anesthesia with sufficient consciousness.</li>
	<li>Let the mouse move freely and allow it to recover for 2 days in the cage. Then, connect the electrode implanted in the mouse brain to a programmable stimulator via leads. Set up the software interface as presented in Figure 2. Set the HFS parameters as 6 series of 6 trains of 6 pulses at 400 Hz (Figure 1B, 100 ms between the trains, 20 s between the series)28. Set the stimulation intensity to 200 &#956;A.</li>
	<li>Deliver an HFS for the desired period of time as set up. Deliver the stimulation 2x a day at 8:00 a.m. and at 8:00 p.m.</li>
	<li>For the control group, implant electrodes in the mice and do not perform a stimulation.</li>
	<li>For the c-fos staining, stimulate the experimental mice acutely (2x per day). For the BrdU labeling, divide the experimental mice into an acute stimulation group (applied 2x per day) and a chronic stimulation group (applied 10x in 5 days, 2x per day). During the course of the stimulation, make sure the mouse is awake. Remember to ensure the leads are not twisted. 
	NOTE: At the last day of the procedure, the mice in the acute stimulation group were stimulated simultaneously.</li>
	<li>When the stimulation is done, carefully disconnect the electrode pin from the electrode holder and the connector of the recording system.</li>
</ol>
3. Immunofluorescence Staining and Bromodeoxyuridine Labeling29
<ol>
	<li>For the c-fos and Notch1 staining, about 3 h after the last HF-DBS stimulation, anesthetize the mice by injecting pentobarbital (20 - 50 mg/kg, I.P.) to achieve a surgical plane of anesthesia.</li>
	<li>For the BrdU labeling, about 12 h after the last HF-DBS stimulation, give 6 injections of BrdU to the control and the stimulated mice at 2-h intervals (50 mg/kg in 0.9% saline, I.P.). 36 h after the last injection, anesthetize the mice by injecting pentobarbital (20 - 50 mg/kg, I.P.) to achieve a surgical plane.</li>
	<li>Make incisions using a scalpel through the rib cage to expose the heart and then pass a 22-gauge blunt perfusion needle to the left ventricle. Make a small incision in the right atrium. Transcardially perfuse it with 50 mL of cold phosphate-buffered saline (PBS) followed by 50 mL of cold 4% paraformaldehyde (PFA) in 1x PBS (pH 7.4). 
	NOTE: Switch the PBS to the PFA when the mouse blood in vivo is replaced and the fluid runs clear. A good perfusion can be judged by the clearing of the liver. The perfusion should be stopped once fixation tremors have been observed30.</li>
	<li>Decapitate the mouse with ophthalmic scissors, make an incision on the skin and expose the skull. Using forceps, chip off the skull slightly and carefully dissect the whole mouse brain. Post-&#64257;x the brain in 4% paraformaldehyde for an additional 2 days and transfer it to a 30% sucrose solution at 4 &#176;C overnight.</li>
	<li>Using a cryostat, cut the brains into 20-&#181;m coronal sections. Transfer and mount the sections to the glass slide with a brush and store them at -20 &#176;C. 
	NOTE: Animals with electrode misplacements or excessive mechanical damage will be excluded from the subsequent analysis.</li>
	<li>Transfer the slides into normal PBS (0.1 M, pH 7.4) containing no fixative. Wash the slices with PBS for 3 - 4x for 5 min each to remove any excess fixative.For the BrdU-labeled group, incubate the sections in 2-N HCl for 30 min at 37 &#176;C, and rinse them in a 0.1-M borate buffer (pH 8.4; 10 min). Then wash the slices 2x for 5 min in PBS. 
	NOTE: Immunostaining is then performed.</li>
	<li>Treat slices by incubation in blocking solution (1 % normal goat serum, 3 % bovine serum albumin &#60;BSA&#62; , 0.5 % Triton X-100 and 0.02 % sodium azide in 1 x PBS) for 1 h at room temperature (RT).</li>
	<li>Incubate the slices with anti-c-fos polyclonal antibody (1:500; rabbit), anti-Notch1 antibody (1:50; goat), or anti-BrdU antibody (1:800; rat) in the blocking solution at 4 &#176;C overnight.</li>
	<li>Wash the slices 3x in PBS, then incubate them with Alexa Fluor 568-conjugated anti-rabbit (1:500), Alexa Fluor 488-conjugated anti-goat (1:1,000), or Alexa Fluor 488-conjugated anti-rat (1:500) secondary antibody for 1 h at RT.</li>
	<li>Wash the samples 3x in PBS (10 min each time). Cover the immuno-labeled brain sections using an anti-fade reagent mounting medium containing DAPI. Let the slices air-dry and store them protected from light at 4 &#176;C.</li>
	<li>Acquire images of the hippocampal areas from the stimulated and unstimulated sides of the brain with a high-resolution multi-channel (sequential) scanning confocal microscope. Set the imaging parameters consistently between the control and experimental groups.</li>
	<li>Perform a mosaic imaging using a high-magnification objective (20X air objective, NA 0.45) to acquire continuous 3D (XYZ) images of adjacent fields of view using the motorized stage and utilize appropriate software to stitch them together to make large composite images. 
	NOTE: Tiling functions include true stitching and smoothing options for improved seamless images. Composite images are quickly and easily prepared using the stitching function, to form an image over a wide area.</li>
	<li>For c-fos and Notch1 staining, perform a positive nuclei quantitative analysis of the immunofluorescent density21 of the stitched images. Randomly select 3 areas in the rostral, dorsal, and ventral hippocampal DG subregions and measure the immunofluorescent density in a double-blinded manner using Image J software (http://rsb.info.nih.gov/ij/)31.</li>
	<li>For a BrdU labeling, analyze the slices around the drilling point through the dorsal hippocampus (-1.7 mm to -2.7 mm relative to the bregma) in a double-blinded manner.</li>
	<li>Count only BrdU-positive neurons in the DG. Include the cells lying within 2-cell diameters of the granule cell in the count. 
	NOTE: Here, counting was performed using a 40X air objective. Randomly, 3 - 5 sections per animal were counted, and the counts were averaged and expressed as means per DG19.</li>
</ol>

