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May 2018

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Three-chamber Social Approach Task with Optogenetic Stimulation (Mice)
配有光遗传学刺激的三间隔社交途径测试(小鼠)   

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Abstract

The formation of social relationships via social interactions and memory are essential for one’s physical and mental health. To date, rodent studies have used the three-chamber social approach test to measure social approach, social novelty, and social memory. In recent years, techniques including optogenetics have been developed to acutely control the activity of genetically defined populations of neurons. Recent studies have even combined optogenetics with advanced temporal gene expression control systems to label certain populations of neurons during learning and subsequently reactivated for memory testing. We combined optogenetic targeting with the three-chamber social approach test to examine particular neural circuits of interest during social memory encoding or retrieval. First, we stereotaxically infected specific brain areas with viral-encoding opsins that acutely activate or inhibit the firing of the neurons. Next, we subjected the mice to the three-chamber behavioral paradigm while delivering light during social memory encoding or retrieval. Lastly, the mice were tested with the delivery of light in a counter-balanced manner which allows each subject to be its own internal control. Thus, the optogenetic stimulation coupled with the three-chamber social approach test is a well-validated paradigm to explore the contribution of diverse brain circuits in various social cognition processes.

Keywords: Social approach (社交途径), Social recognition (社交识别), Social memory (社会记忆), Memory encoding (记忆编码), Memory retrieval (记忆提取), Optogenetics (光遗传学)

Background

Social cognition is essential to our mental health. Deficits in social interaction and memory are hallmark characteristics in numerous brain disorders. Social working memory is an extremely dynamic process that is often unpredictable and requires constant adaptability to the changing stimuli (Lieberman, 2007). Specifically, it is a cognitive process that involves the encoding, storage, and retrieval of socially salient information. Traditional human and primate studies using imaging and lesion experiments, have implicated the medial temporal lobe in social cognitive tasks including social recognition and context evaluation (Insel and Fernad, 2004; Olson et al., 2013; Sandi and Haller, 2015). However, these previous studies employed toxins which caused extensive damage to broad areas in the brain and have limited the control of targeted cell types and subcellular compartments. Recent studies employing acute optogenetic approaches have implicated various brain regions and circuits, including the amygdala, hippocampus, and ventral tegmental area to play a critical role in the different facets of social behavior, including social interactions, approach, and discrimination memory (Felix-Ortiz and Tye, 2014; Gunaydin et al., 2014; Hitti and Siegelbaum, 2014; Okuyama et al., 2016).

To investigate the behavioral significance of a particular brain region in social memory processing, subject mice can be evaluated on their performance in a well-validated three-chamber social approach paradigm (Nadler et al., 2004). In this test, the subject mouse is habituated to the apparatus (stage 1). Then it is first introduced to a novel stranger mouse (S1) to evaluate memory formation and sociability (stage 2), and subsequently presented to a second novel stranger mouse (S2) to test social recognition memory and retrieval (stage 3). The amount of time the mouse spends investigating the stranger mice is recorded. Wild-type mice spend more time exploring a novel stranger mouse compared to a familiar conspecific. Therefore, combined with optogenetic targeting, specific neural circuits can be activated or inhibited during the various stages including memory formation (encoding) or retrieval (discrimination). Furthermore, the use of optogenetics can also identify particular genes in the neural circuits that may be involved in the regulation of sociability and/or social recognition memory in mice (Leung et al., 2018).

Materials and Reagents

  1. Filter paper
  2. Multimode fiber (0.39 NA, high OH, 200 μm Core, Wavelength range: 300-1,200 nm; ThorLabs, catalog number: FT200UMT)
  3. Materials for optogenetic experiments (Table 1)
    1. Multimode fiber (0.39 NA, high OH, 200 μm Core, Wavelength range: 300-1,200 nm; ThorLabs, catalog number: FT200UMT)
    2. 1.25 mm Ceramic Stick Ferrule, 230 μm (Precision Fiber Products, catalog number: MM-FER2007C-2000) 

    Table 1. Summary of the main characteristics of materials used for optogenetic experiments


  4. Injection cannula (5 mm, 26 gauge; Plastics One, catalog number: C315GS-5/SPC)
  5. 1 ml Syringe (BD Sciences, catalog number: 309628)
  6. Tygon tubing 1/16” I.D. 1/8” O.D. and 1/32” wall (US Plastics, catalog number: 57102)
  7. Aluminum foil (Alcan)
    Note: Ceramic ferrules was chosen over metal ferrules, to ensure accurate fiber alignment.
  8. 1.25 mm SM Ceramic split sleeve, 6.60 mm Length (Precision Fiber Products, catalog number: SM-CS125S)
    Note: Ceramic split sleeves were chosen over metal split sleeves, to ensure accurate fiber alignment.
  9. 1.25 mm Ferrule dust cap, white (Precision Fiber Products, catalog number: BCDC-1300-W)
  10. Parafilm M (Pechiney Plastic Packaging, catalog number: PM-996)
  11. Cotton swab (The Lab Depot, catalog number: 394305)
  12. C57BL/6 mice (The Jackson Laboratory, catalog number: 000664)
    Note: Male and female subjects (4 weeks+ old) and strangers (< 3 weeks old) have been successfully trained in this paradigm using this protocol.
  13. AAV Viruses (stored at -80 °C until use with a titer of ~1011 to 1013 pfu/ml)
    For example: AAV-CaMKIIα: eArchT3.0-EYFP (University of North Carolina GTC Vector Core)
    AAV-CaMKIIa-hChR2 (H134R)-mCherry-WFRE-PA (University of North Carolina GTC Vector Core).
  14. Dental cement (Stoelting Co., catalog number: 51458) 
  15. Speed set instant mix epoxy (LePage, https://www.lepage.ca/en/lepage-products/build-things/epoxies/speed_set_instantmixepoxy.html)
  16. Suture kit (Ethicon, catalog number: JJ489)
  17. Tear gel (Novartis, https://well.ca/products/tear-gel-liquid-eye-gel_17561.html?gclid=CjwKCAjw6-_eBRBXEiwA-5zHaVuKltJxR7GYDuMwgeSM5yh_3J6FSiTehp0ekoUOvym2ICm3-YjZTxoCIkoQAvD_BwE)
  18. Betadine
  19. Saline
  20. 70% ethanol 
  21. Hydrogen peroxide
  22. Ice
  23. Analgesics: Metacam (CDMV, 5 mg/ml one daily injection for three consecutive days)

