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Labeling Aversive Memory Trace in Mouse Using a Doxycycline-inducible Expression System
使用多西环素诱导表达系统标记小鼠厌恶记忆痕迹   

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Abstract

A memory trace, also known as a memory engram, is theorized to be a mechanism for physical memory storage in the brain (Silva et al., 2009; Josselyn, 2010) and memory trace is associated with a specific population of neurons (Liu et al., 2012; Ramirez et al., 2013). Labeling and stimulating those neurons will activate the memory trace (Liu et al., 2012; Ramirez et al., 2013). Memory appears to be spread over different regions of the brain rather than being localized to one area. Therefore, the methods used to trace memory have the ability to improve our understanding of neuronal circuits. In this protocol, we introduce a doxycycline-inducible expression system to label the specific neurons associated with the original memory trace.

Keywords: Aversive memory(厌恶记忆), Memory trace(记忆痕迹), TetTag Fos-tTA mouse(TetTag Fos-tTA小鼠 ), AAV9-TRE-mCherry(AAV9-TRE-mCherry), Doxycycline-inducible expression system(多西环素诱导表达系统)

Background

Memory trace is a theoretical means by which memory is stored as a physical or biochemical change in the brain (Ryan et al., 2015). After the concept of memory trace was formulated by German zoologist Richard Semon at the turn of the twentieth century, the specific process involved in memory storage has been an unsolved topic of debate in the field of neuroscience (Poo et al., 2016). Although the mechanism of memory has been a topic of debate for decades, there has been agreement that specific neurons are utilized in the storage of memories (Liu et al., 2012; Ramirez et al., 2013). In addition, it is believed that memory is not localized to one area, but is instead a mechanism that takes place over many different regions of the brain. Early researchers used a combination of behavioral and surgical approaches to identify memory traces (Bruce, 2001). Although we have learned much from these behavioral and surgical approaches, these methods have not provided us the ability to visualize or identify the target neurons used within a memory trace. In the brain, the expression of immediate-early genes such as Fos, is rapidly upregulated in the specific neurons that are associated with learning and memory formation. Recent studies addressed this issue using the mouse TetTag-Fos driven-GFP mouse model (Reijmers et al., 2007) combined with an adeno-associated virus (AAV9) encoding a TRE-mCherry (Liu et al., 2012; Ramirez et al., 2013). In the interest of continuing the work of identifying target neurons involved in memory trace, we generated Fos-tTA mice from TetTag mice by breeding them with C57BL/6J mice and choosing those that carry only the Fos-tTA transgene. The Fos-tTA mice have Fos promoter-driven expression of nuclear-localized, 2-h half-life EGFP. The Fos promoter also drives expression of the tetracycline transactivator (tTA), which can bind to the tetracycline-responsive element (TRE) site on an injected AAV9-TRE-mCherry virus, resulting in the expression of mCherry. In the Fos-tTA mouse system, application of doxycycline (Dox) inhibits binding of the Fos promoter-driven tTA to the TRE site, preventing target gene expression. We inject the AAV9-TRE-mCherry into the amygdala of the Fos-tTA mice. In the absence of Dox, aversive conditioning activates Fos driving mCherry expression in the targeted neurons (Figure 1). We recently employed this technique to determine if CO2/ASICs enhances the memory trace during retrieval (Du et al., 2017). Other studies have utilized the approaches to identify the specific memory engrams and re-activated it using optogenetics (Liu et al., 2012; Ramirez et al., 2013). In summary, this is a powerful technique to identify a memory trace and will strongly benefit the future study of neuronal circuits, learning, and memories.


Figure 1. Schematic showing how neurons are labeled in mice transgenic for TetTag Fos-tTA and microinjected with the viral vector AAV9-TRE-mCherry. A. Neuronal activity activates the Fos promoter. The Fos promoter drives expression of the tetracycline transactivator (tTA) and shEGFP. The shEGFP protein is rapidly degraded with a 2-h half-life. The tTA protein binds to its target, the tetracycline-responsive element (TRE) site on the microinjected AAV9-TRE-mCherry construct, resulting in the expression of mCherry in neurons. The presence of Dox inhibits tTA from binding to the TRE and thus prevents the expression of mCherry. B. The vector map showing how the pAAV-TRE-mCherry plasmid was constructed. AAV9 viruses containing the construct was packaged by the University of Iowa Gene Transfer Vector Core. The vector map was generated by SnapGene 4.0.

