This work is supported in part by American Heart Association Scientist Development Grant and NARSAD Young Investigator Award to Yingwei Mao.

NOTE: All protocols involving animals were followed in accordance with the animal care guidelines of The Pennsylvania State University, IACUC #44057
1. Lentivirus Production
NOTE: The day before transfection, LentiX-293 cells should be at 80% confluency.
<ol>
	<li>Rinse cells with DMEM just before transfection and incubate in 10 ml of DMEM/10% FBS with Penicillin (100 IU/ml) and Streptomycin (100 &#181;g/ml) for 5 min&#160;at RT.</li>
	<li>Dilute DNA and Polyethylenimine (PEI) (1 mg/ml) in 1:3 ratio (1 &#181;g of DNA: 3 &#181;l of PEI) in 1 ml of DMEM and vortex for 1 min. The volume of DNA added is dependent on the concentration of the DNA. Incubate at RT for 10 min.
	<ol>
		<li>For example, use 20 &#181;g of DNA plasmid, comprised of 10 &#181;g of vector plasmid, 5 &#181;g of psPax214 plasmid, 5 &#181;g of vesicular stomatitis virus(VSV) with 60 &#181;l of PEI per dish.</li>
	</ol>
	</li>
	<li>Mix the DNA and PEI solution again by vortexing for 1 min. Incubate at RT for 10 min.</li>
	<li>Add 1/10th volume of total culture medium of DMEM (i.e.&#160;1 ml for 10 ml culture medium in a 100 mm dish). Add 1 ml of DNA-PEI mixture drop by drop into the dish and swirl the dish around until to the culture medium is well-mixed with the DNA.</li>
	<li>Incubate for 6 hr&#160;at RT and remove the supernatant. Add 5 ml of culture medium.</li>
	<li>48 hr&#160;after transfection, collect viral supernatant and spin at 627 x g for 5 min&#160;at 4 &#176;C in a 50 ml tube. Filter 30 ml of viral supernatant through a 0.45 &#181;m syringe filter into an ultracentrifuge tube.</li>
	<li>Add sterile water to balance the tubes, and cover tubes with small piece of parafilm. Spin tubes at 11,249 x g for 120 min&#160;at 4 &#176;C. Remove liquid using a vacuum tip.</li>
	<li>Add 100 &#181;l of cold PBS the tube. Gently rock the tube at 4 &#176;C O/N.</li>
	<li>To re-suspend the virus, pipette the PBS added in step 1.8 over the pellet 10 times, being careful not to touch the pellet with the tip. The pellet will not be re-suspended until this is complete.</li>
	<li>Aliquot the virus at 10-20 &#181;l per tube, flash-freeze in liquid nitrogen and store at -80 &#176;C.</li>
</ol>
2. Stereotactic Injection
2.1) Preparation of Instruments
<ol>
	<li>Place a pair of scissors, blunt-end forceps, a needle holder, scalpel, cotton swabs, and a cloth in a sealed pack with a sterilization indicator, and autoclave prior to surgery. Additionally, obtain 10% povidone iodine, 70% ethyl alcohol, absorbable sutures, a heating pad, artificial tears, a glass bead sterilizer, drill, a drill bit, 2 disposable syringes, an injection syringe, electric razor, analgesic (ketoprofen), anesthetic (avertin), gloves, and a stereotactic apparatus with injection pump.</li>
	<li>Make 1.25% avertin solution fresh that day, filter in a sterile hood using a 0.2 &#181;m sterile syringe filter, and place into a sterile serum vial. Mix 2.5 g of 2,2,2-tribromoethyl alcohol into 5 ml of tert-amyl alcohol, then dissolve in 200 ml water. Keep the pH below 5.15</li>
</ol>
2.2) Preparation of the Mouse
<ol>
	<li>Administer avertin based on the weight of the mouse (375 mg/kg).15 Inject the mouse with the avertin via an intraperitoneal (IP) injection, and then place back into its cage until fully anesthetized.</li>
	<li>Give an analgesic via IP injection (ketaprofen, 5 mg/kg), and place artificial tears on the eyes of the mouse to prevent drying.</li>
	<li>Ensure that the mouse is fully asleep by pinching their foot. If the mouse responds to the foot pinch, then give more anesthetic (in doses of 50 &#181;l). Shave the head of the mouse using an electric razor. Shave the area to be operated on (typically from just behind the ears, to the top of the snout), and the surrounding area.</li>
	<li>Place the mouse into the stereotactic apparatus. Latch the front teeth onto the anterior clamp, and lower the clamp and tighten it so that the jaw is fully secure.</li>
	<li>Insert the ear bars into the ear canal to fully stabilize the head. Be careful not to insert the ear bars in too far, which could damage the inner ear. Once finished, the body of the mouse should be able to be moved slightly without disrupting the position of the head.</li>
	<li>Place a heating pad under the mouse in order to regulate the mouse&#8217;s body temperature throughout the procedure.</li>
	<li>Clean the surgical area thoroughly. Using a cotton swab, rub 10% povidone iodine in a circular motion, starting from the middle of the surgical site and moving outwards. Then, use a cotton swab to rub 70% ethyl alcohol on the surgical site in the same fashion. Repeat two times.</li>
</ol>
2.3) The First Incision
<ol>
	<li>Once the mouse is prepped, open the sterile equipment bag. Change gloves before touching the instruments. Take out the sterile cloth and place the instruments on the cloth. If any instrument touches an un-sterile surface, use the glass bead sterilizer for 15 sec&#160;to sterilize it.</li>
