Name: applying stereotactic injection technique to study genetic effects on animal behaviors
Uploaded: 2015-05-10T14:00:00
Description: Watch this Scientific Journal Video about Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors at JoVE.com

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 Polyethyl...

<ol>
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O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+central+and+basolateral+amygdala+are+critical+sites+of+neuropeptide+Y/Y2+receptor-mediated+regulation+of+anxiety+and+depression.">The central and basolateral amygdala are critical sites of neuropeptide Y/Y2 receptor-mediated regulation of anxiety and depression.</a> Journal of Neuroscience. 30 (18), 6282-6290 (2010).</li><li>Salmon, P., Trono, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+titration+of+lentiviral+vectors.">Production and titration of lentiviral vectors.</a> Current Protoc Hum Gen. 12 (12.10), (2007).</li><li>Carter, D. 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Mouse Behavioral Testing: How to use mice in behavioral neuroscience.</a> Academic Press. , 133-141 (2011).</li><li>Kheirbek, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+control+of+learning+and+anxiety+along+the+dorso-ventral+axis+of+the+dentate+gyrus.">Differential control of learning and anxiety along the dorso-ventral axis of the dentate gyrus.</a> Neuron. 6 (77), 955-968 (2013).</li><li>Snyder, J. S., Soumier, A., Brewer, M., Pickel, J., Cameron, H. 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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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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 ...

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