Materials and Reagents

  1. Pipette tips (USA Scientific, catalog numbers: 1112-1820 ; 1110-1800 ; 1111-3840 )
  2. Iodine wipes (Dynarex, catalog number: 1108 )
  3. Alcohol Prep pads (COVIDIEN, catalog number: 5750 )
  4. Butterfly needles, 23 G (Thermo Fisher Scientific, catalog number: 14-840-35)
    Manufacturer: Exel International, catalog number: 26706 .
  5. 24-well culture plate (Corning, catalog number: 3527 )
  6. Aluminum foil (Thermo Fisher Scientific, catalog number: NC9847171)
    Manufacturer: Reynolds Consumer Products, catalog number: 632 .
  7. Staining basket (PELCO Prep-EzeTM 24-well plate Insert) (Ted Pella, catalog number: 36172 )
  8. Super glue (Loctite® Professional Super Glue, Henkel, catalog number: 1365882 )
  9. Single edge razor blade (Fisher Scientific, catalog number: 12-640 )
  10. Coverslip (Fisher Scientific, catalog number: 12-545M )
  11. C57BL/6J mouse (THE JACKSON LABORATORY, catalog number: 000664 )
    Note: Mice are group housed before and during the experiment.
  12. B6.Cg-Tg(Fos-tTA,Fos-EGFP*)1Mmay/J mouse (THE JACKSON LABORATORY, catalog number: 018306 )
  13. AAV9-TRE-mCherry virus is produced by the University of Iowa Gene Transfer Vector Core and is commercially available in their stock list (Virus name: AAV2/9-TRE-mCherry, CsCl 1st; Lot # AAV 2679; Tier: 1.45E+12 µg/ml) (Figure 1B)
    Note: Aliquots should be stored at -80 °C.
  14. Customized Doxycycline diet, 40 ppm (Envigo, catalog number: TD.10483 )
    Note: Stored at 4 °C.
  15. Tissue adhesive, 3 ml (3M, VetbondTM, catalog number: 70200742529 )
  16. Rabbit polyclonal IgG anti-RFP (Rockland Immunochemicals, catalog number: 600-401-379 )
    Note: Aliquots should be stored at -20 °C.
  17. Chicken IgY anti-GFP (Thermo Fisher Scientific, catalog number: A10262 )
  18. Mouse anti-NeuN, clone A60 (EMD Millipore, catalog number: MAB377X )
  19. Alexa Fluor 488 goat anti-chicken IgG (H+L) (Thermo Fisher Scientific, catalog number: A-11039 )
  20. Alexa Fluor 568 goat anti-rabbit IgG (H+L) (Thermo Fisher Scientific, catalog number: A-11036 )
  21. Alexa Fluor 647 goat anti-mouse IgG (H+L) (Thermo Fisher Scientific, catalog number: A-21235 )
  22. VectaShield H-1500 (Vector Laboratories, catalog number: H-1500 )
  23. Heparin, 10 kU/10 ml (Sagent Pharmaceuticals, catalog number: 25021-400-10 )
  24. Phosphate-buffered saline (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  25. 20% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15713-S )
  26. SuperBlock (PBS) blocking buffer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 37515 )
  27. Triton X-100 (Fisher Scientific, catalog number: BP151-100 )
  28. Ketamine hydrochloride injection, 10 ml-200 mg/ml (Wildlife Pharmaceuticals, ZooPharm compounding pharmacy)
  29. Xylazine HCl, 30 ml-300 mg/ml (Wildlife Pharmaceuticals, ZooPharm compounding pharmacy)
  30. Isoflurane (Henry Schein, catalog number: 029404 )
  31. 100% oxygen cylinder (Airgas, catalog number: UN 1072 )
  32. Alcohol, 140 proof (Decon Labs, catalog number: 2401TP )
  33. PBS/Heparin solution (see Recipes)
  34. PBS/4% paraformaldehyde (PAF) solution (see Recipes)
  35. Blocking solution (see Recipes)
  36. Primary antibody solution (see Recipes)
  37. Ketamine/xylazine cocktail (see Recipes)

Equipment

  1. Anesthesia vaporizer (DRE Medical, model: Drager 19.1 )
  2. Microsyringe, 10 µl, 700 series (Hamilton, catalog number: 80314 )
  3. Small hub removable needles, 33 G, 0.4 inch (Hamilton, catalog number: 7803-05 )
  4. NIR video fear conditioning system for mouse (Med Associates, catalog number: MED-VFC-SCT-M )
  5. Super professional animal clipper (Braintree Scientific, catalog number: CLP-64 800 )
  6. Blunt-ended scissors (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 78702 )
  7. Blunt-ended forceps (Fisher Scientific, catalog number: 13-812-39 )
  8. Iridectomy scissors (Fisher Scientific, catalog number: 50-109-3945 )
    Manufacturer: Sklar Surgical Instruments, catalog number: 642035 .
  9. Point-ended scissors (Fisher Scientific, catalog number: 13-808-2 )
  10. Hemostats Rankin forceps (Fisher Scientific, catalog number: 13-812-45 )
  11. High-speed rotary micromotor drill kit (Blackstone Industries, Foredom, catalog number: K.1070 )
  12. Microsyringe pump (World Precision Instruments, model: UMP3 )
  13. Microsyringe pump controller (World Precision Instruments, model: Micro4 )
  14. Metal spatula (Fisher Scientific, catalog number: 14-374 )
  15. Vibrating blade microtome (Leica Biosystems, model: Leica VT1000 S )
  16. Shaker (Cole-Parmer, Stuart, model: SSL3 )
  17. Pipettes, single channel, 0.2 µl-1 ml (Gibson, model: PiPETMAN® Classic )
  18. Confocal microscope (Olympus, model: FV1000 )
  19. 4 °C refrigerator (Whirlpool, model: WRR56X18FW )
  20. Stereotaxic instrument (KOPF Instruments, model: Model 940 )
  21. Diamond burr grinding bit, Ø0.60 mm (Stoelting, catalog number: 514552 )
  22. Stereo microscope (Amscope, catalog number: SM-5TZ-FRL )
  23. Mini Heat Mat, 4 x 7” (All Living Things®, PETSMART.com)

Software

  1. Imaris Image Analysis Software (Oxford instruments plc.)
  2. ImageJ software (Downloaded from NIH webpage)
  3. SnapGene 4.0 (GSL Biotech LLC)

Procedure

Note: Animal care and procedures met National Institutes of Health standards. Local animal care ethical standards must be adhered to. The University of Iowa Animal Care and Use Committee (ACURF #4041016) and the University of Toledo Institutional Animal Care and Use Committee (Protocol #108791) approved all procedures.

  1. TetTag Fos-tTA mouse generation
    1. The TetTag Fos-tTA mice are generated from the B6.Cg-Tg (Fos-tTA,Fos-EGFP*)1Mmay/J mice by breeding them with C57BL/6J mice and choosing those that carry only the Fos-tTA transgene (Liu et al., 2012; Ramirez et al., 2013). The University of Iowa Genome Editing Core Facility provides mouse genotyping service. The genotyping protocols can be found at https://www.jax.org/strain/018306.
    2. Figure 1 shows how neurons are labeled in mice transgenic for TetTag Fos-tTA and microinjected with the viral vector AAV9-TRE-mCherry.

  2. Viral construct (Figure 1B) and stereotactic injection (Figure 2)


    Figure 2. Schematic showing the procedure of labeling the memory retrieval-induced activation of lateral amygdala neurons in the aversive memory trace. A. Mice are fed Dox for 1 week, and then an AAV9 vector encoding mCherry is microinjected into the amygdala. Dox inhibits mCherry expression. Two weeks later, Dox is discontinued, and mice undergo the aversive conditioning protocol. Under those conditions, the Fos promoter will drive both shEGFP and mCherry expression, but shEGFP will rapidly decay. After aversive conditioning, mice immediately resume Dox treatment. One day later, the mice undergo the retrieval protocol. Thirty minutes after that, brain slices are prepared and the shEGFP- and mCherry-positive neurons in the lateral amygdala are determined. B. The stereotaxic system for the surgery of virus injection.