Equipment

  1. Cleaver (Doric Lenses, catalog number: B600-0002)
  2. Fiber stripping tool (ThorLabs, catalog number: T12S21)
  3. 10 μl Pipette (SARSTEDT, catalog number: 90.1771.002) 
  4. Digital Caliper (ULINE Canada)
  5. Mouse stereotaxic frame (Neurostar) 
  6. Anesthesia system for isoflurane (Kent Scientific Corporation, catalog number: SOMNO-MSEKIT)
  7. Temperature controller (Sunbeam)
  8. Heating pad
  9. Surgical tools including scissors, forceps, scalpels (Fine Science Tools)
  10. 0.6 mm Drill bit connected to the stereotaxic frame (RWD Life Science, catalog number: 78001)
  11. Two-syringe infusion pump (World Precision Instruments, catalog number: SP200iZ)
  12. 10 μl Hamilton Gastight syringe (Hamilton, catalog number: 84875)
  13. White noise machine (Marpac) 
  14. Fiber cable, MM, 200 μm, 0.39 NA, FC/PC-FC/PC (Thorlabs, catalog number: M72L02)
  15. Equipment for optogenetic experiments (Table 2)
    1. 473 nm DPSS Laser system (Laserglow, catalog number: R471003GX)
    2. 532 nm DPSS Laser system (Laserglow, catalog number: R531003GX)
    3. Function/arbitrary waveform generator (BK Precision, catalog number: 4052)

    Table 2. Summary of the main characteristics of the equipment used for optogenetic experiments


  16. Power meter, Si sensor, 400-1,100 nm, 500 nW-500 mW (ThorLabs, catalog number: PM121D)
  17. Laser Glasses, 180-532 nm (ThorLabs, catalog number: LG3) 
  18. Doric mini cube (Doric Lenses, catalog number: B340-0204)
  19. 2 Mono fiberoptic patch cords (Doric Lenses, catalog number: D202-2302)
  20. 45 cm wide x 20 cm long x 30 cm high three-chamber apparatus (ANY-maze)
  21. 8 cm diameter x 17 cm high cylindrical wire cage (ANY-maze)
  22. Autoclave

Software

  1. ANY-maze tracking software (Stoelting Co., http://www.anymaze.co.uk/)

Procedure

  1. Optic fiber generation
    1. Strip a ~5 cm segment of the multimode optic fiber (200 μm diameter, 0.39 NA) using the fiber stripping tool (Figure 1). 


      Figure 1. Optic fiber measurements. A. Schematic of optic fiber thread through a ceramic ferrule. The length (in mm) of the optic fiber depends on the desired targeting region. Add the length of the optic fiber (6.4 mm) and the gap between the ferrule and the skull (0.5 mm) to the Z coordinate of the desired targeting region (denoted as X mm). B. Example of a finished optic fiber.

    2. Use the cleaver to cut a segment of the stripped multimode fiber that is of the appropriate target length as measured using the digital caliper [For example, length = 6.4 mm (ceramic ferrule) + 0.5 mm (gap between ferrule and skull) + desired target depth as determined from a reference atlas in mm] (Figure 2).


      Figure 2. Optic fiber construction. A. Use the digital caliper to measure length of interest. In this example, the Z coordinate of the target region is 3.5 mm. Thus, the total length of the optic fiber is 10.4 mm (3.5 mm + 0.5 mm + 6.4 mm). B. Use the cleaver to cut the appropriate length. C. To secure the optic fiber to the ceramic ferrule, add a drop of epoxy to the middle of the cut optic fiber. D. Push the ferrule over the drop of epoxy to secure it to the optic fiber.

    3. Secure one end of the cut fiber to a ceramic ferrule (230 μm bore size, 1.25 mm outer diameter) using epoxy.
    4. Allow optic fibers to dry overnight.
    5. Autoclave optic fibers prior to implantation. 
    6. Confirm the transmittance of each optic fiber. Compare the laser power measured from the power meter directly from the laser box connected to the fiberoptic patchcord to the laser box connected to the fiberoptic patchcord and the optic fiber. The laser transmittance of each optic fiber must be > 75% to be used for implantation.

  2. Preparation for surgery
    1. Connect the injection cannula to one end of the Tygon tubing while the other end is attached to a 1 ml syringe.
    2. Pipette a desired volume (2 μl/mouse) onto a piece of parafilm.
    3. Fill the injection cannula with the virus by slowly aspirating using the 1 ml syringe. Upon completion, detach the syringe from the Tygon tubing. 
    4. Keep the injection cannula and Tygon tubing with the virus on ice, covered with aluminum foil.
      Note: The injection cannula should be filled with the virus prior to the start of each mouse. The tubing with the virus should be kept on ice for a maximum of 1 h. The virus aliquot should be kept in dry ice.

