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 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.
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.
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.
2. Stereotactic Injection
2.1) Preparation of Instruments
2.2) Preparation of the Mouse
2.3) The First Incision
2.4) Equipment Setup
2.5) Suturing
2.6) Post-operative Care
3. Open Field Test
3.1) Set Up
3.2) Acclimation and Test
3.3) Scoring
4. Forced Swim Test
NOTE: Allow a minimum of five days between behavioral tasks.
4.1) Set Up
4.2) Acclimation and Test
4.3) Scoring
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, the opening in the syringe needle should be placed directly above the intersection. In this study, a lentivirus expressing shRNA against RBM8a, a core factor in the exon junction complex, was injected into the dentate gyrus. The lentivirus also expresses RFP to label infected neurons. Figure 2 shows that the virus was injected into the correct region as indicated by the red signal (Figure 3).
To achieve significance in behavioral tasks, the sample size should range from 12-15 mice per group. Behavioral tasks can be conducted two weeks after stereotactic injections. In the OFT, an anxious phenotype is present if mice spend significantly less time in the center of the open field compared to the control group. The results indicate that knockdown of RBM8a in the dentate gyrus of adult mice leads to anxious behaviors (p <0.05, Figure 4). In the FST, there was no observable difference in immobility between control and experimental mice, suggesting that RBM8a knockdown in the dentate gyrus does not affect anti-depressive behavior (Figure 5). Statistical analysis consisted of performing a two-tailed, two sample-unequal variance t-test.
Figure 1: Intersection of Bregma and the Interaural Line. For stereotactic injections, the syringe needle should be lined up with the intersection of bregma and the interaural line. This point is set at zero.
Figure 2: Schematic Illustrating the Timeline for the Entire Experiment.
Figure 3: Representative brain slice illustrating successful injection of virus into the dentate gyrus. The scale bar = 100 µm.
Figure 4: The Amount of Time that Control and Experimental Mice Spent in the Center of a Novel Open Field. Experimental mice spend significantly less time in the center of the arena, indicating an anxious phenotype (N = 9-11, *, T18 = -2.72, p <0.05, mean SEM)
Figure 5: The Amount of Time that Control and Experimental Mice are Immobile in the FST. Experimental mice do not differ from controls in time spent immobile (N = 9-11, T18 = 1.25, p >0.05, mean SEM)
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 after surgery. Ensuring that the mouse is fully anesthetized throughout the surgery (it should not respond to foot pinches), and giving the correct dose of analgesic (based on the weight of the mouse), should help survival.17 Additionally, the mouse should be kept on a heating pad throughout the surgery process until it wakes up from the anesthesia. Mice cannot regulate their body temperature when they are anesthetized. Their body temperature will drop significantly during the surgery process. Even though the length of stereotactic surgery is relatively short, hypothermia could seriously impair mouse survival. Proper suturing technique is also instrumental in conducting a successful surgery. Mice will try to pick at their sutures, so it is crucial to make sure that the sutures are tight enough to prevent removal, but not too tight as to put too much tension on the wound. Four knots in each stitch should ensure that the mouse is unable to remove the suture. Proper suturing is important as an open wound will increase the susceptibility to infections, which would negatively affect any behavioral experiments.
The OFT is a behavioral paradigm designed to assess locomotion and the anxious phenotype in mice. When testing mouse behavior, it is important to be very careful when handling the mice and setting up the arena. In the OFT, the paradigm is designed to pair the mouse’s apprehension when placed in a new environment, with its natural curiosity and desire to explore novelty. A wild type mouse will initially be hesitant to enter the center of the open field, where it is more vulnerable, but will eventually do so, due to its innate curiosity. In an anxious mouse, the apprehension of crossing a more vulnerable space (the center of the field) will be greater than their natural curiosity and desire to explore, which will result in significantly less time spent in the center. To make sure the paradigm works correctly, it is important to minimize stress associated with the factors other than the open field. Stress can be minimized by allowing the mouse to acclimate to the behavioral room (so the exterior environment is not a possible confounding variable), and cleaning out the field between mice, to guarantee that all smells associated with the previous mouse have been removed.18 These steps should help eliminate any differences that are caused by improperly handling mice. In this study, RBM8a knockdown mice spent significantly less time in the center of a novel open field, compared to controls. This is indicative of an anxious phenotype, since the mice are more reluctant to enter the middle of the novel open field, where they are more vulnerable, compared to controls.