    1. Twelve-week-old mice are fed Dox diet (40 mg/kg) for one week before surgery.
    2. Sterilize the surgery area, instrument, and materials.
    3. Mice are anesthetized using ketamine/xylazine cocktail (see Recipes, 0.1 ml/20 g mouse weight. Intraperitoneal injection) or inhaling 1.5% isoflurane driven by 100% oxygen through an anesthesia machine vaporizer.
      Note: Ketamine/xylazine may cause animal death after surgery. Using an anesthesia machine vaporizer is an ideal option. Before surgery, use 2.5-3.5% isoflurane to anesthetize the animals and then lower down the concentration to 1.5% during the surgery.
    4. Shave the surgical site using an electric clipper and sterilize the site using iodine wipes and alcohol pads alternatively. The skin is opened using a blunt-ended scissors. Then a small hole is made using a high-speed rotary micromotor drill with a sterile diamond burr grinding bit.
    5. In the presence of Dox, 0.5 µl AAV9-TRE-mCherry virus is injected vertically (90° to the skull) into the lateral amygdala bilaterally (relative to the bregma: -1.5 mm anteroposterior; ±  3.5  mm mediolateral; -4.3  mm dorsoventral), using a 10 µl Microliter 700 series syringe through a microsyringe pump (UMP3; WPI) (Figure 2B). A stereotaxic microscope is used for the microinjections to the mouse brain.
    6. The injector is slowly moved to the targeted site and should sit for 5 min before starting virus injection.
    7. A microsyringe pump (UMP3; WPI) with a controller (Micro4; WPI) are used to control the speed of the injection. The speed is set as 0.1 µl per minute.
      Note: The needle tip is easily clogged by dirt after injection. Clean with water and alcohol each time after use.
    8. The needle remains at the site for 5 min after the end of injection.
    9. Slowly extract the microsyringe from the site and mouse brain.
      Note: The microsyringe and needle are wiped with alcohol pads and rinsed with sterile ddH2O after injection.
    10. Glue the skin with Vetbond.
    11. After surgery, mice are housed in their home cages collectively and monitored for two weeks until behavioral testing. The mice will be given Dox diet during the two weeks prior to the behavioral testing.
    12. Anesthetics, Meloxicam and Buprenorphine, are administered to the mice before surgery. Meloxicam is given again 24 h later, and Buprenorphine is given every 12 h for a total 48 h of treatment.
    13. All sites of virus injection are verified histologically based on the immunostaining results after the behavior testing.

  3. Aversive conditioning and memory retrieval (Figure 2)
    1. All the behavioral experiments are performed at the light cycle.
    2. Mice are handled for 30 min on each of 2 days before aversive conditioning.
    3. Twenty-four hours before the TetTag Fos-tTA mice injected with AAV9-TRE-mCherry undergo aversive conditioning, the Dox-containing diet is replaced by a regular diet.
    4. On day 1, mice are habituated to an infrared aversive conditioning chamber (Med Associates Inc.) for 9 min. Then, the mice are presented with six pure tones (80 dB, 2 KHz, 20 sec each). During the last 2 sec of the tone, they receive a foot shock (0.7 mA, 2 sec). The interval between tones is 100 sec. The mice are then returned to their home cage 180 sec after the experiment.
    5. Immediately after aversive conditioning, the Dox-containing diet is restarted.
    6. One day after the aversive conditioning, mice are put into a new context (Figure 3) and receive a retrieval tone or not.


      Figure 3. Contexts for aversive conditioning and memory retrieval experiments. To change the odor in different contexts, the chambers and floors are wiped thoroughly with 1% Bleach or 0.25% peppermint. 

  4. Transcardial perfusion and whole brain fixation
    1. Thirty minutes after retrieval, the mice are euthanized with an intraperitoneal injection of overdose ketamine/xylazine cocktail (0.2 ml/20 g mouse weight).
    2. Place the animal on its back on a dissection board, and pin out all four feet.
    3. Use a blunt-end scissors to open the rib cage and expose the whole heart.
    4. Hold the heart gently with blunt forceps. Hold the butterfly needle with hemostats. Insert the needle into the left ventricle (no more than ¼ inch).
      Note: Gently insert the needle and make sure the needle stays in the left ventricle.
    5. Turn on ice-cold PBS/Heparin solution perfusion (see Recipes). While supporting the heart with the needle and hemostats, snip the right atrium.
    6. Carefully unpin the front feet and skin flap.
    7. Continue the perfusion of PBS/Heparin until blood is invisible in the liver (more than 5 min).
      Note: Make sure blood is completely gone.
    8. Switch the perfusion from PBS/Heparin to ice-cold PBS/4% PAF. Keep running the PBS/4% PAF for at least 5 min.
      Notes:
      1. The mouse tail shows tremble when perfused with PAF efficiently.
      2. 4% PAF/PBS is not stable at 4 °C. Make a fresh solution from 20% PAF every week.
    9. Open the skull with scissors and forceps. Extract the whole brain using a metal spatula.
    10. Immerse the brain in 10 ml of PBS/4% PAF and store at 4 °C for 24 h.

  5. Brain slices preparation
    1. The fixed brain is glued to the plate of the vibratome and rinse with ice-cold PBS.
    2. The brain is cut coronally into 50 µm sections from -1.54 mm to -2.34 mm anteroposterior. The settings of the Leica VT1000 vibratome: Speed: 4; Frequency: 5. Amygdala slices are identified according to a mouse brain atlas book (The Mouse Brain in Stereotaxic Coordinates, 3rd edition, 2008). Six coronal amygdala slices are microdissected from the brain slices using two 33 G syringe needles.