  3. Viral injection
    1. Remove mouse (4-6 weeks of age) for surgery from home cage and place it into an unused cage (single-housed) prior to surgery for ~15 min.
    2. Place the mouse in the induction chamber of the rodent anesthesia machine (induction rate: 4% isoflurane) and wait 3 min until the respiration of the mouse is steady. Toe pinch to ensure mouse is fully anesthetized.
    3. Transfer the mouse from the induction chamber onto the heating pad (set at 37 °C) and connect the anesthesia mask to the nose (maintenance rate: 2.5% isoflurane). Ensure that the flow rate of anesthesia is steady and the mouse is breathing naturally. 
    4. Insert the ear bar of the stereotaxic frame to ensure mouse is parallel to the base panel of the frame. Add tear gel to both eyes (Figure 3A).
    5. Apply 70% ethanol with a cotton swab to clean the site of incision, followed by betadine, and finally repeating with 70% ethanol. 
    6. Make a horizontal incision, from anterior to posterior, with the scalpel to expose the skull and mark Bregma (Figure 3A).
    7. Use hydrogen peroxide on a cotton swab to dry the surface of the exposed skull.
    8. Mark the skull based on the desired bilateral coordinates (AP/ML) for the viral injection sites using the drill.
    9. Position the injection cannula containing the virus into the stereotaxic frame and ensure it is connected to the Hamilton syringe.
    10. Prior to the injection, program the infusion pump to continuously infuse at 0.1 μl/min until a drop of the virus can be observed by eye.
    11. Use filter paper to remove the small drop and program the infusion pump to withdraw 0.1 μl of air at 0.1 μl/min (to form an air bubble between the tip of the infusion cannula and the virus).
    12. Once set up on the stereotaxic frame, re-measure the desired coordinates (AP/ML) with the injection cannula (the injection cannula should sit directly above the mark on the skull generated by the drill).
    13. Lower the infusion cannula until its tip touches the surface of the skull.
    14. Program the infusion pump to infuse 0.1 µl at a rate of 0.1 µl/min. During the infusion (of the air bubble that was previously created from Step C11), gently lower the infusion cannula to the appropriate DV coordinate.
    15. Wait 2 min prior to infusing the virus and program the infusion pump to infuse 0.5 µl of the virus at a rate of 0.1 µl/min. Once infused, leave the internal cannula in place for an additional 5 min prior to raising.
    16. Raise cannula slowly (~0.1 mm/s). Once removed, clean the tip of the internal cannula with ethanol. Ensure that tip is not blocked and repeat Steps C10-C15 for bilateral injections.

  4. Fiber implantation
    1. Following viral injection, remove the injection cannula from the stereotaxic frame. Ensure the injection cannula and tubing with the virus is stored on ice and covered with foil.
    2. Mark the skull based on the desired bilateral coordinates (AP/ML) for the optic fibers using the drill.
    3. Connect the removable arm to the stereotaxic frame and secure a single optic fiber at the tip of the arm using parafilm (Figure 3B).
    4. Once set-up, re-measure the desired coordinates (AP/ML) with the optic fiber (the optic fiber should sit directly above the mark on the skull generated by the drill) (Figure 3B).
    5. Lower the optic fiber to the appropriate DV coordinate. Gently peel off the parafilm that is connecting the optic fiber to the arm using forceps.
    6. Move the stereotaxic arm away from the optic fiber.
    7. Repeat Steps D3-D6 for the bilateral implantation of the optic fibers (Figure 3C).
    8. Once both optic fibers are implanted, secure both optic fibers with dental cement (Figures 3D and 3E).
      Note: Gently apply dental cement around the base of the optic fibers. Ensure optic fibers remain in place using the arm until the dental cement dries.


      Figure 3. Stereotaxic surgery for bilateral optic fiber implantation. A. Horizontal incision to expose the skull. B. A single optic fiber secured on the removable arm with parafilm at the AP/ML coordinate, prior to being implanted. C. The second optic fiber bilaterally implanted using the removable arm. D. Optic fibers implanted bilaterally. E. Bilateral optic fibers secured with dental cement.
      Note: Anesthesia mask is not displayed in these figures.

    9. Suture the skin and clean surgery site with betadine. Inject appropriate analgesics and place mouse on a separate heating pad for 1 h. Ensure mice are fully recovered prior to returning it to a new cage. Mice are single-housed following surgery to prevent fighting. Monitor post-surgical health (Body weight, hydration status, posture/appearance, grimace [Matsumiya et al., 2012] and behavior) for at least 7 days.

  5. Three-chamber social approach test
    1. Following 7 days of post-surgical recovery, handle mice for 10 min over 3 days in the testing room prior to experimentation. Ensure mice are comfortable with being scruffed and connected the patch cords to the implanted optic fibers via the ceramic sleeves in their home cage. 
    2. Prepare 2 juvenile wild-type stranger mice (3-4 weeks of age, to prevent fighting) that are matched by sex to the subject mice. Single house each stranger mouse, 48 h prior to testing.
    3. Set up camera, lighting (20 lux), and white noise generator (65 db).
    4. Clean the three-chamber apparatus thoroughly using 70% ethanol. Set up the three-chamber apparatus by placing the two empty cages in the outer chambers, while leaving the middle chamber empty.
    5. Twenty-four hours prior to testing, connect each mouse to the patch cords and allow a 3-min home cage habituation period, followed by 10 min of exploration within the three-chamber apparatus. Following both habituation periods, return mouse to home cage.
    6. Twenty-four hours prior to testing, habituate the stranger mice in the stranger cages (Figure 4A) for 10 min.
    7. On the day of experimentation, use the power meter to test the strength for both 532 nm and 473 nm lasers (Ensure the power output is consistent between both patch cords).


      Figure 4. Apparatus for the three-chamber social approach test. A. The cage used to place the juvenile strangers for the test. B. The three-chamber social approach apparatus with two empty cages placed on the two outer chambers.

