Name: analysis of gene expression changes in the rat hippocampus after deep brain stimulation of the anterior thalamic nucleus
Uploaded: 2015-03-08T15:00:00.000+00:00
Duration: 9 min 46 s
Description: Watch this Scientific Journal Video about Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus at JoVE.com

We are grateful for the support of the NREF foundation.

NOTE: Ethics Statement: All procedures discussed in this manuscript are in accordance with the NIH guidelines for Animal Research (Guide for the Care and Use of Laboratory Animals) and are approved by the Harvard Medical School IACUC Committee.
1. Pre-surgical Preparation
<ol>
	<li>Make sure that all surgical instruments are sterilized by either autoclaving or by cleansing with antiseptic solution and/or ethanol as necessary. Where possible, use sterile disposable equipment such as scalpels, needle and syringes.</li>
	<li>Cover the workbench with surgical drapes and ensure that there is a biohazard waste disposal available.</li>
	<li>Weigh the rat and calculate anesthesia dose. Use a Ketamine/Xylazine mix (Ketamine 75 mg/kg and Xylazine 10 mg/kg) to anesthetize the rats. 
	NOTE: Between 200-250 g is the optimal weight for proper fixing of the animal in the stereotactic frame as well as for accurate targeting of the ATN. Isoflurane can also be used as the anesthetizing agent.</li>
	<li>Mount and secure two sterile electrodes (sterilized by autoclaving or with ethylene oxide) on the electrode holder &#160;(Figure 2) of a stereotactic surgical frame and with the help of a microscope (10-40X magnification), inspect the tips of the electrode for proper alignment. Secure the two electrodes 3.0 mm apart. 
	NOTE: Take care to avoid damaging the electrode tip by touching on hard surfaces.</li>
</ol>
2. DBS Surgery
<ol>
	<li>Inject the Ketamine/Xylazine mix intraperitoneally to the animal and confirm that the animal has reached a surgical plane of anesthesia (by checking for the toe-pinch reflex, respiratory rate and depth and regularity of breathing). Remove hair from the region of the scalp that will be incised and disinfect the scalp with 3 alternating scrubs each of betadine and either alcohol or sterile saline. Secure and position the animal on the stereotactic frame.</li>
	<li>Use circulating warm water pads&#160;to maintain the animal&#8217;s body temperature at an optimal level.&#160;Apply eye lubricant to the animal&#8217;s eyes to protect from overdrying.&#160;</li>
	<li>Use the following stereotactic coordinates for targeting the ATN (Anterior thalamic nucleus): anterioposterior -1.6 mm, mediolateral 1.5 mm and dorsoventral 5.2 mm1. 
	NOTE: The stereotactic coordinates are based on the Paxinos and Watson (6th edition) rat brain atlas36.</li>
	<li>Make an incision in the scalp sagittally to reveal the skull. Using a pair of retractors secure the incised scalp to expose the skull. Use sterile saline to flush the incision. If incision is too wet, use sterile cotton swabs to dry.&#160;&#160;Locate the bregma and mark with a black marker. To guide the position of the burr holes, make two more marks at approximately 1.5 mm mediolaterally on both sides from the sagittal suture and 1.6 mm posterior to the coronal suture.</li>
	<li>Use a hand-held drill to make the burr holes. Make sure the tip of the burr hole is sterile by sterilizing it with ethanol. Hold the drill at about a 45&#176; angle to the skull surface when drilling. Frequently switch between the two burr holes to avoid excessive heat at the location of any burr hole.</li>