  6. Immunohistochemistry and cell counting
    1. Transfer the amygdala slices into a 24-well culture plate (one slice per well) by brush and wash for 5 min, three times in 4 °C PBS on a shaker.
      Note: The plate is covered with aluminum foil throughout the immunostaining experiment.
    2. Pipette 2 ml of blocking solution (see Recipes) into each well containing the staining basket. Place plate on a shaker at room temperature for one hour.
    3. Transfer each basket into a new well with 1 ml of primary antibody solution (see Recipes). Antibodies: Rabbit polyclonal IgG anti-RFP (Rockland) 1:1,000 dilution; Chicken IgY anti-GFP (Invitrogen) 1:1,000 dilution. Mouse anti-NeuN antibody, clone A60 1:1,000 dilution. Place the plate on a shaker in 4 °C for 12 h.
    4. Transfer each basket into a new well with 2 ml of PBS on a shaker at room temperature. Wash for 10 min, three times.
    5. Transfer each basket into a new well with 1 ml of secondary antibody solution. Antibody: Alexa Fluor 488 Goat anti-chicken IgG (H+L) (Invitrogen); Alexa Fluor 568 Goat anti-rabbit IgG (H+L) (Invitrogen); Alexa Fluor 647 Goat anti-mouse IgG (H+L) (Invitrogen), 1:200 dilution. Place plate on a shaker at room temperature for one hour.
    6. Transfer each basket into a new well with 2 ml of PBS on a shaker at room temperature. Wash for 10 min, three times.
    7. Use a wide mouth pipet to transfer slices to the slide and remove the extra solution. Then apply 3-6 drop mounting solution (VectaShield HardSet Mounting Medium with DAPI) containing DAPI to the edge of the slice, and then cover it with a coverslip.
    8. Check the sample on a confocal microscope and store the slides in 4 °C refrigerator.
    9. Count mCherry-positive and shEGFP-positive neurons from 6 coronal amygdala slices (-1.54 mm to -2.34 mm anterioposterior) for each mouse. Co-localization of shEGFP and mCherry is analyzed by Imaris and ImageJ.

Data analysis

Aversive conditioning labeled amygdala neurons with long-lasting mCherry and with a short-half life (2 h) nuclear-localized EGFP (shEGFP) (Figure 4). Immediately after aversive conditioning, mice began receiving Dox, which prevents expression of mCherry, but not shEGFP. Twenty-four hours later, we delivered a single retrieval tone or not. Thirty minutes after that, we harvested the amygdala and imaged shEGFP- and mCherry-positive cells (Figure 4). Compared to the control, a single retrieval tone increased the percentage of mCherry-positive cells that were also shEGFP-positive (Figure 4). These findings indicate that the retrieval cue reactivated neurons bearing the memory trace.


Figure 4. Representative images of amygdala neurons that are labeled by mCherry after aversive conditioning and by GFP after retrieval as well as NeuN (blue). White dash line circles the area of the amygdala. White arrows indicate examples of colocalization of mCherry and shEGFP. mCherry- and shEGFP-positive cells are also NeuN-positive. Right, data are the percentage of mCherry-positive cells that were also shEGFP-positive. Data are mean ± SEM. n = 5 mice for each group. * indicates P < 0.05 by ANOVA with Tukey’s post hoc multiple comparisons.

Recipes

  1. PBS/Heparin solution (10 U/ml Heparin)
    To make 500 ml PBS/Heparin, add 5 ml Heparin stock (10 kU/10 ml per vial) to 495 ml 1x PBS
  2. PBS/4% paraformaldehyde (PAF) solution
    To make 100 ml 4% PFA in 1x PBS, mix 10 ml 10x PBS and 20 ml 20% PAF. Add double distilled water to make the final volume up to 100 ml
    Note: 4% PAF diluted into PBS can only be stable for one week at 4 °C.
  3. Blocking solution
    SuperBlock blocking buffer
    Triton X-100 (final con. 0.2%)
  4. Primary antibody solution
    Mix equal volume of SuperBlock blocking buffer and PBS, and then add Triton X-100 (final con. 0.2%)
  5. Ketamine/xylazine cocktail
    87.5 mg/kg ketamine
    2.5 mg/kg xylazine

Acknowledgments

We thank Thomas Moninger, Theresa Mayhew, and Sarah Horgen for assistance. We thank Drs. Susumu Tonegawa, Xu Liu, and Steve Ramirez for providing the TRE-mCherry plasmid and their previous works (Liu et al., 2012; Ramirez et al., 2013). JD is supported by the American Heart Association (15SDG25700054) and the University of Toledo start-up fund.

References

  1. Bruce, D. (2001). Fifty years since Lashley’s In search of the Engram: refutations and conjectures. J Hist Neurosci 10(3): 308-318.
  2. Du, J., Price, M. P., Taugher, R. J., Grigsby, D., Ash, J. J., Stark, A. C., Hossain Saad, M. Z., Singh, K., Mandal, J., Wemmie, J. A. and Welsh, M. J. (2017). Transient acidosis while retrieving a fear-related memory enhances its lability. eLife 6.
  3. Josselyn, S. A. (2010). Continuing the search for the engram: examining the mechanism of fear memories. J Psychiatry Neurosci 35(4): 221-228.
  4. Liu, X., Ramirez, S., Pang, P. T., Puryear, C. B., Govindarajan, A., Deisseroth, K. and Tonegawa, S. (2012). Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484(7394): 381-385.
  5. Poo, M. M., Pignatelli, M., Ryan, T. J., Tonegawa, S., Bonhoeffer, T., Martin, K. C., Rudenko, A., Tsai, L. H., Tsien, R. W., Fishell, G., Mullins, C., Goncalves, J. T., Shtrahman, M., Johnston, S. T., Gage, F. H., Dan, Y., Long, J., Buzsaki, G. and Stevens, C. (2016). What is memory? The present state of the engram. BMC Biol 14: 40.
  6. Ramirez, S., Liu, X., Lin, P. A., Suh, J., Pignatelli, M., Redondo, R. L., Ryan, T. J. and Tonegawa, S. (2013). Creating a false memory in the hippocampus. Science 341(6144): 387-391.
  7. Reijmers, L. G., Perkins, B. L., Matsuo, N. and Mayford, M. (2007). Localization of a stable neural correlate of associative memory. Science 317(5842): 1230-1233.
  8. Ryan, T. J., Roy, D. S., Pignatelli, M., Arons, A. and Tonegawa, S. (2015). Memory. Engram cells retain memory under retrograde amnesia. Science 348(6238): 1007-1013.
  9. Silva, A. J., Zhou, Y., Rogerson, T., Shobe, J. and Balaji, J. (2009). Molecular and cellular approaches to memory allocation in neural circuits. Science 326(5951): 391-395.