    8. When ready for testing, connect mouse to the patch cords (which is in turn connected to the waveform generator) and allow a 3-min home cage habituation period, followed by an additional 3-min habituation period in the middle chamber of the three-chamber apparatus. 
    9. Remove the partitions and allow the mouse to freely explore the three-chambers (with 2 empty stranger cages on outer chambers) for 10 min.
    10. Replace partitions following the habituation stage, while ensuring the subject mouse is back in the center chamber for 1 min.
    11. Place the first stranger mouse into one of the two empty stranger cages (the placement of the stranger in the empty cages can be counter-balanced between different subjects, ensure this information is recorded through the software).
    12. Remove the partitions once again and allow the mouse to freely explore the three-chambers for 5 min. The time spent sniffing the stranger mouse in the cage versus the empty cage in the other chamber is recorded manually and through the video tracking system. During this sociability stage, optogenetic manipulations can be applied to test the role of the circuit of interest in social memory encoding. For example: 
      1. For mice expressing ArchT: Bilaterally deliver 532 nm (15 mW and ~119.43 mW/mm2) green-light continuously for 5 min.
        Note: Power can be adjusted accordingly for different regions.
      2. For mice expressing ChR2: Bilaterally deliver 473 nm (20 Hz, 5 ms pulse width, 6.5 mW and ~51.75 mW/mm2) blue-light 30 s light-on followed by 30 s light-off pattern for 5 min.
        Note: Pulse frequency/duration can be adjusted accordingly for different regions. 
    13. Replace partitions following the sociability stage, while ensuring the subject mouse is back in the center chamber for 1 min. 
    14. Place the second stranger mouse into the empty stranger cage.
    15. Remove the partitions and allow the mouse to freely explore the three-chambers once again for 5 min. The time spent sniffing the new stranger mouse versus the familiar stranger mouse is recorded manually and through the video tracking system. During this social recognition memory stage, optogenetic manipulations can be applied to test for memory retrieval (refer to the ArchT and ChR2 conditions in Step E12).
    16. Following testing, lead mice to the middle chamber prior to returning to a new home cage.
    17. Forty-eight hours (minimum) following testing, conduct the counter-balanced test (with/without light delivery) for the same set of mice following Steps E7-E16.
      Note: The counter-balanced behavioral test is conducted 2-7 days after the first test. Previous studies have shown that this paradigm elicits memory only within 24 h following testing (Okuyama et al., 2016). Thus, 48 h is sufficient for the mice for re-testing.

Data analysis

Please refer to Supplemental information from https://doi.org/10.1016/j.celrep.2018.04.073.

  1. All stages were recorded and automatically analyzed using an overhead camera with ANY-maze. The amount of interaction was measured by manually scoring the sniffing time/direct contact when the subject animal oriented its nose or initiated physical contact within 2 cm of the stranger mouse contained in the wired cage (the 2 cm zone surrounding the wired cage was defined as a zone using the ANY-maze software). Climbing on the wire cage was manually excluded from scoring. Data were presented as a Preference (%), which was calculated based on the percentage time spent investigating the target cage over the entire time spent engaged in investigation of either cage (Figure 5).
    Note: Preference (%) calculated through the manually scored interaction time was validated with the Preference (%) determined through the ANY-maze camera software tracking system. Furthermore, this validation helps to address the potential issue of “blind spots” (i.e., top left or bottom right corners); as manual scoring can compensate for these potential issues.


    Figure 5. Zones defined for analysis. A. Zones defined in the three-chamber social approach apparatus. B. Calculation of percentage time spent investigating the target in either the left or right zone, expressed as a sum of both zones.
    Note: Wild-type mice will typically demonstrate a greater Preference (%) for a novel mouse compared to a mouse that they have recently encountered (familiar).

  2. All the data in the graphs were presented as mean ± SEM and statistically evaluated by independent-samples t-tests, paired-samples t-tests or ANOVA (one-way, two-way or repeated measures-RM, wherever appropriate) followed by post-hoc Holm-Sidak's multiple comparisons. P < 0.05 was considered as significant (*P < 0.05, **P < 0.01, ***P < 0.001).

Notes

Please refer to the original publication for examples of the expected results and more applications of this protocol https://doi.org/10.1016/j.celrep.2018.04.073. Although this protocol focuses on the inhibition or activation of neuronal populations, with ArchT or ChR2, it is important to note that alternative systems such as halorhodopsins (NpHR) for inhibition are also effective.

Acknowledgments

This project was supported by grants from the Canadian Institutes of Health Research (CIHR) (MOP119421 and PJT-155959, Canadian Natural Science and Engineering Research Council (NSERC) (RGPIN341498 and RGPIN-2017-06295), and the Hospital for Sick Children. The authors thank Feng Cao for her technical assistance in the protocol images.

Competing interests

The authors declare no conflicts of interest or competing interests.

Ethics

All experimental procedures were conducted in accordance with the guidelines of the Canadian Council on Animal Care (CCAC) and approved by the Animal Care Committees at the Hospital for Sick Children and the University of Toronto.