	<li>Take the scalpel in the dominant hand and blunt-end forceps in the non-dominant hand. Use the blunt-end forceps to gently grip the skin of the mouse, and make an incision using the scalpel. Begin the incision about 1.5 cm above the ears (toward the nose) and extend to about 0.5 cm below the ears. Extend the incision vertically down the middle of the mouse&#8217;s head to expose bregma (Figure 1).</li>
	<li>Once bregma is visualized, take a sterile cotton tip to gently remove any blood covering the surface of the skull. Use two cotton tips to push the skin toward the side of the head.</li>
</ol>
2.4) Equipment Setup
<ol>
	<li>After any blood is removed from the surface of the skull, and bregma is clearly visualized, prepare the syringe (concentration of 108 TU/ml, volume of 1 &#181;l). In the fume hood, fill the syringe with the virus to be injected. Ensure that no bubbles are inside. Place the syringe in the stereotactic apparatus, making sure that it is fully secured.</li>
	<li>Slowly lower the syringe until it is right above the surface of the skull, so that the tip of the sterile syringe needle is set to the intersection of bregma and the interaural line. Set this point as zero, and determine coordinates from this point.</li>
	<li>Depending on the brain area of interest, vary the stereotactic coordinates. Determine these coordinates by utilizing a brain atlas4. Once the coordinates are determined, move the needle of the syringe to match those coordinates. Target the dentate gyrus using the coordinates: M/L= +/- 1 mm, A/P= -1.82 mm. Lower the needle to right above the skull to visualize where the hole needs to be drilled.</li>
	<li>Raise the syringe slightly, take the drill and place the drill bit right above the target drill site at about a 45&#176; angle to the skull. Begin drilling, and keep drilling until the skull gives way. When the skull gives way, a drop in resistance can be detected. Be careful not to drill into the brain, so as to prevent cortical damage.</li>
	<li>Take a sterile cotton tip and wipe away any blood from the hole.</li>
	<li>Lower the syringe so that the tip sits right on the surface of the brain (not the skull). Set the D/V coordinate (depth) to 0. Lower the syringe to the desired depth, based on the brain atlas coordinates. To reach the dentate gyrus, set D/V to -1.79 mm.</li>
	<li>Start the injection at a rate of 0.2 &#181;l/min. Watch to ensure that the syringe does not slip lower than the desired depth. If this occurs, gently raise the syringe to the desired depth again.</li>
	<li>After the injection is finished, wait about 2 min&#160;to ensure that any residual virus has been absorbed. Slowly raise the syringe and use a cotton tip to remove any liquid from the injection site.</li>
	<li>Repeat for the other hemisphere.</li>
</ol>
2.5) Suturing
<ol>
	<li>Remove the sterile suture and needle from its packaging and grip the needle using the needle holders. Face the pointed edge of the needle away from the needle holder. Hold the blunt-end forceps in the non-dominant hand, and use it to grip the skin on the mouse. Start with the dominant hand.</li>
	<li>Gently push the suture through the skin and use the blunt-end forceps to grab the skin on the other side of the incision. Pull the suture material through until about 0.75 inch remains.</li>
	<li>Let go of the needle, and use the blunt-end forceps to wrap the suture around the needle holder one time, then, grab the 0.75 inch remaining suture with the needle holder, and pull the suture through, so that the needle and needle holder end up on the side of the head opposite to that which they were on originally. This should form a knot.</li>
	<li>Repeat this step three more times, alternating the side of the head the needle holder ends up on each time.</li>
	<li>Cut the suture and repeat steps 2.5.1-2.5.3 until the incision is closed.</li>
</ol>
2.6) Post-operative Care
<ol>
	<li>Gently remove the ear bars from the mouse, and remove its jaw from the anterior clamp.</li>
	<li>Keep the mouse on the heating pad until it wakes up and moves around on its own. Monitor the mouse daily, and keep and eye out for signs of pain, such as not grooming, eating or drinking. Give additional analgesic when deemed necessary.</li>
</ol>
3. Open Field Test
3.1) Set Up
<ol>
	<li>Obtain an empty square, plastic arena with dimensions 50 cm x 50 cm, with 50 cm high walls (but can be slightly larger). Clean the arena with 70% ethyl alcohol prior to the beginning of the experiment. Make sure the ethyl alcohol has fully dried before placing a mouse into the arena.</li>
	<li>Place the arena on the floor, and try to ensure the lighting is set to minimize shadows and glare.</li>
	<li>If using tracking software, position the camera directly overhead of the open field, approximately 1.5 ft above the open field (far enough away to full encompass the whole arena, but close enough to have a clear view of the mouse).</li>