简介

存储器跟踪(也称为存储器枚举)被理论化为大脑中物理存储器存储的机制(Silva等人,2009; Josselyn,2010),并且内存跟踪与一个 特定的神经元群体(Liu et al。,2012; Ramirez等人,2013)。 标记和刺激那些神经元将激活记忆痕迹(Liu et al。,2012; Ramirez等人,2013)。 记忆似乎分布在大脑的不同区域,而不是局限于一个区域。 因此,用于跟踪记忆的方法有能力提高我们对神经元电路的理解。 在本协议中,我们引入多西环素诱导表达系统来标记与原始记忆痕迹相关的特定神经元。
【背景】记忆痕迹是记忆被存储为大脑物理或生物化学变化的理论手段(Ryan等人,2015)。在二十世纪初德国动物学家理查德·塞蒙(Richard Semon)制定记忆追踪概念之后,记忆存储的具体过程一直是神经科学领域辩论的一个未解决的话题(Poo et al。,2016)。尽管记忆机制已经成为几十年来的争论焦点,但已经一致认为,特定的神经元被用于记忆的存储(Liu等人,2012; Ramirez等人, ,2013)。另外,据信记忆不是局限于一个区域,而是在脑的许多不同区域发生的机制。早期的研究人员使用行为和手术方法的组合来识别记忆痕迹(Bruce,2001)。虽然我们从这些行为和手术方法中学到了很多,但这些方法并没有为我们提供可视化或识别记忆痕迹中使用的目标神经元的能力。在大脑中,立即早期基因(例如Fos)的表达在与学习和记忆形成相关的特定神经元中被快速上调。最近的研究使用小鼠TetTag-Fos驱动的GFP 小鼠模型(Reijmers等人,2007)与腺相关病毒(AAV 编号TRE-mCherry (Liu等人,2012; Ramirez等人,2013)。为了继续识别涉及记忆痕迹的靶神经元,我们通过用C57BL / 6J小鼠进行育种,从TetTag小鼠中产生了Fos-tTA 小鼠,并选择仅携带 Fos-tTA 转基因。 Fos-tTA 小鼠具有启动子驱动的核定位的2小时半衰期EGFP的表达。 Fos启动子还驱动四环素反式激活因子(tTA)的表达,其可以结合注射的AAV 9 -TRE-mCherry上的四环素应答元件(TRE)位点病毒,导致mCherry的表达。在Fos-tTA 小鼠系统中,多西环素(Dox)的应用抑制了启动子启动子驱动的tTA与TRE位点的结合,从而阻止靶基因的表达。我们将AAV 9 -TRE-mCherry注入Fos-tTA 小鼠的杏仁核。在缺乏Dox的情况下,厌恶性调节激活了在目标神经元中驱动mCherry表达的Fos(图1)。我们最近采用这种技术来确定CO 2 / ASIC在检索过程中是否增强了存储器跟踪(Du等等,2017)。其他研究已经利用这些方法来识别具体的记忆能力,并使用光遗传学重新激活它(Liu et al。,2012; Ramirez等人,2013)。总之,这是一种识别记忆痕迹的强大技术,并将大大有益于未来对神经元电路,学习和记忆的研究。


图1.显示如何在转基因TetTag Fos-tTA的小鼠中标记神经元的示意图,并用病毒载体AAV9 -TRE-mCherry显微注射 A.神经元活动激活了Fos启动子。启动子启动子驱动四环素反式激活因子(tTA)和shEGFP的表达。 shEGFP蛋白质以2小时半衰期迅速降解。 tTA蛋白与其靶标,微注射AAV9 -TRE-mCherry构建体上的四环素应答元件(TRE)结合,导致mCherry在神经元中的表达。 Dox的存在抑制tTA与TRE结合,从而阻止mCherry的表达。 B.显示如何构建pAAV-TRE-mCherry质粒的载体图。含有构建体的AAV 9个病毒由爱荷华大学基因转移载体核心包装。矢量图由SnapGene 4.0生成。