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(94), e52220 (2014).</li><li>Pizzolato, G., Mandat, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+brain+stimulation+for+movement+disorders.">Deep brain stimulation for movement disorders.</a> Frontiers in Integrative Neuroscience. 6, 2 (2012).</li><li>Tierney, T. S., Sankar, T., Lozano, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+recent+trends+and+further+promising+directions+in+functional+neurosurgery.">Some recent trends and further promising directions in functional neurosurgery.</a> Acta Neurochirurgica Supplement. 117 (117), 87-92 (2013).</li><li>Laxton, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+trial+of+deep+brain+stimulation+of+memory+circuits+in+Alzheimer's+disease.">A phase I trial of deep brain stimulation of memory circuits in Alzheimer's disease.</a> Annals of Neurology. 68 (4), 521-534 (2010).</li><li>Min, H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+brain+stimulation+induces+BOLD+activation+in+motor+and+non-motor+networks:+an+fMRI+comparison+study+of+STN+and+EN/GPi+DBS+in+large+animals.">Deep brain stimulation induces BOLD activation in motor and non-motor networks: an fMRI comparison study of STN and EN/GPi DBS in large animals.</a> NeuroImage. 63 (3), 1408-1420 (2012).</li><li>Kukurba, K. R., Montgomery, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RNA sequencing and analysis. 2015 (11), 951-969 (2015).</li><li>Kawashima, T., Okuno, H., Bito, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+era+for+functional+labeling+of+neurons:+activity-dependent+promoters+have+come+of+age.">A new era for functional labeling of neurons: activity-dependent promoters have come of age.</a> Frontiers in Neural Circuits. 8, 37 (2014).</li><li>Liu, J., Solway, K., Messing, R. O., Sharp, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+neurogenesis+in+the+dentate+gyrus+after+transient+global+ischemia+in+gerbils.">Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils.</a> Journal of Neuroscience. 18 (19), 7768-7778 (1998).</li><li>Kuhn, H. G., Dickinson-Anson, H., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+the+dentate+gyrus+of+the+adult+rat:+age-related+decrease+of+neuronal+progenitor+proliferation.">Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation.</a> Journal of Neuroscience. 16 (6), 2027-2033 (1996).</li></ol>

The authors have nothing to declare.