	<li>Set up a zone in the center of the box (approximately an area of 30 cm x 30 cm).16 To set a zone, click on the zone 1 tab in the side panel.
	<ol>
		<li>Select a shape outline in the upper panel, and outline the perimeter of the center zone. Select add zone, and click on the inside of the center zone. Using the program prompts, name the zone (this will be referred to as &#8216;center&#8217; for this procedure). If the behavioral test will be scored by hand, make sure the bottom of the field is divided into 10 cm x 10 cm squares, marked using tape.</li>
	</ol>
	</li>
	<li>Set up the software so that the settings are correct, and the mouse is easily visible on the screen. Accomplish this by adjusting the detection settings on the machine to be compatible with the testing arena (lighting) and mouse color. 
	NOTE: When the settings are correct, the system should track only the mouse, and not any shadows or urine/fecal matter. The specific software settings depend on the lighting of the arena, and the color of the mouse.</li>
</ol>
3.2) Acclimation and Test
<ol>
	<li>Move experimental mice into the behavioral room 1 hr&#160;before the test in order to acclimate them to their surroundings. Leave the mice in their cage for the acclimation.</li>
	<li>Turn on the camera to tape the behavioral task and place the mouse toward the middle front of the arena so that the mouse is facing the wall.</li>
	<li>Set the timer for 5 min, and step away from the arena. If possible, leave the room so as to not accidentally influence the mouse.</li>
	<li>Once 5 min&#160;are up, remove the mouse and place them in their cage.</li>
	<li>Use 70% ethyl alcohol to thoroughly clean out the field before proceeding to the next mouse. Make sure the ethyl alcohol has fully dried.</li>
</ol>
3.3) Scoring
<ol>
	<li>Consider the mouse as being in the center of the field when all four paws are inside the middle 30 cm x 30 cm.</li>
	<li>Quantify the amount of time the mouse spends in the center. Alternatively, determine this time using tracking software (such as Noldus ethovison).
	<ol>
		<li>To do this, go to the analysis tab, and click on &#8220;movement&#8221; under analysis profiles. Delete the categories currently set, and then select &#8220;in zone&#8221; under the location tab. Select the center zone. Next, select &#8220;analysis output&#8221; (under the results tab) to view results. A table listing the time spent immobile for each trial should appear.</li>
	</ol>
	</li>
	<li>Otherwise, score the OFT by hand. The open field should have 25 10 cm x 10 cm squares outlined on the bottom of the open field (as outlined in step 3.1.3). Quantify the amount of time the mouse spends in the center, in addition to the number of entries into the center of the arena.16 
	NOTE: The center 30 cm x 30 cm area is considered the center of the open field. A mouse is regarded as inhabiting the center region if all four paws are inside the 30 x 30 cm area.</li>
</ol>
4. Forced Swim Test
NOTE: Allow a minimum of five days between behavioral tasks.
4.1) Set Up
<ol>
	<li>Obtain a 2 L clear bucket, and fill with 22 &#176;C water. Place the bucket on a table, and try to ensure the lighting is set to minimize shadows and glare.</li>
	<li>If using tracking software, position the camera directly overhead of the bucket.</li>
	<li>Set up software so that the mouse is easily visible on the screen.</li>
</ol>
4.2) Acclimation and Test
<ol>
	<li>Move experimental mice into the behavioral room 1 hr before the test to acclimate the surroundings. Leave the mice in their cage for the acclimation.</li>
	<li>Turn on the camera to tape the behavioral task. Gently, place the mouse in the middle of the bucket of water. Make sure not to drop the mouse in. Slowly lower the mouse, so its front feet touch the water first. Take care to prevent their head from submerging.</li>
	<li>Set the timer for 6 min, and step away from the behavioral area.</li>
	<li>Once 6 min&#160;are up, remove the mouse from the bucket, and wipe off excess water before placing the mouse back in their cage.</li>
</ol>
4.3) Scoring
<ol>
	<li>If using tracking software, set percent immobility to 11%, as suggested by Noldus (this should be the default setting), to quantify time floating vs. swimming.</li>
	<li>To quantify using the tracking system, go to the analysis tab, and click on movement under analysis profiles. Delete the categories currently set, and then select mobility state, on the left side panel (under individual behavior). Set immobility to 11%.</li>
	<li>Select track visualization, under the results tab. Select analysis output (under the results tab) to view results. A table listing the time spent immobile for each trial should appear.</li>
	<li>If hand scoring, measure the time the mouse spends swimming or climbing, and amount of time the mouse spends immobile. In addition, measure the latency to the first time immobile. See Figure 2 in Results Section.</li>
</ol>