	<li>Continue drilling until the dura is exposed. Using a needle with its tip bent resembling the shape of an &#8216;L&#8217;, remove any broken pieces of bone that would obstruct the insertion of the electrode. Take care to avoid damaging the underlying dura and/or brain tissue while removing bone fragments using the bent needle. 
	NOTE: Using a blunted needle or fine blunt forceps is also an option.</li>
	<li>Fix the dual electrode assembly to the rotating handle of the stereotactic frame and fix the handle at a 90&#176; angle. Using the adjustments in the stereotactic frame, position the left electrode exactly above the bregma.</li>
	<li>Using the stereotactic adjustments for mediolateral positioning, precisely move the left electrode 1.5 mm to the left side of bregma such that now there are two electrodes perfectly aligned along the coronal suture but spaced apart at 1.5 mm mediolaterally from the bregma.</li>
	<li>Using the anterioposterior stereotactic adjustments, move the electrodes 1.6 mm posterior to the coronal suture.</li>
	<li>Use the dorsoventral adjustments to lower the electrodes to first check if the burr holes have been made at the right location such that the electrodes can be inserted with ease, without touching the rough edges of the burr holes. If so, insert the electrodes to a depth of 5.2 mm from the surface of the skull.</li>
	<li>Connect the electrodes via leads to a stimulator set at 130 Hz, 2.5 V and 90 &#181;sec pulsewidth1.</li>
	<li>Deliver high frequency stimulation for an hour (or for a desired period of time as per experimental setup). During the course of the stimulation, remember to assure proper anesthetic depth regularly by checking for the absence of foot withdrawal in response to a toe pinch. Supplement anesthesia with approximately half the initial dose used to induce anesthesia. Avoid contamination of your sterile gloves when checking anesthetic depth and change them if necessary.&#160;&#160;Perform unilateral or bilateral stimulation based on one&#8217;s experimental needs. Include controls such as low frequency stimulation (for e.g., 10 Hz) and unstimulated animals (inserting electrodes with no subsequent stimulation).</li>
	<li>After stimulation is done, remove the electrodes carefully and suture the incision with 3-0 sutures or with sterile surgical staples.</li>
	<li>Administer buprenorphine (0.05 mg/kg) subcutaneously as analgesia. Monitor the animal until it returns to normal activity and then return it to the housing facility.</li>
	<li>After a set period of time based on the experimental design (for e.g., 0, 3, 6 or 12 hr post DBS surgery), euthanize the animal with anesthesia overdose. After confirming the absence of vital signs, decapitate the animal.</li>
	<li>Dissect out the brain by first removing the skin using scissors. Cut through the bone along the sagittal suture using dissection scissors. Make two more incisions (about an inch each) through the bone on both the lateral sides. Using forceps, lift the partially severed piece of bone from the top of the skull to expose the brain.</li>
	<li>Using fine scissors or forceps, dislodge the brain from the skull and transfer to a Petri dish with cold PBS on ice. 
	NOTE: Take care to avoid damage to the brain while making the incisions through the bone.</li>
</ol>
3. Hippocampus Isolation