References

  1. Felix-Ortiz, A. C. and Tye, K. M. (2014). Amygdala inputs to the ventral hippocampus bidirectionally modulate social behavior. J Neurosci 34(2): 586-595. 
  2. Gunaydin, L. A., Grosenick, L., Finkelstein, J. C., Kauvar, I. V., Fenno, L. E., Adhikari, A., Lammel, S., Mirzabekov, J. J., Airan, R. D., Zalocusky, K. A., Tye, K. M., Anikeeva, P., Malenka, R. C. and Deisseroth, K. (2014). Natural neural projection dynamics underlying social behavior. Cell 157(7): 1535-1551. 
  3. Hitti, F. L. and Siegelbaum, S. A. (2014). The hippocampal CA2 region is essential for social memory. Nature 508(7494): 88-92. 
  4. Insel, T. R. and Fernald, R. D. (2004). How the brain processes social information: searching for the social brain. Annu Rev Neurosci 27: 697-722. 
  5. Leung, C., Cao, F., Nguyen, R., Joshi, K., Aqrabawi, A. J., Xia, S., Cortez, M. A., Snead, O. C., 3rd, Kim, J. C. and Jia, Z. (2018). Activation of entorhinal cortical projections to the dentate gyrus underlies social memory retrieval. Cell Rep 23(8): 2379-2391. 
  6. Lieberman, M. D. (2007). Social cognitive neuroscience: a review of core processes. Annu Rev Psychol 58: 259-289. 
  7. Matsumiya, L. C., Sorge, R. E., Sotocinal, S. G., Tabaka, J. M., Wieskopf, J. S., Zaloum, A., King, O. D. and Mogil, J. S. (2012). Using the Mouse Grimace Scale to reevaluate the efficacy of postoperative analgesics in laboratory mice. J Am Assoc Lab Anim Sci 51(1): 42-49. 
  8. Nadler, J. J., Moy, S. S., Dold, G., Trang, D., Simmons, N., Perez, A., Young, N. B., Barbaro, R. P., Piven, J., Magnuson, T. R. and Crawley, J. N. (2004). Automated apparatus for quantitation of social approach behaviors in mice. Genes Brain Behav 3(5): 303-314. 
  9. Okuyama, T., Kitamura, T., Roy, D. S., Itohara, S. and Tonegawa, S. (2016). Ventral CA1 neurons store social memory. Science 353(6307): 1536-1541.
  10. Olson, I. R., McCoy, D., Klobusicky, E. and Ross, L. A. (2013). Social cognition and the anterior temporal lobes: a review and theoretical framework. Soc Cogn Affect Neurosci 8(2): 123-133. 
  11. Sandi, C. and Haller, J. (2015). Stress and the social brain: behavioural effects and neurobiological mechanisms. Nat Rev Neurosci 16(5): 290-304.

简介

通过社会互动和记忆形成社会关系对于一个人的身心健康至关重要。迄今为止,啮齿动物研究已经使用三室社会方法测试来衡量社会方法,社会新奇和社会记忆。近年来,已开发出包括光遗传学在内的技术以急性控制遗传定义的神经元群体的活性。最近的研究甚至将光遗传学与先进的时间基因表达控制系统相结合,以在学习期间标记某些神经元群体,并随后重新激活以进行记忆测试。我们将光遗传学靶向与三室社会方法测试相结合,以检查社交记忆编码或检索期间感兴趣的特定神经回路。首先,我们通过病毒编码视蛋白立体定位感染特定大脑区域,这些视蛋白可以急性激活或抑制神经元的激活。接下来,我们让老鼠接受三室行为范式,同时在社交记忆编码或检索过程中提供光线。最后,以反平衡的方式测试小鼠的光照,使每个受试者成为其自身的内部对照。因此,光遗传学刺激与三室社会方法测试相结合是一种经过充分验证的范例,可以探索不同脑回路在各种社会认知过程中的贡献。

【背景】社会认知对我们的心理健康至关重要。社交互动和记忆中的缺陷是许多脑部疾病的标志性特征。社会工作记忆是一个极其动态的过程,往往是不可预测的,需要不断适应变化的刺激(Lieberman,2007)。具体而言,它是一个涉及社会显着信息的编码,存储和检索的认知过程。使用成像和病变实验的传统人类和灵长类研究已经将内侧颞叶与社会认知任务联系起来,包括社会认知和背景评估(Insel和Fernad,2004; Olson et al。,2013; Sandi and哈勒,2015年)。然而,这些先前的研究使用毒素,其对大脑中的广泛区域造成广泛损害并且限制了靶细胞类型和亚细胞区室的控制。最近采用急性光遗传学方法的研究表明,包括杏仁核,海马和腹侧被盖区在内的各种大脑区域和环路在社会行为的不同方面发挥着关键作用,包括社会互动,方法和歧视记忆(Felix-Ortiz)和Tye,2014; Gunaydin et al。,2014; Hitti和Siegelbaum,2014; Okuyama et al。,2016)。

为了研究特定大脑区域在社会记忆处理中的行为意义,可以在经过充分验证的三室社会方法范例中评估受试小鼠的表现(Nadler et al。,2004)。在该测试中,使受试小鼠习惯于该装置(阶段1)。然后首先将其引入新的陌生小鼠(S1)以评估记忆形成和社交性(第2阶段),随后呈现给第二个新颖的陌生小鼠(S2)以测试社交识别记忆和检索(第3阶段)。记录小鼠花在调查陌生小鼠上的时间。与熟悉的同类相比,野生型小鼠花费更多时间探索新奇怪物。因此,结合光遗传学靶向,可以在包括记忆形成(编码)或检索(鉴别)的各个阶段期间激活或抑制特定的神经回路。此外,光遗传学的使用还可以识别神经回路中可能参与小鼠社交性和/或社会识别记忆调节的特定基因(Leung et al。,2018)。

关键字:社交途径, 社交识别, 社会记忆, 记忆编码, 记忆提取, 光遗传学

材料和试剂

  1. 过滤纸
  2. 多模光纤(0.39 NA,高OH,200μm核心,波长范围:300-1,200 nm; ThorLabs,目录号:FT200UMT)
  3. 用于光遗传学实验的材料(表1)
    1. 多模光纤(0.39 NA,高OH,200μm核心,波长范围:300-1,200 nm; ThorLabs,目录号:FT200UMT)
    2. 1.25 mm陶瓷棒套圈,230μm(精密纤维产品,目录号:MM-FER2007C-2000)&nbsp;