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The authors have nothing to disclose.

Successful stereotactic injections rely on three factors: keeping the mouse alive, setting the correct zero point for coordinates (tip of the needle on the intersection of bregma and the interaural line), and setting the right depth to the zero point (tip of the needle just touching the exterior of the brain tissue). The viability of mice is important. Surgery survival can be aided by making sure that the mouse is properly anesthetized and receives adequate analgesic. Pain is known to be a major cause of poor recovery af...

The mouse has been widely used in neurobiology because it is easy to manipulate genetically. Gene knockout techniques allow researchers to investigate how each genetic factor shapes mouse behaviors. Moreover, the cre-loxp system provides a valuable tool for tissue- and cell type- specific gene knockout in mice, which enables researchers to study gene function in different tissues.1 However, in practice, expression patterns of cre promoters are difficult to control, and so far many established cre drivers have not achieved region-specific expression.2,3
Alternatively, stereotactic injection is a method that targets specific brain regions of the adult mouse. By injecting genetically engineered viruses expressing cDNA or shRNA, region-specific modulation of gene expression can be achieved. Although the brain size of each mouse varies, the location of specific brain regions can be determined using stereotactic coordinates set from landmarks on the skull of the mouse brain. The most commonly used landmarks are bregma, lambda, and the interaural line. Using coordinates obtained from a brain atlas,4 the exact location of each brain area can be identified by the antero-posterior (A/P), medial-lateral (M/L), and dorsal-ventral (D/V) axes from bregma/interaural line intersection. Typically viruses injected into the brains of mice are tagged with either red, or green fluorescent protein (RFP or GFP), so that injections can be confirmed by fluorescent microscopy.
Behavioral assessments of mice are especially necessary for basic research of psychiatric disorders. Symptoms of psychiatric disorders in patients typically involve abnormal behaviors. Some of these human behaviors are evolutionarily conserved and can be directly mimicked and observed in the mouse. For example, depression can be modeled in the mouse by measuring behavioral despair. People with depression often feel as though nothing they do will ever help, a symptom that can eventually lead to suicide. In rodents, this can be modeled using the Forced Swim Test (FST), which measures the amount of time a mouse swimming versus floating in a pool of water (viewed as giving up). This paradigm is validated by rescuing the phenotype with anti-depressants.7,8,9 Mice which have received anti-depressants will spend significantly less time immobile compared to untreated controls. Another behavioral test, the Open Field Test (OFT) is designed to assess locomotion in mice, and additionally can be used to analyze the anxious phenotype in mice.5,6 This test is based on the premise that mice feel safer when they are close to the wall in a novel open field. Wild type mice will eventually explore the novel environment, as they are curious animals. However, spending less time in the center of the field indicates anxiety in the mouse, as the mouse will not be able to overcome the initial fear brought on by a novel environment. The anxiety of the mouse, as quantified by the amount of time spent in the center of an open field, can be compared to clinical anxiety in humans, which is present in many psychiatric disorders.
The combination of stereotactic injections with behavioral paradigms is a novel way to alter the expression of a specific gene in a targeted brain area. The effect of modulated gene expression on mouse behaviors can then be determined. In contrast to whole brain knockout, this method is particularly useful as it only targets specific brain areas. In addition, stereotactic injections are typically performed in the adult wild type mouse, therefore, endogenous gene expression has been maintained throughout developmental stages. This method will avoid the confound effect if the gene is required for survival during the embryonic or postnatal stage of development. One major limitation is that the experimental mice need to go through an invasive surgery, in which the skulls of mice have to be opened. Moreover, the degree of gene modulation is determined by the titer and efficiency of the virus. The virus needs to be injected into the correct region using stereotactic coordinates, which requires special instruments. Verification of the correct injection site can only be completed post mortem.