NOTE: Perform all the subsequent steps in this section on ice.
<ol>
	<li>Place the brain on a pre-cooled acrylic brain matrix on ice. Using a razor blade, cut the brain coronally at approximately 7-8 mm from the anterior-most edge of the brain. Make a second cut coronally and posterior to the first cut such that an approximately 5 mm thick brain slice could be removed.</li>
	<li>Transfer the brain slice to a Petri dish with ice-cold PBS. Using razor blade, sever the two hemispherical sections and take care to note which hemisphere corresponds to the left and right sides respectively. This is especially important while performing unilateral stimulations.</li>
	<li>Using fine forceps and scissors remove the hippocampus carefully.</li>
	<li>Flash freeze the hippocampal tissue on dry ice and store in -20 &#176;C freezer until ready for the subsequent RNA extraction steps. 
	NOTE: For long term storage, it is advisable to store tissue in a -80 &#176;C freezer.</li>
</ol>
4. RNA Extraction and Quantitative PCR
<ol>
	<li>Make sure the tissue stays frozen on dry ice until ready for homogenization.</li>
	<li>NOTE: Perform this step in the hood.
	<ol>
		<li>Add 1 ml of Tri reagent to hippocampal tissue in a 1.5 ml centrifuge tube and homogenize it by first pipetting multiple times until the tissue is broken in smaller pieces. Further homogenize the tissue by passing through a syringe with a 25 G needle until there is no unbroken tissue visible. Perform the homogenization steps on ice.</li>
		<li>After homogenizing the tissue, allow the cell suspension to stand at room temperature for 5 min to allow cell lysis. 
		NOTE: After this point the cell suspension can be quick frozen and stored in -70 &#176;C until further processing.</li>
	</ol>
	</li>
	<li>Add 0.2 ml chloroform and mix by vortexing for 20 sec and let the solution rest at room temperature for 15 min.</li>
	<li>Centrifuge the samples at 12,000 x g for 15 min at 4 &#176;C. After centrifugation, remove the tubes carefully without disturbing the three separate layers that will be visible. 
	NOTE: The bottom (red) phase contains proteins, middle cloudy phase contains DNA and the top clear phase contains RNA.</li>
	<li>Transfer the top clear phase (RNA) into a fresh 1.5 ml centrifuge tube (typically 500 &#181;l of the RNA containing phase is obtained). To this add 0.5 ml isopropanol and mix by vortexing. To this add 2-3 &#181;l glycogen (20 mg/ml) as the carrier for the RNA. Allow the sample to rest on the table at room temperature for 10 min.</li>
	<li>Centrifuge the sample at 12,000 x g for 1 hour at 4 &#176;C. Make sure that the RNA pellet is visible at the bottom of the tube.</li>
	<li>Discard the supernatant and wash the RNA pellet by adding 1 ml of 75% ethanol (made with nuclease free water). Invert the sample multiple times until the pellet dislodges from the bottom of the tube and floats in the solution. Centrifuge the solution at 12,000 x g for 10 min at 4 &#176;C. 
	NOTE: The pellet in 75% ethanol can be stored at -20 &#176;C until further steps.</li>
	<li>Allow the RNA pellet to air dry by leaving the tubes open for 5 min at room temperature. Take precaution to avoid over drying the pellet as this might affect its dissolution in water in the subsequent step.</li>
</ol>
5. Removing DNA from the RNA Preparation
<ol>