关键字:厌恶记忆, 记忆痕迹, TetTag Fos-tTA小鼠 , AAV9-TRE-mCherry, 多西环素诱导表达系统

材料和试剂

  1. 移液器提示(USA Scientific,目录号:1112-1820; 1110-1800; 1111-3840)
  2. 碘擦拭巾(Dynarex,目录号:1108)
  3. 酒精制品垫(COVIDIEN,目录号:5750)
  4. 蝴蝶针,23 G(Thermo Fisher Scientific,目录号:14-840-35)
    制造商:Exel International,目录号:26706。
  5. 24孔培养板(Corning,目录号:3527)
  6. 铝箔(Thermo Fisher Scientific,目录号:NC9847171)
    制造商:雷诺消费品,目录号:632。
  7. 染色篮(PELCO Prep-Eze TM 24孔板插入物)(Ted Pella,目录号:36172)
  8. 超级胶(乐泰®专业超级胶,汉高,目录号:1365882)
  9. 单刃剃刀刀片(Fisher Scientific,目录号:12-640)
  10. 盖玻片(Fisher Scientific,目录号:12-545M)
  11. C57BL / 6J小鼠(JACKSON LABORATORY,目录号:000664)
    注意:在实验前和实验过程中,小鼠都是小组。
  12. B6.Cg-Tg(Fos-tTA,Fos-EGFP *)1Mmay / J小鼠(JACKSON LABORATORY,目录号:018306)
  13. AAV 9 -TRE-mCherry病毒由爱荷华大学基因转移载体核心产生,并且可以在它们的库存列表中商购(病毒名称:AAV2 / 9-TRE-mCherry,CsCl 1 st ; Lot#AAV 2679; Tier:1.45E +12μg/ ml)(图1B)
    注意:等分试样应储存在-80°C。
  14. 定制的多西环素饮食,40 ppm(Envigo,目录号:TD.10483)
    注意:存储在4°C。
  15. 3ml(3M,Vetbond TM,目录号:70200742529)的组织粘合剂
  16. 兔多克隆IgG抗RFP(Rockland Immunochemicals,目录号:600-401-379)
    注意:等分试样应保存在-20°C。
  17. 鸡IgY抗GFP(Thermo Fisher Scientific,目录号:A10262)
  18. 小鼠抗NeuN,克隆A60(EMD Millipore,目录号:MAB377X)
  19. Alexa Fluor 488山羊抗鸡IgG(H + L)(Thermo Fisher Scientific,目录号:A-11039)
  20. Alexa Fluor 568山羊抗兔IgG(H + L)(Thermo Fisher Scientific,目录号:A-11036)
  21. Alexa Fluor 647山羊抗小鼠IgG(H + L)(Thermo Fisher Scientific,目录号:A-21235)
  22. VectaShield H-1500(Vector Laboratories,目录号:H-1500)
  23. 肝素10kU / 10ml(Sagent Pharmaceuticals,目录号:25021-400-10)
  24. 磷酸盐缓冲盐水(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
  25. 20%多聚甲醛(Electron Microscopy Sciences,目录号:15713-S)
  26. SuperBlock(PBS)封闭缓冲液(Thermo Fisher Scientific,Thermo Scientific TM,目录号:37515)
  27. Triton X-100(Fisher Scientific,目录号:BP151-100)
  28. 盐酸氯胺酮注射液,10ml-200mg / ml(野生动物医药,动物园复合药剂)
  29. 盐酸西拉嗪,30毫升至300毫克/毫升(野生动物药物,动物园复合药房)
  30. 异氟烷(Henry Schein,目录号:029404)
  31. 100%氧气瓶(Airgas,目录号:UN 1072)
  32. 酒精,140证明(Decon Labs,目录号:2401TP)
  33. PBS /肝素溶液(参见食谱)
  34. PBS / 4%多聚甲醛(PAF)溶液(参见食谱)
  35. 阻塞解决方案(见配方)
  36. 初级抗体溶液(参见食谱)
  37. 氯胺酮/赛利嗪鸡尾酒(见食谱)

设备

  1. 麻醉蒸发器(DRE Medical,型号:Drager 19.1)
  2. 微量注射器,10μl,700系列(Hamilton,目录号:80314)
  3. 小型可拆卸针头,33 G,0.4英寸(Hamilton,目录号:7803-05)
  4. NIR视频恐惧治疗系统(Med Associates,目录号:MED-VFC-SCT-M)
  5. 超级专业动物剪刀(Braintree Scientific,目录号:CLP-64 800)
  6. 平头剪刀(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:78702)
  7. 钝端镊子(Fisher Scientific,目录号:13-812-39)
  8. Iridectomy剪刀(Fisher Scientific,目录号:50-109-3945)
    制造商:Sklar Surgical Instruments,目录号:642035。
  9. 点式剪刀(Fisher Scientific,目录号:13-808-2)
  10. Hemostats Rankin镊子(Fisher Scientific,目录号:13-812-45)
  11. 高速旋转微电机钻具套件(Blackstone Industries,Foredom,目录号:K.1070)
  12. 微量注射泵(世界精密仪器,型号:UMP3)
  13. 微量注射泵控制器(世界精密仪器,型号:Micro4)
  14. 金属刮刀(Fisher Scientific,目录号:14-374)
  15. 振动刀片切片机(Leica Biosystems,型号:Leica VT1000 S)
  16. 振荡器(Cole-Parmer,Stuart,型号:SSL3)
  17. 移液器,单通道,0.2μl-1 ml(Gibson,型号:PiPETMAN Classic)
  18. 共焦显微镜(Olympus,型号:FV1000)
  19. 4°C冰箱(Whirlpool,型号:WRR56X18FW)
  20. 立体定位仪(KOPF仪器,型号:940型)
  21. 金刚石钻头磨头,Ø0.60mm(Stoelting,目录号:514552)
  22. 立体显微镜(Amscope,目录号:SM-5TZ-FRL)
  23. 迷你加热垫,4 x 7“(所有生活事物®,PETSMART.com)

软件

  1. Imaris图像分析软件(Oxford instruments plc。)
  2. ImageJ软件(从NIH网页下载)
  3. SnapGene 4.0(GSL Biotech LLC)

程序

注意:动物护理和手术符合国家卫生研究院的标准。必须遵守当地的动物护理道德标准。爱荷华大学动物护理和使用委员会(ACURF#4041016)和托莱多大学体育动物护理和使用委员会(议定书#108791)批准了所有程序。

  1. TetTag Fos-tTA 鼠标生成
    1. 通过用C57BL / 6J小鼠培养它们,从B6.Cg-Tg(Fos-tTA,Fos-EGFP *)1Mmay / J小鼠产生TetTag Fos-tTA小鼠,并选择仅携带Fos-tTA 转基因(Liu等人,2012; Ramirez等人,2013)。爱荷华大学基因组编辑核心工具提供鼠标基因分型服务。基因分型方案可以在 https://www.jax.org/strain/018306 找到。
    2. 图1显示了如何在转基因TetTag Fos-tTA的小鼠中标记神经元,并用病毒载体AAV 9 -TRE-mCherry显微注射。

  2. 病毒构建体(图1B)和立体定向注射(图2)


    图2.示意图显示了记忆恢复诱导的侧向杏仁核神经元在厌食记忆痕迹中的活化的过程。 A.小鼠喂食Dox 1周,然后将AAV 9 矢量编码mCherry被微量注入杏仁核。 Dox可抑制mCherry的表达。两周后,Dox停药,小鼠经历厌食调理方案。在这种情况下,FOS启动子将驱动shEGFP和mCherry的表达,但是shEGFP会迅速衰减。经过厌恶调理后,小鼠立即恢复Dox治疗。一天后,小鼠经历检索方案。三十分钟后,制备脑切片,确定外侧杏仁核中的shEGFP和mCherry阳性神经元。 B.病毒注射手术的立体定位系统。