This method has been previously used to test the involvement of a specific gene in various neurological diseases. For example, viral mediated RNAi targeting the Th gene (which allows dopamine to be synthesized) was injected into the substantia nigra compacta, and locomotor behavior analysis was conducted.10 Another study used stereotactic injection of a lentivirus silencing DISC1 to assess mouse behavior in relation to schizophrenia. Knockdown of DISC1 led to increased locomotion in response to novelty (parallels positive symptoms in schizophrenia), and greater immobility in the FST.11 Similarly, an additional study found that 5-HT1B overexpression led to increased exploratory behavior in the OFT, consistent with an anti-anxiety phenotype using this method.12 Stereotactic injections can deliver cre virus to induce recombination in cre-loxp mice. This method was used to selectively delete the Y2 receptor in the amygdala and the bed nucleus of the stria terminalis. Upon behavioral analysis, these mice were found to an anti-depressive phenotype when the gene was deleted in the central amygdala, but no phenotype when the gene was deleted in the basolateral amygdala or the bed nucleus of the stria terminalis.13 Thus, this technique provides a unique tool to study the genetic effect on animal behaviors.

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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:107px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="42" style="height:42px;width:216px;">Stereotactic Apparatus item 51725</td>
			<td style="width:107px;">Stoelting co.</td>
			<td align="right" style="width:119px;">51725</td>
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			<td height="42" style="height:42px;width:216px;">Quintessential stereotaxic pump&#160;</td>
			<td style="width:107px;">Stoelting co.</td>
			<td align="right" style="width:119px;">53311</td>
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			<td height="21" style="height:21px;width:216px;">Injection Styringe, 65 RN</td>
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			<td style="width:119px;">7633-01</td>
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			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:107px;">Sigma-Aldrich</td>
			<td style="width:119px;">D5796</td>
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			<td height="42" style="height:42px;width:216px;">Scissors</td>
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			<td height="42" style="height:42px;width:216px;">Blunt end forceps</td>
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			<td height="42" style="height:42px;width:216px;">Needle holder&#160;</td>
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			<td>with pedal</td>
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			<td height="21" style="height:21px;width:216px;">Glass bead sterilizer</td>
			<td style="width:107px;">Inotech&#160;</td>
			<td style="width:119px;">IS-400</td>
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			<td height="21" style="height:21px;width:216px;">Absorbable sutures&#160;</td>
			<td style="width:107px;">Unify</td>
			<td style="width:119px;">M-K518r19</td>
			<td>Absorbable, reverse cutting</td>
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			<td height="21" style="height:21px;width:216px;">Cotton swabs</td>
			<td style="width:107px;">VWR</td>
			<td style="width:119px;">89031-270</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heating pad</td>
			<td style="width:107px;">Gaymar</td>
			<td style="width:119px;">T-pump TP-500 PN11184-000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Artificial tears</td>
			<td style="width:107px;">Rugby</td>
			<td style="width:119px;">NDC 0536-6550-91</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Disposable syringe</td>
			<td style="width:107px;">BD Syringe</td>
			<td align="right" style="width:119px;">309623</td>
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			<td height="21" style="height:21px;width:216px;">Cloth</td>
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			<td height="42" style="height:42px;width:216px;">Avertin or other anesthetic</td>
			<td style="width:107px;">see recipe citation</td>
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			<td height="42" style="height:42px;width:216px;">Ketoprofen or other analgesic</td>
			<td style="width:107px;">see veternarian&#160;</td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:216px;">Tracking software</td>
			<td style="width:107px;">Noldus</td>
			<td style="width:119px;">Ethovision XT</td>
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			<td height="21" style="height:21px;width:216px;">Tracking Camera</td>
			<td style="width:107px;">Noldus</td>
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</table>