	<li>Add 9 &#181;l of nuclease free water to the RNA pellet and make sure the pellet is dissolved before proceeding by gently vortexing the tube. To this add 1 &#181;l of 10x Dnase I Buffer and 1 &#181;l recombinant DNase (from DNase I kit), mix by gently flicking the tubes, briefly spin the tubes and incubate at 37 &#176;C in a water bath for 30 min.</li>
	<li>Add 2 &#181;l DNase inactivation reagent (from DNase I kit) to the sample and incubate at room temperature for 2 min and mix often by gently flicking the tube. Centrifuge at 12,000 x g for 10 min and transfer approximately 10 &#181;l of the clear supernatant to a fresh 1.5 ml centrifuge tube.</li>
	<li>Measure RNA concentration using a nanodrop or spectrophotometer.</li>
</ol>
6. Making cDNA from RNA
<ol>
	<li>Add requisite volume that contains 1 &#181;g of RNA and bring the total volume to 8 &#181;l by adding nuclease free water. To this add 1 &#181;l 10 mM dNTP mix and 1 &#181;l of random hexamers from the Superscript First Strand Synthesis Kit. Gently mix and incubate at 65 &#176;C for 5 min in either a water bath or on a pre-programmed thermocycler.</li>
	<li>Make a premix containing the following reagents: 2 &#181;l of 10x RT Buffer, 4 &#181;l of 25 M MgCl2, 2 &#181;l of 0.1 M DTT and 1 &#181;l of RNAse OUT (40 U/&#181;l). 
	NOTE: Volumes given are per reaction, user would need to scale up according to one&#8217;s experimental needs.</li>
	<li>After removing the RNA containing sample from 65 &#176;C, set the tube at room temperature for a minute. Add 9 &#181;l premix solution and incubate at room temperature for 2 min. Add 1 &#181;l of Superscript II reverse transcriptase enzyme (50 U/&#181;l) and then incubate at room temperature for 10 min.</li>
	<li>Incubate the samples at 42 &#176;C for 50 min for the reverse transcription to occur.</li>
	<li>Incubate the samples at 70 &#176;C for 15 min to terminate the reaction.</li>
	<li>Place the samples on ice briefly and add 1 &#181;l RNAseH (2 U/&#181;l) and incubate at 37 &#176;C for 20 min.</li>
	<li>Briefly centrifuge the tubes at 3,000 x g to spin down the condensate liquid. 
	NOTE: This is the cDNA that will be used for quantitative PCR. cDNA can be stored in -20 &#176;C freezer until PCR setup. cDNA can also be diluted with nuclease free water before proceeding to PCR.</li>
</ol>
7. Quantitative PCR
<ol>
	<li>Make a master mix containing the following reagents (volumes given are per reaction):6.3 &#181;l of 2x Sybr Green Mix, 0.6 &#181;l of Forward Primer (10 &#181;M), 0.6 &#181;l of Reverse Primer (10 &#181;M) and 0.5 &#181;l of Nuclease free water. 
	NOTE: Primer design was done using the &#8216;Pick Primers&#8217; option in NCBI&#8217;s nucleotide sequence page for the gene of interest.</li>
	<li>Add 10 &#181;l mastermix to each well of a 96 well- PCR Plate. Add 2.5 &#181;l of cDNA made earlier to the well containing the mastermix. Make sure to set up triplicate PCR reactions for each sample.</li>
	<li>After completing the addition of mastermix and cDNA, cover the PCR plate using an optical adhesive sheet to seal the wells. Spin the plate briefly for 5 min at 500 x g in a tabletop centrifuge to settle all the liquid to the bottom of the well.</li>
	<li>Load the PCR plate onto a pre-programmed RT-PCR machine. Use the PCR parameters provided in Table 1.</li>
	<li>Data analysis: Use the Ct values from the qPCR output for further calculations. Along with the test gene, set up PCR reactions for internal control genes, 18S rRNA (to ensure equal input) and &#946;-actin (negative control). Calculate fold changes based on &#916;&#916;Ct method 37.</li>
</ol>

