Labeling Aversive Memory Trace in Mouse Using a Doxycycline-inducible Expression System

[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.

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.
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. 7 www.bio-protocol.org/e2578 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.  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.

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.  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. E. 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, 3 rd edition, 2008). Six coronal amygdala slices are microdissected from the brain slices using two 33 G syringe needles. 8. Check the sample on a confocal microscope and store the slides in 4 °C refrigerator.

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.