    表1.用于光遗传学实验的材料的主要特征总结


  4. 注射插管(5 mm,26 gauge; Plastics One,目录号:C315GS-5 / SPC)
  5. 1毫升注射器(BD Sciences,目录号:309628)
  6. Tygon管1/16“I.D。 1/8“O.D。和1/32“墙(美国塑料,目录号:57102)
  7. 铝箔(Alcan)
    注意:陶瓷插芯选用金属插芯,以确保准确的光纤对准。
  8. 1.25 mm SM陶瓷开口套管,长6.60 mm(精密纤维产品,产品目录号:SM-CS125S)
    注意:陶瓷开口套管选用金属开口套管,以确保准确的纤维对齐。
  9. 1.25 mm套圈防尘盖,白色(Precision Fiber Products,目录号:BCDC-1300-W)
  10. Parafilm M(Pechiney塑料包装,目录号:PM-996)
  11. 棉签(The Lab Depot,目录号:394305)
  12. C57BL / 6小鼠(The Jackson Laboratory,目录号:000664)
    注意:使用此协议已成功训练男性和女性受试者(4周+年龄)和陌生人(<3周龄)。
  13. AAV病毒(储存于-80°C直至使用,滴度为~10 11 至10 13 pfu / ml)
    例如:AAV-CaMKIIα:eArchT3.0-EYFP(北卡罗来纳大学GTC矢量核心)
    AAV-CaMKIIa-hChR2(H134R)-mCherry-WFRE-PA(北卡罗来纳大学GTC载体核心)。
  14. 牙科水泥(Stoelting Co.,目录号:51458)&nbsp;
  15. 速度设置即时混合环氧树脂(LePage, http://www.lepage.ca/en/lepage-products/epoxy-glue/speed-set-instant-mix-epoxy.html )
  16. 缝合套件(Ethicon,目录号:JJ489)
  17. 撕裂凝胶(诺华,https: //well.ca/products/tear-gel-liquid-eye-gel_17561.html?gclid=CjwKCAjw6-_eBRBXEiwA-5zHaVuKltJxR7GYDuMwgeSM5yh_3J6FSiTehp0ekoUOvym2ICm3-YjZTxoCIkoQAvD_BwE )
  18. 聚维酮碘
  19. 盐水
  20. 70%乙醇&nbsp;
  21. 过氧化氢
  22. 镇痛药:Metacam(CDMV,连续三天每日注射5 mg / ml)

设备

  1. Cleaver(Doric Lenses,目录号:B600-0002)
  2. 光纤剥线工具(ThorLabs,目录号:T12S21)
  3. 10μl移液器(SARSTEDT,目录号:90.1771.002)&nbsp;
  4. 数字卡尺(ULINE Canada)
  5. 小鼠立体定位框架(Neurostar)&nbsp;
  6. 异氟醚的麻醉系统(Kent Scientific Corporation,目录号:SOMNO-MSEKIT)
  7. 温度控制器(Sunbeam)
  8. 加热垫
  9. 手术工具,包括剪刀,镊子,手术刀(精细科学工具)
  10. 0.6 mm钻头连接到立体定位框架(RWD Life Science,目录号:78001)
  11. 双注射器输液泵(World Precision Instruments,目录号:SP200iZ)
  12. 10μlHamiltonGastight注射器(汉密尔顿,目录号:84875)
  13. 白噪声机(Marpac)&nbsp;
  14. 光纤电缆,MM,200μm,0.39 NA,FC / PC-FC / PC(Thorlabs,目录号:M72L02)
  15. 光遗传学实验设备(表2)
    1. 473 nm DPSS激光系统(Laserglow,目录号:R471003GX)
    2. 532 nm DPSS激光系统(Laserglow,目录号:R531003GX)
    3. 功能/任意波形发生器(BK Precision,目录号:4052)

    表2.用于光遗传学实验的设备的主要特征总结


  16. 功率计,Si传感器,400-1,100 nm,500 nW-500 mW(ThorLabs,目录号:PM121D)
  17. 激光眼镜,180-532 nm(ThorLabs,目录号:LG3)&nbsp;
  18. Doric迷你立方体(Doric Lenses,目录号:B340-0204)
  19. 2个单声道光纤跳线(Doric Lenses,目录号:D202-2302)
  20. 45厘米宽x 20厘米长x 30厘米高的三室设备(ANY-maze)
  21. 8厘米直径x 17厘米高圆柱形线笼(ANY-maze)
  22. 高压灭菌器

软件

  1. ANY-maze跟踪软件(Stoelting Co., http://www.anymaze.co.uk/ )

程序

  1. 光纤生成
    1. 使用光纤剥离工具(图1)剥去多模光纤(直径200μm,0.39 NA)的~5 cm区段。&nbsp;


      图1.光纤测量。 A.光纤穿过陶瓷插芯的示意图。光纤的长度(以mm为单位)取决于所需的靶向区域。将光纤的长度(6.4 mm)和套圈与颅骨之间的间隙(0.5 mm)添加到所需瞄准区域的Z坐标(表示为X mm)。 B.成品光纤的例子。

    2. 使用切割刀切割剥离的多模光纤的一段,该光纤具有使用数字卡尺测量的适当目标长度[例如,长度= 6.4 mm(陶瓷套圈)+ 0.5 mm(套圈和颅骨之间的间隙)+所需目标深度由参考地图集(mm)确定(图2)。


      图2.光纤结构。 A.使用数字卡尺测量感兴趣的长度。在该示例中,目标区域的Z坐标是3.5mm。因此,光纤的总长度为10.4毫米(3.5毫米+ 0.5毫米+6.4毫米)。 B.使用切割刀切割适当的长度。 C.为了将光纤固定到陶瓷插芯上,在切割光纤的中间添加一滴环氧树脂。 D.将套圈推到环氧树脂滴上以将其固定到光纤上。

    3. 使用环氧树脂将切割光纤的一端固定到陶瓷套圈(孔径230μm,外径1.25 mm)。
    4. 让光纤干燥过夜。
    5. 植入前高压灭菌光纤。&nbsp;
    6. 确认每根光纤的透射率。将功率计测量的激光功率直接从连接到光纤跳线的激光盒到连接到光纤跳线和光纤的激光盒进行比较。每根光纤的激光透射率必须> 75%用于植入。

  2. 准备手术
    1. 将注射插管连接到Tygon管的一端,而另一端连接到1 ml注射器。
    2. 将所需体积(2μl/小鼠)移液到一片封口膜上。
    3. 通过使用1ml注射器缓慢吸出,向注射套管中注入病毒。完成后,将注射器从Tygon管上拆下。&nbsp;
    4. 将注射插管和Tygon管与病毒一起放在冰上,用铝箔覆盖。
      注意:在每只小鼠开始之前,注射插管应该充满病毒。带有病毒的管道应保持在冰上最多1小时。病毒等分试样应保存在干冰中。