The authors have no disclosures.

Following the landmark work by Benabid et al. in using deep brain stimulation to treat Parkinson&#8217;s disease and essential tremor, the DBS surgical technique has been investigated with much interest over the past decade to treat many neurological disorders6,10,43. DBS studies targeting various neuro-anatomical regions of the brain circuitry are currently performed by many groups to address major neuronal diseases and are in various stages of clinical trials. Stimulation of the subthalamic nucleus ...

The history behind the development of Deep Brain Stimulation as a neurosurgical technique dates back to the 1870s when the possibility of electrically stimulating the brain circuitry was explored2. The use of chronic high-frequency stimulation as treatment for neuronal disorders started in the 1960s3. Later in the 1990s with the advent of chronic implantation DBS electrodes4-6, the number of neuronal disorders that were treated by DBS continued to increase. Deep Brain Stimulation was first used in the United States as a treatment for essential tremor6. Today the surgery is used widely to treat neuronal disorders that are currently untreatable by pharmacological intervention. DBS is currently used to treat movement disorders of Parkinson&#8217;s disease and dystonia7-9. Alzheimer&#8217;s type dementia, Huntington&#8217;s disease, epilepsy, pain and neuropsychiatric diseases such depression, OCD, Tourette&#8217;s syndrome and addiction are some of the conditions amenable to treatment by DBS10-12. While DBS surgery is FDA approved for treating Parkinson&#8217;s disease, dystonia and essential tremor, the use of DBS for treating other conditions mentioned above are in various stages of lab and clinical studies offering much promise to patients13,14.
Clinically, DBS surgery is performed in two stages. The first stage involves surgically positioning the DBS electrodes at the targeted anatomical location using a combination of radiological positioning, CT, MRI as well as microelectrode readings for enhanced precision. The second stage involves implanting a pulse generator in the patient&#8217;s upper chest and installing extension leads from the scalp to the pulse generator. Based on the neurological condition, several programming schemes for the pulse generator have been standardized and will be used to deliver the desired voltage. The final voltage is reached in a stepwise fashion so as to receive the best clinical response with minimal voltage15. However, in our studies, unlike the chronic DBS implants used clinically, for the sake of simplicity, we have resorted to studying a one-time high frequency stimulation (for 1 hr) in our animal models.
Part of our group&#8217;s research focuses on investigating the use of DBS surgery for treatment-refractory epilepsy. Stereotactic surgical approaches using high frequency stimulation has been explored by many others as an effective option to treat medically-refractory epilepsy which constitutes about 30% of all incidences of epilepsy10,16,17. Cerebellar stimulation targeting the cortical surface as well as the deep cerebellar nuclei have been used in the past as targets to treat epilepsy10,18,19. In addition, hippocampus stimulation has also been tried but with mixed results20,21. Some of the other investigated DBS targets for epilepsy include the cerebral cortex, thalamus, subthalamic nucleus and vagus nerve8. However, following results from several studies in the past few years, the anterior thalamic nucleus (ATN) has emerged as the most common DBS target for epilepsy treatment10,22. Based on knowledge about neuroanatomical circuitry and findings from animal models, several studies have focused on the therapeutic effect of deep brain stimulation of the ATN in treating epilepsy 23-26. The ATN is part of the limbic circuit and is located in the region of the brain that affects seizure frequency. Studies by Hamani et al., have tested the efficacy of ATN-DBS in a pilocarpine induced epilepsy model and found that bilateral ATN stimulation prolonged latencies for pilocarpine-induced seizures and status epilepticus24. Furthermore, high frequency stimulation of the ATN was found to reduce seizure frequency in a pentylenetetrazol (PTZ) model of epilepsy25,27-29. Lee et al., have reported a mean reduction in seizure frequency by about 75% upon chronic deep brain stimulation of the ATN in treating refractory partial epilepsy30.
A recent clinical study on treatment-refractory epilepsy has shown promising results after DBS surgery targeting the anterior thalamic nucleus (ATN)22. A multicenter randomized clinical trial with 110 patients undergoing bilateral DBS of the ATN for treatment refractory epilepsy (SANTE trial) indicated a drop in seizure frequency by approximately 40%31. The results from this study also hinted on a delayed optimal anti-epileptic effect observed at 2-3 months post surgery. Further studies by Toda et al., corroborated with these findings where they demonstrated neurogenesis happening at a later time post DBS (days 3-5) in animal models1. In addition, Encinas et al., have reported hippocampal neurogenesis in the adult mouse dentate gyrus after high frequency stimulation of the ATN32. Previous studies33-35 have reported declining hippocampal neurogenesis in certain epileptic cases such as chronic temporal lobe epilepsy and an association with learning deficits, memory impairment and spontaneous recurrent motor seizures. Furthermore, there was a reduction in neural stem cell progenitor factors such as FGF2 and IGF-1 in the chronically epileptic hippocampus in animal models 33. Considering this, interventional strategies such as DBS that show an augmentation of neurogenesis in the dentate gyrus are exciting avenues for research. These findings have encouraged us to explore further deeply into the mechanism underlying neurogenesis post-DBS treatment for epilepsy. We have targeted the ATN both unilaterally (data not reported) as well as bilaterally (in representative results) and seen elevated neurotrophin (BDNF) expression in the rat dentate gyrus. Our current hypothesis is that BDNF expression initiates a gene expression cascade that culminates in neurogenesis that translates to the anti-epileptic effect of DBS surgery. In this paper, we present our methods for DBS surgery targeting the ATN in rats followed by gene expression analysis as an attractive approach to study the mechanism underlying the benefits of DBS.