    1. 在手术前一周,给十二周龄的小鼠喂食Dox饮食(40mg / kg)。
    2. 消毒手术区域,仪器和材料。
    3. 使用氯胺酮/赛拉嗪混合物麻醉小鼠(参见食谱,0.1ml / 20g小鼠体重,腹膜内注射)或吸入由100%氧气驱动的1.5%异氟烷通过麻醉机蒸发器。
      注意:氯胺酮/赛拉嗪可能在手术后导致动物死亡。使用麻醉机蒸发器是理想的选择。手术前,使用2.5-3.5%的异氟烷麻醉动物,然后在手术中将浓度降低至1.5%。
    4. 使用电动剪刀剃刮外科手术部位,并使用碘擦拭巾和酒精垫对其进行消毒。使用钝头剪刀打开皮肤。然后使用带有无菌金刚石钻头磨头的高速旋转微电机钻头制作一个小孔。
    5. 在Dox存在下,将0.5μlAAV9 -TRE-mCherry病毒垂直注射(90°到颅骨)到双侧扁桃体中(相对于前白斑:-1.5mm前后±3.5° mm内侧; -4.3 mm背神经),使用10μlMicroliter 700系列注射器通过微注射泵(UMP3; WPI)(图2B)。立体定位显微镜用于对小鼠脑的显微注射。
    6. 注射器缓慢移动到目标部位,应该在开始病毒注射前静置5分钟。
    7. 使用具有控制器(Micro4; WPI)的微注射泵(UMP3; WPI)来控制注射速度。速度设置为每分钟0.1μl。
      注意:针尖在注射后容易被污垢堵塞。使用后每次用水和酒精清洁。
    8. 针头在注射结束后保持5分钟。
    9. 从现场和小鼠脑缓慢提取微量注射器。
      注意:微量注射器和针头用酒精垫擦拭,注射后用无菌ddH 2 O冲洗。
    10. 用Vetbond胶粘皮肤。
    11. 手术后,将小鼠集体饲养在家庭笼中,并监测两周,直到行为测试。在行为测试前的两周内,小鼠将获得Dox饮食。
    12. 麻醉剂,美洛昔康和丁丙诺啡,在手术前给予小鼠。 24小时后给予美洛昔康,每12小时给予丁丙诺啡48小时。
    13. 病毒注射的所有部位基于行为测试后的免疫染色结果进行组织学验证。

  3. 厌恶调理和记忆检索(图2)
    1. 所有的行为实验都是在光周期进行的
    2. 在厌食调理前的2天内,小鼠处理30分钟。
    3. 在注射AAV 9 -TRE-mCherry的小鼠TetTag Fos-tTA 小鼠前24小时,经过厌恶调理,含有Dox的饮食被正常饮食所代替。
    4. 在第1天,将小鼠习惯于红外线厌恶性调理室(Med Associates Inc.)9分钟。然后,给予小鼠六种纯音(80dB,2KHz,各20秒)。在最后2秒的音调中,他们会受到脚部震动(0.7 mA,2秒)。音调间隔为100秒。然后在实验180秒后将小鼠返回家中笼。
    5. 在厌恶调理后立即重新启动含Dox的饮食。
    6. 在厌恶调理后的一天,将小鼠放入新的环境中(图3),并收到检索音。


      图3.反向条件和内存检索实验的上下文。 为了改变不同情况下的气味,用1%的漂白剂或0.25%的薄荷清洗室内和地板。&nbsp;

  4. 经心脏灌注和全脑固定
    1. 取出三十分钟后,将小鼠用腹膜内注射过量氯胺酮/赛拉嗪混合物(0.2ml / 20g小鼠重量)来安乐死。
    2. 将动物放在背部的解剖板上,并将所有四只脚打出。
    3. 使用平头剪刀打开肋骨笼并暴露整个心脏。
    4. 用钝镊子轻轻握住心脏。用止血针握住蝴蝶针。将针插入左心室(不超过1/4英寸)。
      注意:轻轻插入针头,确保针头留在左心室。
    5. 打开冰冷的PBS /肝素溶液灌注(见食谱)。在用针和止血液支撑心脏的同时,剪断右心房。
    6. 仔细取下前脚和皮瓣。
    7. 继续灌注PBS /肝素直到血液在肝脏中看不见(超过5分钟)。
      注意:确保血液完全消失。
    8. 将PBS /肝素灌注切换成冰冷的PBS / 4%PAF。保持运行PBS / 4%PAF至少5分钟。
      注意:
      1. 当PAF灌注时,鼠尾巴显示颤抖。
      2. 4%PAF / PBS在4℃下不稳定。每周从20%的PAF获得新的解决方案。
    9. 用剪刀和镊子打开头骨。使用金属刮刀提取整个大脑。
    10. 将大脑浸入10 ml PBS / 4%PAF中,并在4°C储存24 h

  5. 脑切片准备
    1. 将固定的脑粘贴在vibratome的板上,用冰冷的PBS冲洗。
    2. 将大脑从冠状切割成从1.54mm至前后-2.34mm的50μm切片。 Leica VT1000 vibratome的设置:速度:4;频率:5.根据小鼠脑图集书(“立体定位坐标下的小鼠脑”,2008年第3期)确定杏仁核切片。使用两根33 G注射针,从脑切片显微切割六个冠状扁桃体切片
  6. 免疫组织化学和细胞计数
    1. 通过刷子将杏仁核切片转移到24孔培养板(每孔一片)中,并在摇床上在4℃PBS中洗涤5分钟,三次。
      注意:在整个免疫染色实验过程中,该板被铝箔覆盖。
    2. 将2ml封闭溶液(见食谱)吸入含有染色篮子的每个孔中。将板放在振荡器上室温1小时
    3. 使用1 ml的一级抗体溶液将每个筐转移到一个新的孔中(参见食谱)。抗体:兔多克隆IgG抗RFP(Rockland)1:1,000稀释;鸡IgY抗GFP(Invitrogen)1:1,000稀释。小鼠抗NeuN抗体,克隆A60 1:1,000稀释。将板放在振荡器上4℃12小时。
    4. 每个篮子在室温下用振荡器的2ml PBS转移到新的孔中。洗涤10分钟,三次。
    5. 用1ml二抗溶液将每个筐转移到新的孔中。抗体:Alexa Fluor 488山羊抗鸡IgG(H + L)(Invitrogen); Alexa Fluor 568山羊抗兔IgG(H + L)(Invitrogen); Alexa Fluor 647山羊抗小鼠IgG(H + L)(Invitrogen),1:200稀释。将板放在振荡器上室温1小时
    6. 每个篮子在室温下用振荡器的2ml PBS转移到新的孔中。洗涤10分钟,三次。
    7. 使用宽口吸管将切片转移到载玻片上,并移除额外的解决方案。然后将含有DAPI的含有DAPI的ViteraShield HardSet安装介质(VectaShield HardSet安装介质,DAPI)应用到切片的边缘,然后用盖玻片覆盖。
    8. 在共焦显微镜上检查样品,并将载玻片存放在4°C冰箱中。
    9. 从每只小鼠的6个冠状扁桃体切片(-1.54mm至-2.34mm前后)计数mCherry阳性和shEGFP阳性神经元。通过Imaris和ImageJ分析shEGFP和mCherry的共定位