applying stereotactic injection technique to study genetic effects on animal behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Accurate stereotactic injection relies heavily on setting the correct coordinates. The tip of the needle used to inject the virus should be set directly on the intersection of bregma and the interaural line (Figure 1). It is helpful to use a stereomicroscope to ascertain whether or not the needle is placed correctly. When looking through the microscope, the needle should be positioned so that if the virus were injected, it would land directly on the intersection of bregma and the interaural line. That is...

Watch this Scientific Journal Video about Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors at JoVE.com

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

applying-stereotactic-injection-technique-to-study-genetic-effects-on

Research

JoVE Journal

Behavior

14.1K Views.  The Pennsylvania State University. Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

Video: Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

A Within-subjects Experimental Protocol to Assess the Effects of Social Input on Infant EEG

Despite the importance of social interactions for infant brain development, little research has assessed functional neural activation while infants socially interact. Electroencephalography (EEG) power is an advantageous technique to assess infant functional neural activation. However, many studies record infant EEG only during one baseline condition. This protocol describes a paradigm that is designed to comprehensively assess infant EEG activity in both social and nonsocial contexts as well as tease apart how different types of social inputs differentially relate to infant EEG. The within-subjects paradigm includes four controlled conditions. In the nonsocial condition, infants view objects on computer screens. The joint attention condition involves an experimenter directing the infant's attention to pictures. The joint attention condition includes three types of social input: language, face-to-face interaction, and the presence of joint attention. Differences in infant EEG between the nonsocial and joint attention conditions could be due to any of these three types of input. Therefore, two additional conditions (one with language input while the experimenter is hidden behind a screen and one with face-to-face interaction) were included to assess the driving contextual factors in patterns of infant neural activation. Representative results demonstrate that infant EEG power varied by condition, both overall and differentially by brain region, supporting the functional nature of infant EEG power. This technique is advantageous in that it includes conditions that are clearly social or nonsocial and allows for examination of how specific types of social input relate to EEG power. This paradigm can be used to assess how individual differences in age, affect, socioeconomic status, and parent-infant interaction quality relate to the development of the social brain. Based on the demonstrated functional nature of infant EEG power, future studies should consider the role of EEG recording context and design conditions that are clearly social or nonsocial.

This novel protocol is designed to assess the neural bases of social interaction in infants. The paradigm is designed to tease apart how various social inputs such as language, joint attention, and face-to-face interaction relate to infant neural activation. Infant EEG power is recorded during both social and nonsocial conditions.

Despite the importance of social interactions for infant brain development, little research has assessed functional neural activation while infants ...

Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral science, conflicting outcomes are often provided from different laboratories. To identify reasons for the different outcomes, we previously focused on arm features, which differ between laboratories, most notably the presence/absence of ledges at the sides of open arms and the transparency/opaqueness of closed arm walls. In a previous report, we used a custom designed container to compare rat behavior on different combinations of open and closed arm designs under otherwise identical experimental conditions, and showed that differences in arm features interfere with experimental outcomes. In brief, open arm ledges significantly increased anxiety-like behavior in rats, while transparent arms may also have decreased this behavior. Furthermore, we verified a higher detection sensitivity of the effect of an anxiolytic drug in a combination of no-ledged open arm + opaque walled closed arm compared with a combination of ledged open arm + transparent walled closed arm. In this report, we introduce our protocol for the elevated plus maze test, together with discussion of the key results from the previous report and our experimental experience. We believe this report will provide useful information for researchers who have employed or who plan to use the elevated plus maze in their studies.