  3. 病毒注射
    1. 从家笼中取出小鼠(4-6周龄)进行手术,并在手术前将其放入未使用的笼子(单个容器)中约15分钟。
    2. 将小鼠置于啮齿动物麻醉机的诱导室中(诱导率:4%异氟烷)并等待3分钟直至小鼠呼吸稳定。脚趾夹紧,以确保鼠标完全麻醉。
    3. 将鼠标从诱导室转移到加热垫(设置在37°C)并将麻醉面罩连接到鼻子(维持率:2.5%异氟烷)。确保麻醉流速稳定,小鼠自然呼吸。&nbsp;
    4. 插入立体定位框架的耳杆,确保鼠标平行于框架的底板。在双眼上加入泪液凝胶(图3A)。
    5. 用棉签涂抹70%乙醇清洁切口部位,然后用聚维酮碘,最后用70%乙醇重复。&nbsp;
    6. 用手术刀从前到后做一个水平切口,露出颅骨并标记前囟(图3A)。
    7. 在棉签上使用过氧化氢来干燥暴露的头骨表面。
    8. 使用钻头根据病毒注射部位所需的双侧坐标(AP / ML)标记颅骨。
    9. 将含有病毒的注射插管定位到立体定位框架中并确保其连接到Hamilton注射器。
    10. 在注射之前,将输注泵编程为以0.1μl/ min连续输注,直到可以通过眼睛观察到一滴病毒。
    11. 使用滤纸去除小滴,并对输液泵进行编程,以0.1μl/ min的速度取出0.1μl空气(在输液插管尖端和病毒之间形成气泡)。
    12. 一旦设置在立体定位框架上,用注射套管重新测量所需的坐标(AP / ML)(注射套管应直接位于钻头产生的颅骨上的标记上方)。
    13. 降低输液套管,直到其尖端接触颅骨表面。
    14. 对输液泵进行编程,以0.1μl/ min的速率注入0.1μl。在输注(先前从步骤C11产生的气泡)期间,将输注套管轻轻降低到适当的DV坐标。
    15. 在输注病毒前等待2分钟,然后对输液泵进行编程,以0.1μl/ min的速率注入0.5μl病毒。注入后,将内部插管在放置前再放置5分钟。
    16. 慢慢抬起插管(~0.1 mm / s)。取下后,用乙醇清洁内部插管的尖端。确保尖端未被堵塞,并重复步骤C10-C15进行双侧注射。

  4. 纤维植入
    1. 病毒注射后,从立体定位框架中取出注射套管。确保带有病毒的注射插管和管道储存在冰上并用箔覆盖。
    2. 使用钻头根据所需的双边坐标(AP / ML)标记颅骨。
    3. 将可拆卸臂连接到立体定位框架,并使用封口膜将单根光纤固定在手臂末端(图3B)。
    4. 设置完成后,用光纤重新测量所需的坐标(AP / ML)(光纤应直接位于钻头产生的颅骨上的标记上方)(图3B)。
    5. 将光纤降低到适当的DV坐标。使用镊子轻轻剥离将光纤连接到手臂的封口膜。
    6. 将立体定位臂远离光纤。
    7. 重复步骤D3-D6以进行光纤的双侧植入(图3C)。
    8. 一旦植入两根光纤,用牙科粘固剂固定两根光纤(图3D和3E)。
      注意:在光纤底部周围轻轻涂抹牙科粘固剂。使用手臂确保光纤保持在原位,直到牙科粘固剂干燥。


      图3.双侧光纤植入的立体定向手术。 :一种。水平切口暴露头骨。 B.在植入之前,单个光纤固定在可移除臂上,在AP / ML坐标处具有封口膜。 C.使用可移除臂双侧植入的第二光纤。 D.双侧植入的光纤。 E.用牙科粘固剂固定的双侧光纤。
      注意:这些图中未显示麻醉面罩。

    9. 用betadine缝合皮肤和清洁手术部位。注射适当的镇痛药并将鼠标放在单独的加热垫上1小时。确保小鼠在将其放回新笼子之前完全恢复。小鼠在手术后单独饲养以防止搏斗。监测手术后的健康状况(体重,水合状态,姿势/外观,鬼脸[Matsumiya 等,2012]和行为)至少7天。

  5. 三室社会方法测试
    1. 在手术后恢复7天后,在实验前在测试室中处理小鼠3天,持续10分钟。确保老鼠能够舒适地进行揉搓,并通过家笼中的陶瓷套管将跳线连接到植入的光纤上。&nbsp;
    2. 准备2只与受试小鼠性别匹配的幼年野生型陌生小鼠(3-4周龄,以防止战斗)。单个房子每个陌生人的老鼠,测试前48小时。
    3. 设置摄像头,照明(20 lux)和白噪声发生器(65 db)。
    4. 使用70%乙醇彻底清洁三室装置。通过将两个空笼放置在外室中来设置三室装置,同时使中间室留空。
    5. 在测试前24小时,将每只小鼠连接到接插线并允许3分钟的家庭笼子适应期,然后在三室装置内进行10分钟的探索。在两个适应期后,将鼠标归还到家笼。
    6. 在测试前24小时,使陌生笼中的陌生小鼠习惯(图4A)10分钟。
    7. 在实验当天,使用功率计测试532 nm和473 nm激光器的强度(确保两个跳线之间的功率输出一致)。