<table><tbody><tr><td>Deep Brain Stimulation Surgery</td><td></td><td></td><td></td></tr><tr><td>Stereotactic frame</td><td>Kopf Instruments</td><td>Model 900</td><td></td></tr><tr><td>Drill&amp;nbsp;</td><td>Dremmel</td><td>7700, 7.2 V</td><td></td></tr><tr><td>Scalpel</td><td>BD</td><td>372610</td><td></td></tr><tr><td>Ketamine</td><td>Patterson Veterinary</td><td>07-803-6637</td><td>Schedule III Controlled Substance, procurement, use and storage according to institutional rules</td></tr><tr><td>Xylazine</td><td>Patterson Veterinary</td><td>07-808-1947</td><td></td></tr><tr><td>Buprenorphine</td><td>Patterson Veterinary</td><td>07-850-2280</td><td>Schedule III Controlled Substance, procurement, use and storage according to institutional rules</td></tr><tr><td>Surgical staples</td><td>ConMed Corporation</td><td>8035</td><td></td></tr><tr><td>Sutures (3-0)&amp;nbsp;</td><td>Harvard Apparatus</td><td>72-3333</td><td></td></tr><tr><td>Syringe (1 ml, 29 1/2 G)</td><td>BD</td><td>329464</td><td>Sterile, use for Anesthesia administration intraperitoneally</td></tr><tr><td>Syringe (3 ml, 25 G)</td><td>BD</td><td>309570</td><td>Sterile, use for Analgesia administration subcutaneously</td></tr><tr><td>Needles</td><td>BD</td><td>305761</td><td>Sterile, use for clearing broken bone pieces from the burr holes</td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>S25309B</td><td>Use for general sterilization&amp;nbsp;</td></tr><tr><td>Eye Lubricant</td><td>Fisher Scientific</td><td>19-898-350</td><td></td></tr><tr><td>Stimulator</td><td>Medtronic</td><td>Model 3628</td><td></td></tr><tr><td>DBS electrodes</td><td>Rhodes Medical Instruments, CA</td><td>SNEX100x-100 mm</td><td>Electrodes are platinum, concentric and bipolar</td></tr><tr><td>Betadine (Povidone-Iodine)&amp;nbsp;</td><td>PDI</td><td>S23125</td><td>Single use swabsticks, use for sterilizing the scalp before making incision&amp;nbsp;</td></tr><tr><td>&amp;nbsp;Brain Dissection and Hippocampal tissue isolation</td><td></td><td></td><td></td></tr><tr><td>Acrylic Rodent Brain Matrix</td><td>Electron Microscopy Sciences</td><td>175-300</td><td>www.emsdiasum.com</td></tr><tr><td>Razor Blade</td><td>V W R</td><td>55411-050</td><td></td></tr><tr><td>Guillotine Scissors</td><td>Clauss</td><td>18039</td><td>For decapitation, make sure these scissors are maintained in clean and working condition</td></tr><tr><td>Scissors</td><td>Codman Classic</td><td>34-4098</td><td>Use for removing the brain from the skull</td></tr><tr><td>Forceps</td><td>Electron Microscopy Sciences</td><td>72957-06</td><td>Use for removing the brain from the skull and for handling during dissection</td></tr><tr><td>Phosphate Buffered Saline&amp;nbsp;</td><td>Boston Bioproducts</td><td>BM-220</td><td></td></tr><tr><td>&amp;nbsp;RNA Extraction and cDNA Preparation</td><td></td><td></td><td></td></tr><tr><td>Tri Reagent</td><td>Sigma</td><td>T9424</td><td>Always use in a fume hood and wear protective goggles while handling; avoid contact with skin</td></tr><tr><td>Syringe (3 ml, 25 G)</td><td>BD</td><td>309570</td><td>Use for tissue homogenization</td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>BP1145-1</td><td>Always use in a fume hood and wear protective goggles while handling; avoid contact with skin</td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>A416-1</td><td></td></tr><tr><td>Glycogen</td><td>Thermo Scientific</td><td>R0561</td><td></td></tr><tr><td>Dnase I Kit</td><td>Ambion</td><td>AM1906</td><td></td></tr><tr><td>Superscript First Strand Synthesis Kit</td><td>Invitrogen</td><td>11904-018</td><td></td></tr><tr><td>Tabletop Microcentrifuge</td><td>Eppendorf</td><td>5415D</td><td></td></tr><tr><td>&amp;nbsp;Quantitative PCR</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>SYBR Green PCR Kit</td><td>Qiagen</td><td>204143</td><td></td></tr><tr><td>Custom Oligos</td><td>Invitrogen</td><td>10668051</td><td></td></tr><tr><td>PCR Plates (96 wells)</td><td>Denville Scientific</td><td>C18080-10</td><td></td></tr><tr><td>Optical Adhesive Sheets</td><td>Thermo Scientific</td><td>AB1170</td><td></td></tr><tr><td>Nuclease free Water</td><td>Thermo Scientific</td><td>SH30538-02</td><td></td></tr><tr><td>Real Time PCR Machine</td><td>Applied Biosystems</td><td>7500</td><td></td></tr></tbody></table>

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.