The HF-DBS technique has been widely used as a powerful tool for the treatment of many neurological disorders since the 1990s. So far, the landmark work of HF-DBS is for the treatment of Parkinson&#39;s disease and essential tremor, which has attracted much attention and interest both in the clinic and scientific community. There are various types of ongoing HF-DBS studies by many groups for HF-DBS&#39;s therapeutic application in certain neurological and psychiatric disorders32,<sup cl...

HF-DBS is a neurosurgical technology for electrical stimulation in the brain, which has been developed since the 1870s1. In the late 1980s, HFS was first used as a potential therapeutic intervention for Parkinson&#39;s disease and other movement disorders2. In the past few decades, HF-DBS has been more and more widely used in the treatment of brain disorders which are currently untreatable by a traditional therapeutic strategy. Particularly, due to the accuracy improvement of the HFS electrode, the highly effective outcomes, and minimal side effects, the number of brain disorders treated by HF-DBS has significantly increased over the past decades3,4,5. For example, HF-DBS has been approved by the US Food and Drug Administration (FDA) for the treatment of Parkinson&#39;s disease (PD), Alzheimer&#39;s type dementia, essential tremor, and other types of movement disorders2,6,7. In PD patients, the dopaminergic medication is reduced up to 50% during HF-DBS8. In addition to the successful treatment of movement disorders, HF-DBS has also demonstrated its powerful effects in the treatment of psychiatric diseases in the clinic, and for cognitive augmentation as well2,9,10,11. It should be noted that the research of HFS for the treatment of other psychiatric disorders are in various stages, offering much promise to patients12.
Although many studies have demonstrated that a focal HFS has both local and remote effects throughout the brain13, the neurological and molecular mechanisms of the effects remain elusive2,14. In the clinic, therapeutic HF-DBS is usually applied in a long-term manner for the treatment of Parkinson&#39;s disease and chronic pain, etc. Many opinions are raised to explain the improvement generated by an HF-DBS treatment, among which one possibility that the HFS current modulates the neuronal network activity, probably by a repetitive depolarization of the axons in the vicinity of the implanted HFS electrode. Or, HF-DBS may change the discharge rate of the output neurons and the projected targets. Also, HF-DBS may lead to long-term synaptic changes, including long-term potentiation (LTP) and long-term depression (LTD), which may contribute to a symptomatic improvement. So far, it is still unclear whether HFS in&#64258;uences the key molecular events that regulate cellular processes such as adult neurogenesis in vivo. Several lines of studies have demonstrated that HFS in rodents could mimic similar neural responses of clinically applied DBS15,16. To understand the underlying cellular mechanisms of HF-DBS, in this study, we first set up an in vivo HFS methodology in mice in an acute (one day) or chronic (five days) manner. Secondly, we set up an activation analysis methodology to determine the alteration of the neuronal activity and neurogenesis after an HF-DBS delivery.
Given that the neuronal production from neural stem cells is abundant during the embryonic development but continues throughout adult life, the hippocampal subgranular zone is one of the major areas where the neurogenesis occurs. The process of neurogenesis is influenced by many physiological and pathological factors. In certain epileptic cases, the hippocampal neurogenesis is dramatically decreased17,18. In addition, a single electroconvulsive therapy could significantly increase the neuronal production in the dentate gyrus19. These observations suggest that the electrophysiological activity plays a critical role in the regulation of adult neurogenesis and synaptic plasticity in hippocampal neurons. Therefore, to further demonstrate the effects of HF-DBS on neuronal activity and neurogenesis, we first carry out an immunostaining assay of the immediate early gene (IEG) c-fos which is a well-known marker of short-term neuronal activity resulting from experience20. Notch1 signaling is also detected to monitor the signaling activation after the HFS delivery21,22. Moreover, we also detect the neuronal production by a BrdU labeling analysis after the HF-DBS induction in various manners, though BrdU staining can also be a marker for gliogenesis.
In the present study, two HFS methodologies are adapted to target the activation of the hippocampal DG directly and indirectly. The electrode is implanted into the DG directly or implanted into the medial perforant path (PP) which sends projections to activate the DG neurons. For the HF-DBS induction, a programmable stimulator is presented for a continuous stimulation via the fixed electrode onto the mouse head. To determine the effects of HFS on neuronal activation and neurogenesis, we detect the expression of c-fos and Notch1 by immunofluorescent staining and the number of BrdU-incorporated positive neurons in the hippocampal DG region, respectively, after the HFS treatment. Particularly, the effects of the HF-DBS on the neurogenesis in the DG are compared between an acute and a chronic stimulation manner, or between a direct and an indirect stimulation manner, respectively.