Rats' anxiety-like behavior tested by the elevated plus maze varies highly depending on the testing environment. Here we show how apparatus settings, especially arm structure, affect behavior on the maze, and we explain a reliable protocol to detect effects of anxiolytic drugs. This report will contribute to anxiety research.

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral ...

Assessment of Social Transmission of Food Preferences Behaviors

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy aging groups. For example, olfactory dysfunction in AD can present as impairment in olfactory recognition, emerging during early stages of the disease and worsening while the disease progresses. The social transmission of food preferences (STFP) task is based on a rudimentary form of communication between rodents concerning distant foods dependent on the transmission of olfactory cues. Healthy wild-type mice would prefer to eat a novel, flavored food that was previously cued by a conspecific, and this food preference would be hampered in transgenic AD mice, such as the APP/PS1 model. Indeed, a strong preference for the cued food in C57Bl6/J mice of 3 months of age was found, and this was reduced in 3 months old transgenic APP/PS1 mice. In summary, STFP task could be a powerful measure to be integrated in present subclinical detection assays of AD.

This paper presents a protocol for the investigation of social transmission of food preference in mice. The advantages and possible applications for this procedure, for instance, in detecting early changes in AD mouse models, are highlighted. To conclude, interpretation of the results in light of critical details are discussed.

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy ...

Tickling, a Technique for Inducing Positive Affect When Handling Rats

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk for both the animals and the hands of their handlers, decreased animal welfare, and less valid research data. To minimize negative effects on experimental results and human-animal relationships, research animals are often habituated to being handled. However, the methods of habituation are highly variable and often of limited effectiveness. More potently, it is possible for humans to mimic aspects of the animals&#39; playful rough-and-tumble behavior during handling. When applied to laboratory rats in a systematic manner, this playful handling, referred to as tickling, consistently gives rise to positive behavioral responses. This article provides a detailed description of a standardized rat tickling technique. This method can contribute to future investigations into positive affective states in animals, make it easier to handle rats for common husbandry activities such as cage changing or medical/research procedures such as injection, and be implemented as a source of social enrichment. It is concluded that this method can be used to efficiently and practicably reduce rats&#39; fearfulness of humans and improve their welfare, as well as reliably model positive affective states.

This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk ...

Using a Classroom-Based Deese Roediger McDermott Paradigm to Assess the Effects of Imagery on False Memories

Associated word list procedures can elicit false memories in predictable ways by inducing associative processing, thus making it harder to monitor the accuracy of memories. The purpose of the method presented here was to induce false memories using lists of either semantically or phonologically related words and to assess the effects of imagery instructions on the recall and recognition of those false memories. To do this, we used a modified version of the Deese Roediger McDermott (DRM) paradigm. We adapted word lists from previous DRM studies to suit imagery procedures and created an automated presentation to present the word lists in classroom settings. We then recruited undergraduate classes and instructed some of the classes to create mental images of the list words as they were being presented, while instructing the other classes to simply remember the words. The automated presentation presented word lists to participants, one word at a time, alternating between phonologically and semantically related lists. Participants used paper-pencil recall packets to immediately recall list items, complete a distractor activity, and take a subsequent final recognition test. Often, participants immediately recalled and later recognized words that were related to the list items but were not actually presented; these are known as critical lures and indicate a false memory. The protocol detailed here describes a four-step procedure - list presentation, immediate recall, distractor phase, and final recognition - that can assess the effects of list type and imagery instruction within the DRM paradigm on memory. The automated nature of the list presentation provides the ability to systematically vary variables of interest, and the paper and pencil method of data collection affords an easily accessible method for collecting data in classroom settings. The protocol also offers options to modify the procedure to a more traditional DRM paradigm without imagery and/or list type manipulations. The use of this protocol can provide results relevant to both classroom learning and cognitive science principles.

The method presented here induced false memories using lists of related words and also assessed the effects of imagery instructions on the recall and recognition of those false memories. This protocol details a modified version of the Deese Roediger McDermott (DRM) paradigm.

Associated word list procedures can elicit false memories in predictable ways by inducing associative processing, thus making it harder to monitor the ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...