      图4.用于三腔室社交方法测试的设备。 A.用于放置少年陌生人进行测试的笼子。 B.三室社会进场装置,在两个外室上放置两个空笼。

    8. 准备好进行测试时,将鼠标连接到接插线(然后连接到波形发生器)并允许3分钟的家庭笼子适应期,然后在三个中间室中再加上3分钟的适应期。腔室装置。&nbsp;
    9. 取下隔板,让鼠标自由探索三室(外室有2个空陌生的笼子)10分钟。
    10. 在适应阶段后更换隔板,同时确保主体鼠标回到中心室1分钟。
    11. 将第一个陌生人的鼠标放入两个空的陌生人笼子中的一个(陌生人在空笼中的位置可以在不同的主体之间进行平衡,确保通过软件记录此信息)。
    12. 再次移除分区,让鼠标自由探索三个腔室5分钟。在手中并通过视频跟踪系统记录在笼子中嗅探陌生人鼠标与在另一个房间中空笼子所花费的时间。在这个社交阶段,可以应用光遗传学操作来测试感兴趣的电路在社交记忆编码中的作用。例如:&nbsp;
      1. 对于表达ArchT的小鼠:连续5分钟地双峰递送532nm(15mW和~119.43mW / mm 2 )绿光。
        注意:可以针对不同地区相应调整功率。
      2. 对于表达ChR2的小鼠:双侧递送473nm(20Hz,5ms脉冲宽度,6.5mW和~51.75mW / mm 2 )蓝光30秒点亮,然后30秒点亮模式5分钟。
        注意:可以针对不同地区相应地调整脉冲频率/持续时间。&nbsp;
    13. 在社交阶段之后更换分区,同时确保主题鼠标回到中心室1分钟。&nbsp;
    14. 将第二个陌生人的老鼠放入空陌生的笼子里。
    15. 取下分区,让鼠标再次自由探索三个腔室,持续5分钟。通过手动和视频跟踪系统记录嗅探新陌生鼠标与熟悉的陌生鼠标的时间。在该社会识别记忆阶段期间,可以应用光遗传学操作来测试记忆检索(参见步骤E12中的ArchT和ChR2条件)。
    16. 在测试之后,在返回新的笼子之前将小鼠带到中间室。
    17. 测试后48小时(最小),按照步骤E7-E16对同一组小鼠进行平衡测试(有/无光传递)。
      注意:在第一次测试后2-7天进行平衡行为测试。以前的研究表明,这种范式仅在测试后24小时内引发记忆(Okuyama 等 ,2016)。因此,48小时足以让小鼠重新测试。

数据分析

请参阅 https://doi.org/10.1016/j.celrep.2018.04的补充信息0.073 。

  1. 记录所有阶段并使用具有ANY-maze的顶置相机自动分析。当受试动物定向其鼻子或在有线笼子中包含的陌生小鼠2cm内开始物理接触时,通过手动评分嗅探时间/直接接触来测量相互作用量(围绕有线笼子的2cm区域被定义为使用ANY-maze软件的区域)。手动将绳子上的攀爬从评分中排除。数据以偏好(%)表示,其基于在调查任一笼子的整个时间内调查目标笼子所花费的时间百分比来计算(图5)。
    注意:通过手动评分的交互时间计算的偏好(%)通过ANY-maze相机软件跟踪系统确定的偏好(%)进行验证。此外,这种验证有助于解决潜在的“盲点”问题(即左上角或右下角);因为人工评分可以弥补这些潜在的问题。


    图5.为分析定义的区域。 :一种。在三室社会进场设备中定义的区域。 B.计算在左侧或右侧区域调查目标所花费的百分比时间,表示为两个区域的总和。
    注意:与他们最近遇到的(熟悉的)小鼠相比,野生型小鼠通常会表现出更高的偏好(%)。

  2. 图中的所有数据均以平均值±SEM表示,并通过独立样本 t - 测试,配对样本 t - 测试或ANOVA(单向,双向或重复测量-RM,在适当的情况下),然后进行事后Holm-Sidak的多重比较。 P &lt; 0.05被认为是显着的(* P <0.05,** P <0.01,*** P <0.001)。

笔记

有关此协议的预期结果和更多应用的示例,请参阅原始出版物 https:/ /doi.org/10.1016/j.celrep.2018.04.073 。尽管该方案着重于抑制或激活神经元群体,但使用ArchT或ChR2,重要的是要注意用于抑制的替代系统如halorhodopsin(NpHR)也是有效的。

致谢

该项目得到了加拿大卫生研究院(CIHR)(MOP119421和PJT-155959,加拿大自然科学和工程研究理事会(NSERC)(RGPIN341498和RGPIN-2017-06295)以及病童医院的资助。作者感谢Feng Cao在协议图像方面的技术帮助。

利益争夺

作者声明没有利益冲突或竞争利益。

伦理

所有实验程序均按照加拿大动物保护协会(CCAC)的指导方针进行,并经病童医院动物护理委员会和多伦多大学批准。

参考

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  2. Gunaydin,LA,Grosenick,L.,Finkelstein,JC,Kauvar,IV,Fenno,LE,Adhikari,A.,Lammel,S.,Mirzabekov,JJ,Airan,RD,Zalocusky,KA,Tye,KM,Anikeeva,P 。,Malenka,RC和Deisseroth,K。(2014年)。 社会行为背后的自然神经投影动态。 Cell 157 (7):1535-1551。&nbsp;
  3. Hitti,F。L.和Siegelbaum,S。A.(2014)。 海马CA2区域对社交记忆至关重要。 自然 508(7494):88-92。&nbsp;
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  5. Leung,C.,Cao,F.,Nguyen,R.,Joshi,K.,Aqrabawi,AJ,Xia,S.,Cortez,MA,Snead,OC,3rd,Kim,JC and Jia,Z。(2018) 。 激活对齿状回的内嗅皮质投射是社会记忆检索的基础。 Cell Rep 23(8):2379-2391。&nbsp;
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引用:Leung, C., Kim, J. C. and Jia, Z. (2018). Three-chamber Social Approach Task with Optogenetic Stimulation (Mice). Bio-protocol 8(24): e3120. DOI: 10.21769/BioProtoc.3120.
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