The FST seeks to investigate the anti-depressive phenotype in mice. When placed in a bucket of water, mice that spend significantly less time floating versus swimming are considered to have an anti-depressive phenotype. Immobility in the forced swim test is interpreted as behavioral despair (e.g. giving up). This paradigm is validated by anti-depressant treatment.7,8,9 Mice that receive anti-depressant drugs will spend significantly less time floating compared to controls, which is indicative of an anti-depressive phenotype. In this study, RBM8a knockdown mice did not significantly differ from control mice in time spent immobile. This indicates that RBM8a knockdown in the dentate gyrus of adult mice does not elicit a depressive, or anti-depressive phenotype. In our previous study, RBM8a overexpression in the dentate gyrus of adult mice significantly decreases time immobile compared to controls, which indicates an anti-depressive effect.16
The stereotactic injections used for representation in this paper were targeted to the dentate gyrus. The dentate gyrus was chosen as the target site, since the gene of interest is highly expressed there in the adult mouse. In addition, the dentate gyrus has been associated with depression and anxiety. For example, one study used POMC-Chr2 mice to selectively activate the granule cells in either the dorsal or ventral dentate gyrus. Activation of the both the dorsal and ventral dentate gyrus led to increased exploration of a novel environment, indicating a potential role of the dentate gyrus in anxiety.19 A different study found that mice with impaired neurogenesis had increased immobility in the FST, indicating a depressive phenotype.20 Moreover, activation of Ap oa1 in the forebrain leads to an anti-depressive effect in the FST, novelty suppressed feeding, and sucrose consumption test. When VEGF was knocked-down in the dentate gyrus of Ap oa1, this anti-depressive phenotype was lost.21 These data suggest that the dentate gyrus has a role in the pathophysiology of anxiety and depression, and indicated that the dentate gyrus may be a good area to target for our genetic modulation and behavioral experiments. A lentivirus was used specifically, so that all cells in the dentate gyrus would be infected, as opposed to only dividing cells (retrovirus).
A combination of these three experimental designs (in addition to other behavioral tasks) is a novel way to assess behavioral phenotypes in mice, since a virus can be injected into a specific brain area at specific developmental times. Using stereotactic injections of a lentivirus paired with behavioral tasks only takes a few months to complete and allows researchers to gather preliminary data on the behavioral effects of gene expression in a specific brain area. In contrast, conditional knockout mice often take years to generate. Moreover, cell type preference of viruses can be used to further target cells. For example, retroviruses can only infect dividing cells (such as neural stem cells in the dentate gyrus) whereas a lentivirus infects all cell types.22,23 Thus, together with co-injection, these widely used and individually validated experimental protocols provide a quick but comprehensive way to test the role of specific gene in a targeted brain area, on mouse behaviors.
The authors have nothing to disclose.
This work is supported in part by American Heart Association Scientist Development Grant and NARSAD Young Investigator Award to Yingwei Mao.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Stereotactic Apparatus item 51725 | Stoelting co. | 51725 | |
Quintessential stereotaxic pump | Stoelting co. | 53311 | |
Injection Styringe, 65 RN | Hamilton | 7633-01 | |
DMEM | Sigma-Aldrich | D5796 | |
PEI | Polysciences Inc. | 23966 | |
Scissors | Fine Surgical Tools | 14084-08 | |
Blunt end forceps | Fine Surgical Tools | 11002-12 | |
Needle holder | Fine Surgical Tools | 12001-13 | |
Drill | Ram Products Inc | Microtorque control box, Tech2000 handpiece | with pedal |
Glass bead sterilizer | Inotech | IS-400 | |
Absorbable sutures | Unify | M-K518r19 | Absorbable, reverse cutting |
Cotton swabs | VWR | 89031-270 | |
Heating pad | Gaymar | T-pump TP-500 PN11184-000 | |
Artificial tears | Rugby | NDC 0536-6550-91 | |
Disposable syringe | BD Syringe | 309623 | |
Cloth | |||
Gloves | Ansell | Senseitouch #7823 | |
Avertin or other anesthetic | see recipe citation | ||
Ketoprofen or other analgesic | see veternarian | ||
Tracking software | Noldus | Ethovision XT | |
Tracking Camera | Noldus | Media Recorder |