Applying an eMASS Customization Program as a Research Tool to Evaluate Consumer Benefits

As many scholars and practitioners study personalization and relationship marketing, it is important to provide personalization such as mass customization through marketing technology. The purpose of this study is to examine how to conduct consumer research using an online survey and analysis of data. This study examines consumers&#39;&#160;perceived benefits while customizing a product as well as emotional product attachment, attitudes toward a customization program, and loyalty intentions in the context of online retailing. In addition, this study investigates how consumer responses are different based on individual characteristics such as fashion innovativeness. An online survey company in South Korea recruited 290 female apparel shoppers who purchased apparel online. To enhance external validity, this study used an existing retail website with a well-established mass customization program. After completing the customization program, participants complete the online questionnaire. Structural equation modeling (SEM) and latent mean analyses (LMAs) are then performed for analyses. This study stresses the importance of testing measurement invariance for mean comparisons. Before the SEM and LMA, this study follows the hierarchy of invariance tests (configural invariance test, metric invariance test, and scalar invariance test), which are not considered by traditional approaches such as ANOVA. These statistical analyses provide applicability of the invariance test procedures and LMA to consumer behaviors. The conclusions of mean differences have integrity and validity because they are guided by a sophisticated statistical procedure to ensure measurement invariance.

Presented here is a protocol to examine consumer responses toward mass customization in the context of online retailing. The protocol details the online survey procedure and how to analyze data using structural equation modeling and group differences using latent mean analyses.

As many scholars and practitioners study personalization and relationship marketing, it is important to provide personalization such as mass customization ...

Effects of Surgical Masks on Cardiopulmonary Function in Healthy Subjects

We study the effect of surgical masks on cardiopulmonary function based on a cardiopulmonary exercise test (CPET). This study shows that surgical masks reduce cardiopulmonary exercise capacity and ventilation in healthy young subjects and wearing masks might affect aerobic exercise capacity more in female subjects than in male subjects.

We study the effect of surgical masks on cardiopulmonary function based on a cardiopulmonary exercise test (CPET). This study shows that surgical masks reduce cardiopulmonary exercise capacity and ventilation in healthy young subjects and wearing masks might affect aerobic exercise capacity more in female subjects than in male subjects.

We study the effect of surgical masks on cardiopulmonary function based on a cardiopulmonary exercise test (CPET). This study shows that surgical masks ...

Animal Models of Depression - Chronic Despair Model (CDM)

Major depressive disorder is one of the most prevalent forms of mental illnesses and causes tremendous individual suffering and socioeconomic burden. Despite its importance, current pharmacological treatment is limited, and novel treatment options are urgently needed. One key factor in the search for potential new drugs is evaluating their anti-depressive potency in appropriate animal models. The classical Porsolt forced swim test was used for this purpose for decades to induce and assess a depressive-like state. It consists of two short periods of forced swimming: the first to induce a depressed state and the second on the following day to evaluate the antidepressant effect of the agent given in between the two swim sessions. This model might be suitable as a screening tool for potential antidepressive agents but ignores the delayed onset of action of many antidepressants. The CDM was recently established and represented a modification of the classical test with notable differences. Mice are forced to swim for 5 consecutive days, following the idea that in humans, depression is induced by chronic rather than by acute stress. In a resting period of several days (1-3 weeks), animals develop sustained behavioral despair. The standard read-out method is the measurement of immobility time in an additional delayed swim session, but several alternative methods are proposed to get a broader view of the mood status of the animal. Multiple analysis tools can be used targeting behavioral, molecular, and electrophysiological changes. The depressed phenotype is stable for at least 4 weeks, providing a time window for rapid but also subchronic antidepressant treatment strategies. Furthermore, alterations in the development of a depressive-like state can be addressed using this approach. CDM, therefore, represents a useful tool to better understand depression and to develop novel treatment interventions.

Animal Models of Depression - Chronic Despair Model CDM

The chronic despair mouse model (CDM) of depression consists of repetitive forced swim sessions and another delayed swim phase as a read-out. It represents a suitable model for induction of a chronic depressive-like state stable for at least 4 weeks, amendable to evaluate subchronic and acute treatment interventions.

Major depressive disorder is one of the most prevalent forms of mental illnesses and causes tremendous individual suffering and socioeconomic burden. ...