数据分析

厌食调理标记的杏仁核神经元与持久的mCherry和短半衰期(2小时)核定位EGFP(shEGFP)(图4)。在厌食调理之后,小鼠开始接受Dox,其阻止mCherry的表达,但不阻止shEGFP的表达。二十四小时后,我们提供了单一的检索音。三十分钟后,我们收获了杏仁核并成像了shEGFP和mCherry阳性细胞(图4)。与对照相比,单个检索色调增加了也是shEGFP阳性的mCherry阳性细胞的百分比(图4)。这些发现表明检索线索重新激活了具有记忆痕迹的神经元

图4.杏仁核神经元的代表性图像,在厌食调理后由mCherry标记,在GFP检索后标记为NeuN(蓝色)。 白色划线圈住了杏仁核的区域。白色箭头表示mCherry和shEGFP共定位的实例。 mCherry-和shEGFP阳性细胞也是NeuN阳性。对,数据是mCherry阳性细胞也是shEGFP阳性的百分比。数据为平均值±SEM。每组n = 5只小鼠。 *表示 P 0.05的ANOVA与Tukey的事后多重比较。

食谱

  1. PBS /肝素溶液(10U / ml肝素) 为了制备500毫升PBS /肝素,加入5毫升肝素原液(每瓶10公斤/ 10毫升)至495毫升1x PBS
  2. PBS / 4%多聚甲醛(PAF)溶液
    在1×PBS中制备100ml 4%PFA,混合10×10×PBS和20%20%PAF。加入双蒸水,使最终体积达到100 ml
    注意:稀释到PBS中的4%PAF只能在4℃下稳定一周。
  3. 阻塞解决方案
    超级块阻塞缓冲区
    Triton X-100(最终0.2%)
  4. 初级抗体溶液
    混合等体积的SuperBlock封闭缓冲液和PBS,然后加入Triton X-100(最终浓度为0.2%)
  5. 氯胺酮/赛利嗪鸡尾酒
    87.5 mg / kg氯胺酮
    2.5 mg / kg赛拉嗪

致谢

我们感谢Thomas Moninger,Theresa Mayhew和Sarah Horgen的协助。我们感谢Drs。 Susumu Tonegawa,Xu Liu和Steve Ramirez提供TRE-mCherry质粒及其以前的作品(Liu et al。,2012; Ramirez等人,2013)。 JD由美国心脏协会(15SDG25700054)和托莱多大学创业基金提供支持。

参考

  1. Bruce,D。(2001)。 拉什利在寻找Engram五十年之后:驳斥和推测。 J Hist Neurosci 10(3):308-318。
  2. Du,J.,Price,MP,Taugher,RJ,Grigsby,D.,Ash,JJ,Stark,AC,Hossain Saad,MZ,Singh,K.,Mandal,J.,Wemmie,JA and Welsh,MJ(2017 )。 在检索与恐惧有关的记忆的瞬间酸中毒增强其不耐性。 eLif e 6.
  3. Josselyn,S.A。(2010)。 继续搜索engram:检查恐惧记忆的机制。 J Psychiatry Neurosci 35(4):221-228。
  4. Liu,X.,Ramirez,S.,Pang,P.T.,Puryear,C.B.,Govindarajan,A.,Deisseroth,K.and Tonegawa,S。(2012)。 海马engram的激发刺激激活恐惧记忆回忆 自然 484(7394):381-385。
  5. Poo,MM,Pignatelli,M.,Ryan,TJ,Tonegawa,S.,Bonhoeffer,T.,Martin,KC,Rudenko,A.,Tsai,LH,Tsien,RW,Fishell,G.,Mullins, Goncalves,JT,Shtrahman,M.,Johnston,ST,Gage,FH,Dan,Y.,Long,J.,Buzsaki,G。和Stevens,C。(2016)。 什么是记忆? engram的现状。 BMC Biol 14:40.
  6. Ramirez,S.,Liu,X.,Lin,P.A.,Suh,J.,Pignatelli,M.,Redondo,R.L.,Ryan,T.J。和Tonegawa,S。(2013)。 在海马中创造虚假记忆。 科学 341 (6144):387-391。
  7. Reijmers,L.G.,Perkins,B.L.Matsuo,N。和Mayford,M。(2007)。 关联记忆的稳定神经相关性的本地化 科学 317(5842):1230-1233。
  8. Ryan,T.J.,Roy,D.S.,Pignatelli,M.,Arons,A.andTonegawa,S。(2015)。 内存。 Engram细胞在逆行遗忘症下保留记忆。 科学 348(6238):1007-1013。
  9. Silva,A.J.,Zhou,Y.,Rogerson,T.,Shobe,J.and Balaji,J。(2009)。 神经回路内存分配的分子和细胞方法科学 326(5951):391-395。
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Copyright Du and Koffman. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Du, J. and Koffman, E. E. (2017). Labeling Aversive Memory Trace in Mouse Using a Doxycycline-inducible Expression System. Bio-protocol 7(20): e2578. DOI: 10.21769/BioProtoc.2578.
  2. Du, J., Price, M. P., Taugher, R. J., Grigsby, D., Ash, J. J., Stark, A. C., Hossain Saad, M. Z., Singh, K., Mandal, J., Wemmie, J. A. and Welsh, M. J. (2017). Transient acidosis while retrieving a fear-related memory enhances its lability. eLife 6.
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