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			<td height="26" style="height:26px;width:255px;">Brain stereotaxic instrument</td>
			<td style="width:215px;">Stoelting</td>
			<td style="width:199px;">51730D</td>
			<td style="width:307px;">Stereotactic intracranial implantation for mouse</td>
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		<tr height="24">
			<td height="24" style="height:24px;width:255px;">Stimulator</td>
			<td style="width:215px;">A-M systems</td>
			<td style="width:199px;">Model 3800</td>
			<td>MultiStim 8-Channel programmable stimulator</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:255px;">Dental driller</td>
			<td style="width:215px;">Saeshin Precision Co., Ltd</td>
			<td style="width:199px;">STRONG 90</td>
			<td>For drilling and crainiotomy&#160;</td>
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		<tr height="24">
			<td height="24" style="height:24px;width:255px;">Burr</td>
			<td style="width:215px;">Meisinger</td>
			<td style="width:199px;">HM1 005#</td>
			<td>For drilling and crainiotomy&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:255px;">Digidata 1550 Digitizer</td>
			<td style="width:215px;">Molecular Devices</td>
			<td style="width:199px;">AXON 1550</td>
			<td>High-resolution data acquisition</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Cryotome</td>
			<td style="width:215px;">Thermo Fisher Scientific</td>
			<td style="width:199px;">Thermo Cryotome FSE</td>
			<td>Cutting frozen sections of specimens</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Confocal microscope</td>
			<td style="width:215px;">Olympus</td>
			<td style="width:199px;">FV-1200</td>
			<td>Japan, with 20x Objective (NA 0.45)</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:255px;">Mouse surgery tools</td>
			<td style="width:215px;">F.S.T.</td>
			<td style="width:199px;">14084-08,11254-20,16109-14</td>
			<td style="width:307px;">Scissors, forceps, bone cutter, holders etc.</td>
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			<td height="21" style="height:21px;width:255px;">Pentobarbital sodium</td>
			<td style="width:215px;">R&#38;D systems</td>
			<td style="width:199px;">4579</td>
			<td>20-50mg/kg for i.p. injection</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Penicillin G&#160;</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:199px;">P3032</td>
			<td>75,000 U for i.m. injection</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Carprofen</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:199px;">SML1713</td>
			<td>5-10mg/kg, for s.c. injection</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">4% Paraformaldehyde (PFA)</td>
			<td style="width:215px;">Beijing Solarbio Sci-Tech Co.&#160;</td>
			<td style="width:199px;">P1110</td>
			<td>stocking solution for tissue fixation</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Phosphate buffer (PBS)</td>
			<td style="width:215px;">Invitrogen</td>
			<td style="width:199px;">10010023</td>
			<td>pH7.4, 500ml in stocking</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:215px;">Sakura</td>
			<td style="width:199px;">4583</td>
			<td>Formulation of water-soluble glycols and resins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">anti-BrdU antibody</td>
			<td style="width:215px;">Abcam</td>
			<td style="width:199px;">ab6326</td>
			<td>Dilutions:1/800</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">anti-c-fos antibody</td>
			<td style="width:215px;">Abcam</td>
			<td style="width:199px;">ab209794</td>
			<td>Dilutions:1/500</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:255px;">Goat Anti-Rabbit IgG (Alexa Fluor 568)</td>
			<td style="width:215px;">Thermo Fisher Scientific</td>
			<td style="width:199px;">A11036</td>
			<td>Dilutions:1/500</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Donkey Anti-Rat IgG (Alexa Fluor 488)</td>
			<td style="width:215px;">Jackson ImmunoResearch</td>
			<td style="width:199px;">712-546-150</td>
			<td>Dilutions:1/500</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:255px;">Antifade mounting medium with DAPI</td>
			<td style="width:215px;">Vector Laboratories</td>
			<td style="width:199px;">H-1200</td>
			<td>Counterstaining with DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">anti-Notch1 antibody (C-20)</td>
			<td style="width:215px;">Santa Cruz Biotech</td>
			<td style="width:199px;">sc-6014</td>
			<td>Dilutions:1/50</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:255px;">Donkey Anti-Goat IgG (Alexa Fluor 488)</td>
			<td style="width:215px;">Abcam</td>
			<td style="width:199px;">ab150073</td>
			<td>Dilutions:1/1000</td>
		</tr>
	</tbody>
</table>