Figures 1A and 1B show the relative expression of BDNF and GABRD relative to the control gene &#946;-actin. BDNF, a neurotrophin is often associated with neuroprotective effects in many neuronal diseases38-41. It is therefore interesting to analyze the expression profile of BDNF in response to stimulation of the ATN which yields therapeutic benefits to epileptic patients. In Figure 1A which shows the gene expression profile of BDNF across the indicated time-po...

Watch this Scientific Journal Video about Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus at JoVE.com

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

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 ...

analysis-gene-expression-changes-rat-hippocampus-after-deep-brain

Research

JoVE Journal

Neuroscience

10.9K Views.  Brigham & Women's Hospital, Harvard Medical School. 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.

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

Intra-Operative Behavioral Tasks in Awake Humans Undergoing Deep Brain Stimulation Surgery

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through implanted electrodes. Deep brain stimulation was first implemented as a therapeutic modality by Benabid et al. in the late 1980s, when he used this technique to stimulate the ventral intermediate nucleus of the thalamus for the treatment of tremor 1. Currently, the procedure is used to treat patients who fail to respond adequately to medical management for diseases such as Parkinson's, dystonia, and essential tremor. The efficacy of this procedure for the treatment of Parkinson's disease has been demonstrated in well-powered, randomized controlled trials 2. Presently, the U.S. Food and Drug Administration has approved DBS as a treatment for patients with medically refractory essential tremor, Parkinson's disease, and dystonia. Additionally, DBS is currently being evaluated for the treatment of other psychiatric and neurological disorders, such as obsessive compulsive disorder, major depressive disorder, and epilepsy. 
DBS has not only been shown to help people by improving their quality of life, it also provides researchers with the unique opportunity to study and understand the human brain. Microelectrode recordings are routinely performed during DBS surgery in order to enhance the precision of anatomical targeting. Firing patterns of individual neurons can therefore be recorded while the subject performs a behavioral task. Early studies using these data focused on descriptive aspects, including firing and burst rates, and frequency modulation 3. More recent studies have focused on cognitive aspects of behavior in relation to neuronal activity 4,5. This article will provide a description of the intra-operative methods used to perform behavioral tasks and record neuronal data with awake patients during DBS cases. Our exposition of the process of acquiring electrophysiological data will illuminate the current scope and limitations of intra-operative human experiments.

Deep brain stimulation surgery offers a unique opportunity to examine information encoding in the awake human brain. This article will describe intra-operative methods used to perform cognitive and behavioral tasks while simultaneously acquiring physiological data such as EMG, single-unit neuronal activity and/or local field potentials.

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through ...

Medicine

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

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, ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

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.

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 ...

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, ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

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 ...

Transcranial Direct Current Stimulation (tDCS) in Mice

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for several neuropsychiatric diseases. The biological effects of tDCS are not fully understood, which is in part explained due to the difficulty in obtaining human brain tissue. This protocol describes a tDCS mouse model that uses a chronically implanted electrode allowing the study of the long-lasting biological effects of tDCS. In this experimental model, tDCS changes the cortical gene expression and offers a prominent contribution to the understanding of the rationale for its therapeutic use.

Transcranial Direct Current Stimulation tDCS in Mice

Transcranial direct current stimulation (tDCS) is a therapeutic technique proposed to treat psychiatric diseases. An animal model is essential for understanding the specific biological alterations evoked by tDCS. This protocol describes a tDCS mouse model that uses a chronically implanted electrode.

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for ...

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

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Chapters in this video

Intra-Operative Behavioral Tasks in Awake Humans Undergoing Deep Brain Stimulation Surgery

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Deep Brain Stimulation with Simultaneous fMRI in Rodents

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

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

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

Transcranial Direct Current Stimulation (tDCS) in Mice