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.

Following the HF-DBS stimulation to the hippocampal DG subregion directly or the PP subregion to activate the DG indirectly via inserted electrodes using the stereotactic adjustments, the rodents were anesthetized with pentobarbital and sampled 3 h after the last HF-DBS stimulation for the c-fos and Notch1 immunostaining. For the BrdU staining, 36 h after the last BrdU injection after 1 day or 5 days of HF-DBS stimulation, the rodents were anesthetized with pentobarbital...

Watch this Scientific Journal Video about An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation at JoVE.com

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

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 ...

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Research

JoVE Journal

Neuroscience

7.6K Views.  Beijing Institute of Basic Medical Sciences. 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.

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

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A protocol is provided to generate a model for studying CA1 &#947; oscillations (20 - 80 Hz). These &#947; oscillations are stable for at least 30 min&#160;and depend upon excitatory and inhibitory synaptic activity in addition to activation of pacemaker currents. Tetanically stimulated oscillations have a number of reproducible and easily quantifiable characteristics including spike count, oscillation duration, latency and frequency that report upon the network state. The advantages of the electrically stimulated oscillations include stability, reproducibility and episodic acquisition enabling robust characterization of network function. This model of CA1 &#947; oscillations can be used to study cellular mechanisms and to systematically investigate how neuronal network activity is altered in disease and by drugs. Disease state pharmacology can be readily incorporated by the use of brain slices from genetically modified or interventional animal models to enable selection of drugs that specifically target disease mechanisms.

Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 &#947; oscillations.

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. Through such combinatorial coding, organisms can detect and discriminate between a myriad of volatile odor molecules. Thus, an odor at a given concentration can be described by an activation pattern of ORs, which is specific to each odor. In that sense, cracking the mechanisms that the brain uses to perceive odor requires the understanding odorant-OR interactions. This is why the olfaction community is committed to &#34;de-orphanize&#34;&#160;these receptors. Conventional in vitro systems used to identify odorant-OR interactions have utilized incubating cell media with odorant, which is distinct from the natural detection of odors via vapor odorants dissolution into nasal mucosa before interacting with ORs. Here, we describe a new method that allows for real-time monitoring of OR activation via vapor-phase odorants. Our method relies on measuring cAMP release by luminescence using the Glosensor assay. It bridges current gaps between in vivo and in vitro approaches and provides a basis for a biomimetic volatile chemical sensor.

Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase.

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.

Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...

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

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Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Generation of Local CA1 γ Oscillations by Tetanic Stimulation

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation