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May 2018

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Assessing Classical Olfactory Fear Conditioning by Behavioral Freezing in Mice
通过小鼠冻结行为检测经典嗅觉条件恐惧   

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Abstract

Classical fear conditioning typically involves pairing a discrete cue with a foot shock. Quantifying behavioral freezing to the learned cue is a crucial assay for neuroscience studies focused on learning and memory. Many paradigms utilize discrete stimuli such as tones; however, given mice are odor-driven animals and the wide variety of odorants commercially available, using odors as conditioned stimuli presents advantages for studies involving learning. Here, we describe detailed procedures for assembling systems for presenting discrete odor cues during single-day fear conditioning and subsequent analysis of freezing behavior to assess learning.

Keywords: Classical conditioning (经典条件作用), Olfactory conditioning (嗅觉条件作用), Fear learning (恐惧学习), Behavior (行为), Freezing (冻结), Olfactory fear (嗅觉恐惧)

Background

Associative fear learning, the root of several anxiety disorders, involves pairing a neutral stimulus with an aversive outcome. This pairing produces robust behavioral fear responses, in the form of freezing (LeDoux, 2003), to the conditioned stimulus, which can be quantified as a measure of fear learning and memory. Discrete stimuli, such as tones, are often used as conditioned stimuli for fear conditioning; however, olfactory cues are also highly effective at inducing learned freezing (Pavesi et al., 2012; Ross and Fletcher, 2018). This method of associative fear learning differs from those utilizing predator odors, which produces instinctive behaviors rather than learned behaviors, making it ideal for rapidly assessing olfactory learning. Behavioral freezing, defined as absence of all voluntary movements (Blanchard and Blanchard, 1969; Fanselow, 1980), can be measured through automated software that compares pixel differences on a frame-by-frame basis. We developed a protocol that uses the automated FreezeFrame software to deliver discrete olfactory cues during training and testing. This protocol supports standard fear conditioning and subsequent testing but also provides flexibility for expansion to fit broad experimental needs such as extinction, discriminate conditioning, and generalization paradigms or experimental manipulations (e.g., optogenetics or chemogenetics). In addition, olfactory fear conditioning provides a rapid method of studying mechanisms of olfactory associative learning given that training requires few trials in a single day with learning assessed the next day.

Materials and Reagents

  1. Parafilm
  2. Tubing
    1. 1/16” ID Kflex tubing (United States Plastic, catalog number: 65170 )
    2. 1/16” ID Tygon tubing (Fisher Scientific, catalog number: 14-171-129 )
    3. 1/8” ID Masterflex Tygon E-Lab (E-3603) Pump Tubing (Cole-Parmer Instrument, catalog number: EW-06509-16 )
    4. 1/4” ID Tygon tubing (Fisher Scientific, catalog number: 14-171-221 )
  3. Luers and connectors
    1. Stopcock 1-way male lock (Cole-Parmer Instrument, catalog number: EW-30600-00 )
    2. Male Luer x 1/16” hose barb (Cole-Parmer Instrument, catalog number: EW-45518-00 )
    3. Male Luer Lock Plug (Cole-Parmer Instrument, catalog number: EW-30800-30 )
    4. Female Luer x 1/8” hose barb (Cole-Parmer Instrument, catalog number: SI-30800-08 )
    5. Masterflex Y-connector (Cole-Parmer Instrument, catalog number: EW-30614-04 )
    6. Female Luer 1/16” x 1/16” hose barb (Cole-Parmer Instrument, catalog number: EW-45508-00 )
    7. (2) 1/4” straight barbed connectors (Cole-Parmer Instrument, catalog number: EW-30612-13 )
    8. 1/8” NPT male adapter to 1/16” barb (Cole-Parmer Instrument, catalog number: EW-06365-41 )
    9. 1/8” NPT male adapter to 1/4” barb (Cole-Parmer Instrument, catalog number: EW-30704-09 )
    10. 1/2” NPT Male adapter to 1/4” Hose Barb (Cole-Parmer Instrument, catalog number: EW-30704-17 )
  4. Pipette tips (Eppendorf, catalog numbers: 0030073061 , 0030073100 )
  5. 20 ml glass scintillation vial with polypropylene cap (Sigma-Aldrich, catalog number: Z190535 )
  6. 16 G 1½ needle (Surgo Surgical Supply, catalog number: 150-305198)
    Manufacturer: BD, catalog number: 305198 .
  7. Mice (C57BL/6J; THE JACKSON LABORATORY, catalog number: 000664 ), preferably > 6 weeks old
    Notes: 
    1. This protocol is suitable for use with other strains, but we recommend users test and determine suitability with their strains.
    2. If using fear conditioning in conjunction with other techniques, e.g., optogenetics, prepare mice in advance and allow sufficient recovery time prior to training.
  8. Epoxy (Thorlabs, catalog number: G14250 )
  9. Mineral oil (Sigma-Aldrich, catalog number: M5904 )
  10. Odorants, e.g., Ethyl Valerate (Sigma-Aldrich, catalog number: 290866 )
  11. Alconox (Sigma-Aldrich, catalog number: 242985 )

Equipment

  1. Single-channel pipettes (Eppendorf, catalog numbers: 3121000074 and 3121000120 )
  2. Training chamber (Figure 1B)
    1. Shock floor (Coulbourn Instruments, catalog number: H10-11M-TC-SF )
    2. Training cage (Coulbourn Instruments, catalog number: H10-11M-TC )
    3. Metal wall panels (Coulbourn Instruments, catalog number: H90-00M-M-KT01 )
    4. 25 ft shock cable (Coulbourn Instruments, catalog number: H93-01-25 )
    5. Precision animal shocker (Coulbourn Instruments, catalog number: H13-15 )
  3. Testing chamber (custom-made clear Plexiglas chamber, Figure 1C)
    1. Outer dimensions: 11” x 6” x 5.5” (L x W x H) with 1/2” thick walls.
    2. A 1/4” thick wall with 2 rows of 6 holes (1/2” dia) beginning ~1/2” from the top to allow air/odor diffusion across the middle wall divides the 11” chamber length in half, such that there are two inner compartments measuring 5” x 5” x 5.5”.
    3. The chamber has a 1/2” thick lid that can be secured to the chamber by screws.
    4. The chamber has one 1/2” dia hole on either side of the cage ~1/2” from the top for odor and vacuum lines, respectively.
  4. USB Cameras (Coulbourn Instruments, catalog number: ACT-VP-02 )
  5. Actimetrics USB Digital Interface (Coulbourn Instruments, catalog number: ACT-712 )
  6. FreezeFrame 4 adapter cable for Coulbourn hardware (Actimetrics, catalog number: ACT-INTF )
  7. Isolation cubicle (Coulbourn Instruments, catalog number: H10-24T )
  8. Infrared illuminator (Stoelting, catalog number: 60540 )
  9. Acrylic Flowmeter, 0.1-1 LPM (Cole-Parmer Instrument, catalog number: EW-32460-42 )
  10. Aquarium air pump, non-UL (Spectrum Brands, Tetra, Whisper®, catalog number: 77851 )
  11. 2-Channel SPDT Relay Board (Winford Engineering, catalog number: RLY102-12V-DIN )
  12. 12 V wall power supply for 2-Channel SPDT Relay Board (Winford Engineering, catalog number: WSD050-10-0 )
  13. Pinch Valves
    1. 1 Tube Normally Open pinch valve (NResearch, catalog number: 225P021-21 )
    2. 1 Tube Normally Open pinch valves (NResearch, catalog number: 648P021-82 )
    3. Olfactory stimulus control (Coulbourn Instruments, catalog number: H15-03 )
  14. True HEPA Air Purifier, 390 sq. ft. (Honeywell, model: 50250 )
  15. Cool Moisture Console Humidifier (Honeywell, model: HCM-6009 )
  16. Humidity and Temperature Monitor (such as FisherbrandTM TraceableTM Thermometer/Clock/Humidity Monitor, Fisher Scientific, catalog number: 06-662-4 )


    Figure 1. Odor delivery and behavioral chambers. A. The aquarium pump provides air to the flowmeter through Tygon tubing, which is then routed to the olfactory stimulus control for programming delivery. Kflex tubing is attached to the olfactory stimulus control and the non-odorized air stopcock to provide air flow to the assembled odor vial to make odorized air. The odorized air will flow out of the odorized air stopcock and to the attached chamber via additional Kflex tubing. B. The training chamber is modified to add small holes at the left and right of the cage. An NPT pipe is fitted through each hole and secured to the inside of the chamber with a nut. Odorized air lines are attached to the barbed end of the NPT pipe at the left of the cage while a vacuum line is attached to the barbed end of the NPT pipe at the right of the cage, just beneath the shock floor. A camera is positioned above the cage for recording. C. The testing chamber is placed inside the isolation cubicle with an infrared light source positioned on the right of the chamber and a camera mounted above the chamber. Separate tubing carrying clean and odorized air enter the chamber on the left and a vacuum is attached to the right of the chamber to facilitate odor clearance.

Software

  1. FreezeFrame 4 Software (Coulbourn Instruments, catalog number: ACT-100A)

Procedure

Notes:

  1. Assemble behavioral training chamber according to the manufacturer’s instructions and optimize for your needs.
  2. Set up cameras and program the delivery of stimuli [odor(s), shock(s), air, vacuum, etc.] and duration of training and testing protocols into FreezeFrame by following the instructions provided with the software.
  3. This protocol will detail training and testing murine olfactory fear learning with a single odor, ethyl valerate (E5), but can be adapted to test multiple odors by adding additional odor vials and lines to the testing chamber (see Ross and Fletcher, 2018). It could be expanded for use with discriminate conditioning by adding additional odor vials and lines to the training chamber. Users can also add consecutive testing days to assess extinction if desired.

  1. Modify the training and testing chambers
    1. Use a drill to make a hole into one side of the training chamber for the vacuum line. The hole should be situated at the middle of the chamber between the shock floor and drop pan. Insert the 1/2” NPT male pipe adapter with 1/4” barb, which should fit snugly, into the drilled hole from the outside of the chamber and secure with a 1/2” NPT nut. The nut should be on the inside of the chamber and the barb should extend from the outside of the chamber (Figure 1B).
    2. Use a drill to make a second smaller hole in the opposite side of the training chamber for the odor line. The hole should also be situated at the middle of the chamber approximately 1/2-1” above the shock floor. Insert the 1/8” NPT male pipe adapter with 1/16” barb, which should fit snugly, into the drilled hole from the outside of the chamber and secure with a 1/8” NPT nut. The nut should be on the inside of the chamber, and the barb should extend from the outside of the chamber (Figure 1B).
    3. Place the testing chamber inside the isolation cubicle and run all tubing/cords through the holes on the back of the chamber to the inside (Figure 1C).
    4. Place the infrared light source inside the cubicle towards the side of the testing chamber that will not hold the mouse/the side that odorized air will enter the chamber. Situate to minimize glare on the chamber/camera by pointing the light source directly at a wall of the isolation cube rather than the camera or testing chamber (Figure 1C).
    5. Insert and thread the NPT end of the 1/2” NPT Male Adapter to 1/4” Hose Barb into one of the holes on the sides of the testing chamber. Attach the vacuum line to the barbed end (Figure 1).

  2. Assemble odor vial(s)
    1. Remove 16 G 1½ needles from individual packaging and lock a stopcock onto the female Luer side of each needle. 
    2. If not already separated, remove the plastic cap from the 20 ml scintillation vial. Drill two holes into the top of the cap, leaving enough space between the holes for the needles with attached stopcocks. The holes should be as close to the size of the needles as possible. Secure the cap with drilled holes onto the bottle.
    3. Carefully uncap the needles and insert the beveled end through the holes on the cap, so that the needles are inside the bottle. Needles should fit snugly in drilled holes.
    4. Mix epoxy and apply to the cap of the bottle liberally to secure the needles to the cap. Make sure not to get epoxy on the stopcocks or the bottle itself. Let cure overnight.
    5. Use a sharpie to mark one of the stopcocks. This stopcock will act as the “odorized air” side of the odor vial. The other stopcock will receive non-odorized air from the Olfactory Stimulus Control. 
    6. Cut a length of Kflex tubing that will fit your specific setup (long enough to stretch from bottles to chambers without excessive tension). In one end of the Kflex tubing, insert the 1/16” hose barb end of a male Luer. Attach the male end of the Luer to the top of the marked stopcock and attach the other end of the Kflex tubing to the training or testing chamber as desired.
    Notes:
    1. It can be difficult to detach tubing from barbed luers; therefore, is advantageous to have a separate length of tubing from the odor vial(s) to each chamber. 
    2. If using more than one odor for testing, Kflex tubing should be similar length for all odor vials to testing chamber. Multiple tubes can be secured to one another with parafilm and inserted into the hole on the side of the testing chamber.

  3. Assemble clean air and odorized air delivery system
    1. Attach one end of the Masterflex Tygon tubing to one of the aquarium air pump outputs and the other end of the Tygon tubing to the input of the flowmeter. On a second piece of Tygon tubing, connect the 1/8” female Luer and cap it off with the corresponding male Luer lock plug. Attach the other end to the opposing flowmeter input.
    2. Attach one end of a short piece of the 1/16” Tygon tubing to the flowmeter output, in the other, place the Masterflex Y-connector. Cut two more pieces of the 1/16” Tygon tubing, approximately equal lengths, and join to the remaining barbs on the Y-connector. One of these lines will supply the air for the “clean air” line while the other will supply air for the “odor” line(s).
    3. The tubing designated for “clean air” should be paired with a Normally Open pinch valve with 1/16” ID tubing, while the tubing for the “odorized air” line should be paired with the Olfactory Stimulus Control. Fit the end of the “clean air” line with Luers to connect to the pinch valve tubing. Use a combination of 1/16” male and female Luers to adjoin (optionally, you can use straight barbed connectors with 1/16” barbs on both sides).
    4. After attaching the “odorized air” tubing to the back of the Olfactory Stimulus Control, connect another length of tubing to one of the three ports on top of the Olfactory Stimulus Control. Place a 1/16” male Luer to the other end of the tubing so that it can be connected directly to the unmarked stopcock on the odor vial(s).
    5. Wire the pinch valve to 2-Channel SPDT Relay Board, making sure to match the pinch valve type (NO or NC for Normally Open and Normally Closed, respectively) to the correct contact.
      Notes: 
      1. For more information regarding wiring of relay boards, please refer to the schematics provided on the Winford website.
      2. In order to minimize noise emitted by the pinch valve during training and testing, we place the pinch valve inside a styrofoam or plastic (such as a used pipette tip) box with foam or styrofoam peanuts. We drill small holes in the side of the box for the pinch valve wires and mount the relay board to the top (lid) of the box.
    6. Using a combination of 1/16’ male and female Luers (or 1/16” straight barbed connectors), connect the remaining “clean air” pinch valve tubing to 1/16” Kflex tubing. Direct and attach this tubing to the training or testing chamber as needed.
      Note: Again, it may be advantageous to have separate “clean air” lines for the training and testing chambers that can be attached to the pinch valve separately.

  4. Assemble vacuum system
    1. Wire the 1/4” ID tubing pinch valve to a 2-Channel SPDT Relay Board (as before) and enclose in a container for noise reduction if desired.
    2. Fit one end of 1/4” ID Tygon tubing directly onto laboratory vacuum nozzle. Place a 1/4” straight barbed connector into the other end and attach directly to the pinch valve tubing.
    3. Place the second 1/4” straight barbed connector into the opposite end of the pinch valve tubing and connect it to another length of 1/4” ID Tygon tubing.
    4. Attach this end of the vacuum line to the training or testing chamber as needed.
      Note: It can be difficult to detach tubing from barbed connectors. Optionally, you can cut two lengths of 1/4” ID Tygon tubing and designate one for the training chamber and one for testing chamber. In this case, use a combination of 1/4” male and female Luers to join the pinch valve and vacuum tubing in Step D3. Attach each length of vacuum tubing to the appropriate chamber and connect to the pinch valve as needed.

  5. Program FreezeFrame protocols for stimulus delivery
    1. Open the FreezeFrame Recorder and create a training protocol
      Note: Refer to the FreezeFrame software manual provided by Coulbourn for additional information regarding FreezeFrame setup.
    2. The training protocol should consist of 6 odor-shock pairings that occur every other minute starting in the second minute of the training protocol (Figure 2A). The odor presentations should last 10 sec each with the shock occurring 0.5 sec before the end of the odor presentation and lasting until the end of the odor presentation (i.e., Odor 60-70 sec, shock 69.5-70 sec; Odor 180-190 sec, shock 189.5-190 sec, etc.). Clean air and vacuum should be on at all times except during odor presentations (i.e., Clean air/vacuum 0-60 sec; 70-179 sec, etc.). Approximate training time = 720 sec.
    3. To test learned fear for just the training odor, the protocol should consist of 2 presentations of the conditioned odor (Figure 2B). Presentations should last for 20 sec every 240 sec starting in the second minute of the testing protocol (i.e., #1 60-80 sec; #2 300-320 sec). Clean air and vacuum should be on at all times except during odor presentations (i.e., Clean air/vacuum should be on from 0 sec to 59 sec and 120 sec to 299 sec but programmed to be off from 60 to 119 and 300 to 359)
      Note:
      1. The testing protocol can be adapted to increase the number of presentations and/or to test multiple odors. Adding epochs 240 sec apart with 20 sec presentations as needed.
      2. Remember, the clean air and vacuum lines are attached to NO pinch valves, meaning the trigger from the computer will signal to close the pinch valve, thus shutting the air and vacuum off.

  6. Prepare odor(s) for training/testing
    Note: Prepare odor(s) fresh daily. 
    1. Empty any waste odorant into an appropriate container.
    2. Inside a fume hood, dilute E5 in mineral oil to ~200 ppm (64 μl E5 in 1,936 μl mineral oil) inside the odor vial. Gently shake to mix.
      Note: Any additional odorants should also be intensity matched to ~200 ppm, which will require different ratios of odor: mineral oil, to be determined by users. 
    3. Place the non-odorized air line (from the Olfactory Stimulus Control) on the unmarked stopcock and the “odorized air” Kflex tubing on the marked stopcock. Make sure to open stopcocks during training and testing when odor should reach chambers.

  7. Olfactory fear conditioning (training)
    1. Turn on humidifier and HEPA filter. If humidity is low, allow enough time before training to ensure humidity is > 35%. Leave both running throughout training.
    2. Ensure that the appropriate camera is plugged into the computer and open the FreezeFrame Recorder.
    3. Turn on vacuum, aquarium pump, pinch valves, and Precision Animal Shocker and make sure all necessary tubing is attached to the training chamber. Prepare fresh odor (above), attach necessary tubing, and open the stopcocks on the odor vial.
    4. Set the Shocker to deliver 0.8 mA shocks with 8 pole scanned output. Make sure that the shocker switch is positioned to “Remote” (under “Operate”) and “Subject.”
      Note: Other shock intensities may be used, to be determined by the user. We have elicited fear learning using 0.4 mA and 0.6 mA in addition to 0.8 mA.
    5. Use the Stimulus Configuration Panel under the Settings tab to ensure all stimuli are functioning and will be triggered appropriately during training.
    6. Select the training protocol from the dropdown menu.
    7. Specify a data file for storing data.
    8. Enter a unique name for each mouse (ideally this should relate to a physical identifier such as tail mark, ear punch, etc.) and take the reference frame.
      Note: If transporting mice from animal facility, allow at least 30 min before subjecting to training.
    9. Place mouse in the chamber and allow 5 min for habituation.
    10. Press start and observe mice during training for signs of shock experience (jumping, squeaking, increased freezing, etc.).
    11. Remove mouse after completion of training protocol and return to the cage.
      Note: We wait approximately 24 h after training before testing mice; however, different time points could be useful for determining short-term vs. long-term memory or retention.
    12. Clean training chamber with 1% Alconox solution and water, making sure to wipe down the shock floor and drop pan before starting next mouse.

  8. Olfactory fear testing
    1. Ensure that the appropriate camera is plugged into the computer and open the FreezeFrame Recorder.
    2. Turn on vacuum, aquarium pump, pinch valves, and infrared light and make sure all necessary tubing is attached to the testing chamber. Prepare fresh odor (above), attach necessary tubing, and open the stopcocks on the odor vial.
      Note: If using multiple odors during testing, move the non-odorized air line to each odor vial shortly before presenting that odor and open stopcocks. The non-odorized air line provides air to the odor vial that will become your odorized air for odor presentations. Make sure all stopcocks on other odor vials are closed when not being presented.
    3. Use the Stimulus Configuration Panel under the Settings tab to ensure all stimuli are functioning and will be triggered appropriately during testing.
    4. Select the testing protocol from the dropdown menu.
    5. Specify a data file for storing data.
    6. Enter the same unique name for each mouse as the previous day (to allow matching between training and testing data if desired) and take the reference frame.
      Note: If transporting mice from animal facility, allow at least 30 min before subjecting to training.
    7. Place mouse in the chamber and allow 5-10 min for habituation. During this time, observe mice for behaviors indicative of comfort and exploration, such as rearing and grooming. Mice that exhibit excessing freezing during the first minute of testing will be removed from the analysis (see below), so it is important to provide sufficient time during habituation for any novel context-induced freezing to subside.
    8. Press start and allow the mouse to complete testing protocol.
    9. Remove mouse after completion of training protocol and return to the cage.
    10. Clean testing chamber with 1% Alconox solution and water before starting next mouse.

Data analysis

  1. Open the FreezeFrame Trial Viewer and locate the appropriate testing data file.
  2. Select the desired mouse from the Animal box to the left of the screen (Video 1).

    Video 1. Example of testing data from FreezeFrame4 Trial Viewer. This video depicts 120 sec of olfactory fear testing 24 h after olfactory fear conditioning with all of the data provided from FreezeFrame4 (video playback in real time). The conditioned odor is presented at 60 sec for 20 sec and the clean air and vacuum are turned off at 60 sec for a total of 60 sec. The Animal menu on the left shows all Animal IDs recorded under the same data file, with the selected trial highlighted in blue. The Motion Index graph indicates the recorded activity (y-axis; yellow line) across time (x-axis). The vertical blue line moves in real time with the video while the horizontal blue line shows the user-defined threshold for freezing. The rectangle below shows bouts of freezing, as determined by user-defined threshold and bout, as yellow boxes. Periods of non-freezing are not colored. The next rectangle below provides a visual for the protocol and shows the onset/offset of all stimuli during the protocol (blue rectangle: odor onset, yellow rectangle: vacuum offset, green rectangles: clean air offset). The motion index histogram should be used to set threshold. The bout length can be set to fit specific needs; in this case, 2 sec. The video to the right of the histogram shows the behavior of the mouse throughout testing, with bouts of freezing (determined through the user-defined threshold and bout length) indicated by a green light above. In this example, the mouse freezes for 0% of the 1st 60 sec interval (baseline freezing) and 74.28% during the 2nd 60 sec interval during the presentation of the conditioned stimulus. (All experimental protocols were approved by the University of Tennessee Institutional Animal Care and Use Committee.) 

  3. Look at the Motion Index graph in the lower left corner. Use your cursor to drag the blue vertical line inside the graph to the first trough of the yellow line. This will set your freezing threshold.
    Note: In our hands when sampling at 7.5 Hz, thresholds typically occur at motion index values between 2 and 5, and no higher than 10 (Video 1).
  4. Manually set the bout to 2 sec (Video 1).
    Note: This indicates the motion index must be below the set threshold for a total of 2 sec to be counted as a bout of freezing.
  5. Navigate to the Analysis tab and record the % Freezing in 60 sec intervals for the duration of the testing protocol for each mouse.
    Note: Optionally, you may set the same threshold for all mice and export all the analysis for all mice into a single spreadsheet.
  6. Exclude any mice that freeze excessively in the first minute of the testing protocol from analysis. Additionally, mice should freeze during odor cue but should decrease freezing behaviors as the odor is evacuated from the chamber before the next cue (Figure 2C).
    Note: Based on data collected in our lab, we exclude any mice that freeze more than 20% in the first minute, but this can be adjusted based on specific needs of individual users. High freezing during the first minute indicates high generalized anxiety and can make it difficult to determine whether freezing during odor presentations is specific to the odor cue.
  7. For each mouse, average together the % freezing values during presentations of E5 (i.e., if you presented E5 twice during testing from 60 sec to 120 sec and again from 300 sec to 360 sec, average the two corresponding values together to get an average % freezing for each mouse).
    Note: If you presented two separate odors twice each during testing, average the two values for the 1st odor together and the two values for the 2nd odor together separately so you have an average % freezing for each mouse for each odor; if you presented multiple odors only once each, do not average.
  8. Average the E5 % freezing values for all mice together to generate a group average.
  9. Average the % freezing during the first minute of testing for all mice together to generate a group average of baseline freezing.
  10. Use a paired samples t-test to compare the E5-induced freezing to the baseline freezing. Significant results demonstrate learned fear as a result of fear conditioning to E5 (Figure 2D).
    Note: If you are interested in comparing acquired freezing between different experimental groups, you may use appropriate tests to statistically compare E5-induced freezing between groups. Additionally, if you test freezing to multiple odors, you may use appropriate tests to compare freezing to one odor with that to others.


    Figure 2. Training and testing schematics and representative testing analysis. A. Schematic for training paradigm illustrating timing of each stimulus. B. Schematic for testing paradigm illustrating timing of each stimulus. C. Representative testing data demonstrating % freezing behaviors of B6 mice (n = 10) during 60 sec bins throughout testing paradigm. Colored bars represent 60 sec bins during which E5 was filling chamber. Uncolored bars represent 60 sec bins during which air and vacuum are on and odor is not filling chamber. Freezing does not differ significantly between the first and second presentations of E5. D. Representative testing data demonstrating average freezing behaviors during baseline 1st minute and for both 60 sec E5 presentations (n = 10). Mice freeze significantly more during presentations of E5 than during the baseline minute, indicating acquired fear to the conditioned stimulus. Error bars denote SEM, *P < 0.05.

Notes

  1. Rarely, control mice will unexpectedly exhibit very low freezing to the conditioned stimulus; therefore, we do not include control mice in analysis that do not freeze at least 25% to the first presentation of the conditioned stimulus. These mice are deemed non-learners.
  2. Aged mice typically freeze less but we observe consistent freezing of at least 50% to the conditioned stimulus up to 8 months of age in control mice.

Acknowledgments

This publication was supported by the NIDCD awards R01DC013779 to MLF and F31DC016485 to JMR. An adapted form of this protocol for testing fear generalization was used in Ross and Fletcher (2018), which utilized multiple intensity matched odors (~200 ppm as measured by a photoionization detector, Rae Systems, catalogue number: 059-4020-000) during testing (Sigma-Aldrich, catalogue numbers: E15701, 148962, 418099, 537683, 00790).

Competing interests

The authors declare that they have no conflict of interest.

Ethics

All experimental protocols were approved by the University of Tennessee Institutional Animal Care and Use Committee.

References

  1. Blanchard, R. J. and Blanchard, D. C. (1969). Crouching as an index of fear. J Comp Physiol Psychol 67(3): 370-375.
  2. Fanselow, M. S. (1980). Conditioned and unconditional components of post-shock freezing. Pavlov J Biol Sci 15(4): 177-182.
  3. LeDoux, J. (2003). The emotional brain, fear, and the amygdala. Cell Mol Neurobiol 23(4-5): 727-738.
  4. Pavesi, E., Gooch, A., Lee, E. and Fletcher, M. L. (2012). Cholinergic modulation during acquisition of olfactory fear conditioning alters learning and stimulus generalization in mice. Learn Mem 20(1): 6-10.
  5. Ross, J. M. and Fletcher, M. L. (2018). Learning-dependent and -independent enhancement of mitral/tufted cell glomerular odor responses following olfactory fear conditioning in awake mice. J Neurosci 38(20): 4623-4640.

简介

经典的恐惧条件反射通常涉及将离散的线索与脚部震动配对。 量化对学习线索的行为冻结是针对学习和记忆的神经科学研究的关键分析。 许多范例利用诸如音调之类的离散刺激; 然而,鉴于小鼠是气味驱动的动物和商业上可获得的各种气味剂,使用气味作为条件刺激对于涉及学习的研究具有优势。 在这里,我们描述了用于组装系统的详细程序,该系统用于在单日恐惧条件反射期间呈现离散的气味线索并随后分析冷冻行为以评估学习。

【背景】联想恐惧学习是几种焦虑症的根源,涉及将中性刺激与厌恶结果配对。这种配对产生强烈的行为恐惧反应,以冷冻(LeDoux,2003)的形式,对条件刺激,可以量化为恐惧学习和记忆的量度。离散刺激,如音调,通常被用作恐惧条件反射的条件刺激;然而,嗅觉提示在诱导学习冷冻方面也非常有效(Pavesi et al。,2012; Ross and Fletcher,2018)。这种联想恐惧学习方法不同于利用捕食者气味的方法,这种方法产生本能行为而非学习行为,使其成为快速评估嗅觉学习的理想选择。行为冻结,定义为缺乏所有自愿运动(Blanchard和Blanchard,1969; Fanselow,1980),可以通过自动化软件进行测量,该软件逐帧地比较像素差异。我们开发了一种协议,该协议使用自动FreezeFrame软件在训练和测试期间提供离散的嗅觉提示。该协议支持标准的恐惧条件反射和随后的测试,但也提供扩展的灵活性,以适应广泛的实验需要,如灭绝,区分条件,泛化范例或实验操作(例如,光遗传学或化学遗传学)。此外,嗅觉恐惧条件反射提供了一种研究嗅觉联想学习机制的快速方法,因为培训需要在一天内进行少量试验,第二天进行学习评估。

关键字:经典条件作用, 嗅觉条件作用, 恐惧学习, 行为, 冻结, 嗅觉恐惧

材料和试剂

  1. 封口膜
  2. 油管
    1. 1/16“ID Kflex管(美国塑料,目录号:65170)
    2. 1/16“ID Tygon管(Fisher Scientific,目录号:14-171-129)
    3. 1/8“ID Masterflex Tygon E-Lab(E-3603)泵管(Cole-Parmer仪器,目录号:EW-06509-16)
    4. 1/4“ID Tygon管(Fisher Scientific,目录号:14-171-221)
  3. Luers和连接器
    1. 旋塞式单向公锁(Cole-Parmer仪器,目录号:EW-30600-00)
    2. Male Luer x 1/16“软管倒钩(Cole-Parmer仪器,目录号:EW-45518-00)
    3. 公Luer锁插头(Cole-Parmer仪器,目录号:EW-30800-30)
    4. 母Luer x 1/8“软管倒钩(Cole-Parmer仪器,目录号:SI-30800-08)
    5. Masterflex Y型连接器(Cole-Parmer仪器,产品目录号:EW-30614-04)
    6. 母Luer 1/16“x 1/16”软管倒钩(Cole-Parmer仪器,产品目录号:EW-45508-00)
    7. (2)1/4“直带式连接器(Cole-Parmer仪器,产品目录号:EW-30612-13)
    8. 1/8“NPT公头适配器至1/16”倒钩(Cole-Parmer仪器,目录号:EW-06365-41)
    9. 1/8“NPT公头适配器至1/4”倒钩(Cole-Parmer仪器,目录号:EW-30704-09)
    10. 1/2“NPT公头适配器至1/4”软管倒钩(Cole-Parmer仪器,目录号:EW-30704-17)
  4. 移液器吸头(Eppendorf,目录号:0030073061,0030073100)
  5. 带聚丙烯盖的20毫升玻璃闪烁瓶(Sigma-Aldrich,目录号:Z190535)
  6. 16G1½针(Surgo Surgical Supply,目录号:150-305198)
    制造商:BD,目录号:305198。
  7. 小鼠(C57BL / 6J; THE JACKSON LABORATORY,目录号:000664),优选> 6周大了
    注意:&nbsp;
    1. 此协议适用于其他菌株,但我们建议用户测试并确定其菌株的适用性。
    2. 如果将恐惧条件与其他技术结合使用,例如光遗传学,请事先准备好小鼠并在训练前留出足够的恢复时间。
  8. 环氧树脂(Thorlabs,目录号:G14250)
  9. 矿物油(Sigma-Aldrich,目录号:M5904)
  10. 气味剂,例如,乙酸乙酯(Sigma-Aldrich,目录号:290866)
  11. Alconox(Sigma-Aldrich,目录号:242985)

设备

  1. 单通道移液器(Eppendorf,目录号:3121000074和3121000120)
  2. 训练室(图1B)
    1. 冲击地板(Coulbourn Instruments,目录号:H10-11M-TC-SF)
    2. 训练笼(Coulbourn Instruments,目录号:H10-11M-TC)
    3. 金属墙板(Coulbourn Instruments,目录号:H90-00M-M-KT01)
    4. 25英尺防震电缆(Coulbourn Instruments,目录号:H93-01-25)
    5. 精密动物减震器(Coulbourn Instruments,目录号:H13-15)
  3. 测试室(定制透明有机玻璃室,图1C)
    1. 外形尺寸:11“x 6”x 5.5“(长x宽x高),壁厚1/2”。
    2. 一个1/4“厚的壁,有2排6孔(1/2”直径)从顶部开始~1 / 2“,允许空气/气味扩散穿过中间壁,将11”腔室长度分成两半,这样有两个内部隔间,尺寸为5“x 5”x 5.5“。
    3. 腔室有一个1/2“厚的盖子,可以用螺丝固定在腔室上。
    4. 该腔室在笼子的两侧各有一个1/2英寸的直径孔,从顶部到1/2英寸,分别用于气味和真空管线。
  4. USB相机(Coulbourn Instruments,目录号:ACT-VP-02)
  5. Actimetrics USB数字接口(Coulbourn Instruments,目录号:ACT-712)
  6. 用于Coulbourn硬件的FreezeFrame 4适配器电缆(Actimetrics,目录号:ACT-INTF)
  7. 隔离隔间(Coulbourn Instruments,目录号:H10-24T)
  8. 红外照明器(Stoelting,目录号:60540)
  9. 丙烯酸流量计,0.1-1 LPM(Cole-Parmer仪器,目录号:EW-32460-42)
  10. 水族馆空气泵,非UL(Spectrum Brands,Tetra,Whisper ®,目录号:77851)
  11. 双通道SPDT继电器板(Winford Engineering,目录号:RLY102-12V-DIN)
  12. 用于双通道SPDT继电器板的12 V墙壁电源(Winford Engineering,目录号:WSD050-10-0)
  13. 夹管阀门
    1. 1管常开夹管阀(NResearch,目录号:225P021-21)
    2. 1管常开夹管阀(NResearch,目录号:648P021-82)
    3. 嗅觉刺激控制(Coulbourn Instruments,目录号:H15-03)
  14. 真正的HEPA空气净化器,390平方英尺(霍尼韦尔,型号:50250)
  15. Cool Moisture Console加湿器(霍尼韦尔,型号:HCM-6009)
  16. 湿度和温度监测器(如Fisherbrand TM Traceable TM 温度计/时钟/湿度监测仪,Fisher Scientific,目录号:06-662-4)


    图1.气味输送和行为室。:一种。水族箱泵通过Tygon管道为流量计提供空气,然后将其输送到嗅觉刺激控制器以进行编程输送。 Kflex管连接到嗅觉刺激控制器和非加臭空气旋塞阀,以向组装的气味瓶提供气流以产生加臭空气。加臭的空气将通过额外的Kflex管流出加臭的空气旋塞阀并流到附属的腔室。 B.修改训练室以在笼子的左侧和右侧增加小孔。通过每个孔安装NPT管并用螺母固定到腔室内部。在笼子左侧的NPT管道的带倒钩的末端连接有气味的空气管路,而真空管线连接在笼子右侧的NPT管道的带倒钩的末端,正好位于冲击地板的下方。相机位于笼子上方以进行记录。 C.将测试室放置在隔离室内,其中红外光源位于室的右侧,摄像机安装在室的上方。带有清洁和加臭空气的独立管道进入左侧腔室,腔室右侧有一个真空吸尘器,以便清除气味。

软件

  1. FreezeFrame 4软件(Coulbourn Instruments,目录号:ACT-100A)

程序

注意:

  1. 根据制造商的说明组装行为训练室,并根据您的需求进行优化。
  2. 按照软件提供的说明,设置摄像机并将刺激[气味,冲击,空气,真空等]的传递和训练和测试协议的持续时间编程到FreezeFrame中。
  3. 该协议将详细介绍用单一气味,戊酸乙酯(E5)进行小鼠嗅觉恐惧学习的训练和测试,但可以通过在测试室中添加额外的气味小瓶和线来调整多种气味(参见Ross和Fletcher, 2018)。通过向训练室添加额外的气味小瓶和管线,可以扩展其用于区分调节。如果需要,用户还可以添加连续的测试日来评估灭绝。

  1. 修改培训和测试室
    1. 使用钻头在真空线的训练室的一侧打孔。孔应位于冲击地板和下水盘之间的腔室中间。将带有1/4“倒钩的1/2”NPT公管接头插入腔室外部的钻孔中,并用1/2英寸NPT螺母固定。螺母应位于腔室内部,倒钩应从腔室外部伸出(图1B)。
    2. 使用钻头在训练室的另一侧为气味线制作第二个小孔。该孔也应位于室的中间,大约在震动地板上方1 / 2-1“处。将带有1/16“倒钩的1/8”NPT公管接头插入腔室外部的钻孔中,并用1/8“NPT螺母固定。螺母应位于腔室内部,倒钩应从腔室外部延伸(图1B)。
    3. 将测试室放入隔离室内,并将所有管道/电线穿过室背面的孔向内(图1C)。
    4. 将红外光源放置在隔间内,朝向测试室的侧面,该侧面不会抓住鼠标/加臭的空气将进入室的一侧。通过将光源直接指向隔离立方体的墙壁而不是相机或测试室(图1C),最大限度地减少腔室/摄像机的眩光。
    5. 将1/2“NPT公头适配器的NPT端插入并穿过1/4”软管倒钩到测试室两侧的一个孔中。将真空管连接到带倒钩的一端(图1)。

  2. 组装气味小瓶
    1. 从单个包装中取出16G1½针,并将旋塞锁定在每根针的母鲁尔侧。&nbsp;
    2. 如果尚未分离,请从20 ml闪烁瓶中取出塑料盖。在帽顶部钻两个孔,在孔之间留有足够的空间用于带有连接旋塞的针。孔应尽可能接近针的尺寸。将带有钻孔的盖子固定在瓶子上。
    3. 小心地打开针头,将斜面端部穿过盖子上的孔,使针头在瓶子内部。针应紧贴钻孔。
    4. 混合环氧树脂并松散地涂在瓶盖上,将针头固定在盖子上。确保不要在旋塞阀或瓶子本身上涂上环氧树脂。让治疗过夜。
    5. 使用尖锐物标记其中一个旋塞。这个旋塞阀将充当气味瓶的“加臭空气”一侧。另一个旋塞将从嗅觉控制中获得无异味的空气。&nbsp;
    6. 切割一段适合您特定设置的Kflex管(长度足以从瓶子伸展到腔室而不会产生过大的张力)。在Kflex管的一端,插入公鲁尔接头的1/16英寸软管倒钩端。将Luer的公端连接到标记的旋塞的顶部,并根据需要将Kflex管的另一端连接到训练或测试室。
    注意:
    1. 从带倒钩的管道上拆下管子可能很困难;因此,有利的是从气味瓶到每个腔室具有单独长度的管道。&nbsp;
    2. 如果使用多种气味进行测试,Kflex管的长度应与所有气味样品瓶的测试室相似。多个管可以用封口膜相互固定,并插入测试室侧面的孔中。

  3. 组装清洁空气和加臭空气输送系统
    1. 将Masterflex Tygon管的一端连接到其中一个水族箱空气泵输出端,将Tygon管的另一端连接到流量计的输入端。在第二根Tygon管上,连接1/8“母鲁尔接头,并用相应的公鲁尔锁定塞将其盖住。将另一端连接到相对的流量计输入端。
    2. 将一小段1/16“Tygon管的一端连接到流量计输出端,另一端连接Masterflex Y型连接器。再切两片1/16“Tygon管,长度大致相等,并连接到Y型连接器上的剩余倒钩。其中一条线路将为“清洁空气”线路提供空气,而另一条线路将为“气味”线路提供空气。
    3. 指定用于“清洁空气”的管道应与带有1/16“内径管的常开夹管阀配对,而用于”加臭空气“管线的管道应与嗅觉控制管道配对。用Luers安装“清洁空气”管线的末端,以连接夹管阀管。使用1/16“公头和母头Luers的组合连接(可选,您可以使用两侧带1/16”倒钩的直带倒钩连接器)。
    4. 将“加臭空气”管连接到嗅觉控制器背面后,将另一段管连接到嗅觉控制器顶部的三个端口之一。将1/16“公鲁尔接头放置在管道的另一端,使其可以直接连接到气味样品瓶上未标记的旋塞阀。
    5. 将夹管阀连接到2通道SPDT继电器板,确保将夹管阀类型(分别为常开和常闭的NO或NC)与正确的触点匹配。
      注意:&nbsp;
      1. 有关继电器板接线的更多信息,请参阅 Winford网站<上提供的原理图。 / A>
      2. 为了最大限度地减少夹管阀在训练和测试期间发出的噪音,我们将夹管阀放入发泡胶或塑料(如用过的移液器吸头)盒内,泡沫或泡沫聚苯乙烯花生。我们在箱体侧面钻有小孔,用于夹管阀线,并将继电器板安装到箱子的顶部(盖子)。
    6. 使用1/16英尺公母L(或1/16英寸直带倒钩连接器)的组合,将剩余的“清洁空气”夹管阀连接到1/16英寸Kflex管。根据需要将该管道直接连接到培训或测试室。
      注意:同样,为培训和测试室提供单独的“清洁空气”管线可能是有利的,可以单独连接到夹管阀。

  4. 组装真空系统
    1. 将1/4“内径管夹管阀连接到2通道SPDT继电器板(如前所述),如果需要,将其封装在容器中以降低噪音。
    2. 将1/4“ID Tygon管的一端直接安装到实验室真空吸嘴上。将1/4“直带倒钩连接器放入另一端,并直接连接到夹管阀管。
    3. 将第二个1/4“直带倒钩连接器放入夹管阀管的另一端,并将其连接到另一长度为1/4”ID的Tygon管。
    4. 根据需要将真空管的这一端连接到培训或测试室。
      注意:从带倒钩的连接器上拆下管子可能很困难。您可以选择切割两段1/4“内径的Tygon管,并指定一个用于训练室,另一个用于测试室。在这种情况下,在步骤D3中使用1/4“公头和母头Luers的组合来连接夹管阀和真空管。将每段真空管连接到适当的腔室,并根据需要连接到夹管阀。

  5. 程序FreezeFrame协议用于刺激传递
    1. 打开FreezeFrame Recorder并创建一个培训协议
      注意:请参阅由 FreezeFrame软件手册提供的有关FreezeFrame设置的其他信息,请参阅Coulbourn。
    2. 训练方案应包括6个气味 - 冲击配对,从训练方案的第二分钟开始每隔一分钟发生一次(图2A)。气味呈现应持续10秒,在气味呈现结束前0.5秒发生休克,持续至气味呈现结束(即,气味60-70秒,休克69.5-70秒;气味180-190秒,震惊189.5-190秒,等)。除气味介绍外,应始终打开洁净空气和真空(即,清洁空气/真空0-60秒; 70-179秒,等)。近似训练时间= 720秒。
    3. 为了测试对训练气味的学习恐惧,该方案应该包括两种有条件气味的呈现(图2B)。从测试方案的第二分钟开始,演示应该每240秒持续20秒(即,#1 60-80秒;#2 300-320秒)。除气味介绍外,应始终打开清洁空气和真空(即,清洁空气/真空应在0秒至59秒和120秒至299秒之间打开,但程序设定为从60秒开始到119和300到359)
      注意:
      1. 可以调整测试协议以增加演示的数量和/或测试多种气味。添加240秒的纪元,并根据需要进行20秒的演示。
      2. 请记住,清洁空气和真空管路连接到NO夹管阀,这意味着计算机的触发器将发出信号以关闭夹管阀,从而关闭空气和真空。

  6. 准备气味进行培训/测试
    注意:每天准备新鲜的气味。
    1. 将任何废气味清空剂倒入适当的容器中。
    2. 在通风橱内,将气味油中的E5稀释至气味小瓶内约200 ppm(在1936μl矿物油中的64μlE5)。轻轻摇动混合。
      注意:任何其他添加剂的强度也应与~200 ppm强度匹配,这需要不同比例的气味:矿物油,由用户决定。
    3. 将未加味的空气管路(来自嗅觉控制器)放在未标记的旋塞阀上,并在标记的旋塞阀上放置“加臭空气”Kflex管。确保在训练和测试期间打开旋塞阀,当气味到达室时。

  7. 嗅觉恐惧条件(训练)
    1. 打开加湿器和HEPA过滤器。如果湿度低,在训练前留出足够的时间以确保湿度> 35%。让两者都在整个训练中运行
    2. 确保将相应的摄像头插入计算机并打开FreezeFrame Recorder。
    3. 打开真空,水族箱泵,夹管阀和Precision Animal Shocker,确保所有必要的管道都连接到培训室。准备新鲜气味(上图),连接必要的管道,并打开气味瓶上的旋塞。
    4. 设置Shocker以通过8极扫描输出提供0.8 mA冲击。确保将电击开关定位到“远程”(在“操作”下)和“主题”。
      注意:可以使用其他冲击强度,由用户确定。除0.8 mA外,我们还使用0.4 mA和0.6 mA引发恐惧学习。
    5. 使用“设置”选项卡下的“刺激配置”面板可确保所有刺激都正常运行,并在训练期间适当触发。
    6. 从下拉菜单中选择培训协议。
    7. 指定用于存储数据的数据文件。
    8. 为每只鼠标输入一个唯一的名称(理想情况下,这应该与物理标识符相关,如尾标,耳塞,等。)并获取参考框架。
      注意:如果从动物设施中运送小鼠,请在接受培训前至少30分钟。
    9. 将小鼠置于室中并允许5分钟适应。
    10. 按下开始并在训练期间观察小鼠是否有休克体验(跳跃,吱吱声,增加冰冻,等)。
    11. 完成训练方案后移除鼠标并返回笼子。
      注意:我们在训练后大约24小时等待测试小鼠;但是,不同的时间点可用于确定短期记忆与长期记忆或保留。
    12. 使用1%Alconox溶液和水清洁训练室,确保在开始下一只鼠标之前擦拭震动地板和平底锅。

  8. 嗅觉恐惧测试
    1. 确保将相应的摄像头插入计算机并打开FreezeFrame Recorder。
    2. 打开真空,水族箱泵,夹管阀和红外灯,确保所有必要的管道连接到测试室。准备新鲜气味(上图),连接必要的管道,并打开气味瓶上的旋塞。
      注意:如果在测试过程中使用多种气味,请在出现异味和打开旋塞之前不久将无气味的空气管路移至每个气味样品瓶中。无气味的空气管路为气味小瓶提供空气,气味小瓶将成为气味空气,用于气味呈现。确保其他气味小瓶上的所有旋塞在未出现时关闭。
    3. 使用“设置”选项卡下的“激励配置”面板可确保所有刺激都正常运行,并在测试期间适当触发。
    4. 从下拉菜单中选择测试协议。
    5. 指定用于存储数据的数据文件。
    6. 为每个鼠标输入与前一天相同的唯一名称(如果需要,可以在训练和测试数据之间进行匹配)并获取参考框架。
      注意:如果从动物设施中运送小鼠,请在接受培训前至少30分钟。
    7. 将小鼠置于室中并允许5-10分钟适应。在此期间,观察老鼠指示舒适和探索的行为,例如饲养和梳理。在测试的第一分钟期间表现出过度冷冻的小鼠将从分析中移除(见下文),因此在适应期间提供足够的时间以使任何新的背景诱导的冷冻消退是重要的。
    8. 按开始并允许鼠标完成测试协议。
    9. 完成训练协议后移除鼠标并返回笼子。
    10. 在开始下一只小鼠之前,用1%Alconox溶液和水清洁测试室。

数据分析

  1. 打开FreezeFrame试用查看器并找到相应的测试数据文件。
  2. 从屏幕左侧的“动物”框中选择所需的鼠标(视频1)。


    视频1.从FreezeFrame4试验查看器测试数据的示例。此视频描述了在使用FreezeFrame4提供的所有数据(实时视频播放)后嗅觉恐惧条件反射24小时后120秒的嗅觉恐惧测试。经调节的气味在60秒时出现,持续20秒,清洁空气和真空在60秒时关闭,总共60秒。左侧的Animal菜单显示在同一数据文件下记录的所有Animal ID,所选试用以蓝色突出显示。运动索引图表指示跨时间(x轴)记录的活动(y轴;黄线)。垂直蓝线与视频一起实时移动,而水平蓝线显示用户定义的冻结阈值。下面的矩形显示了由用户定义的阈值和回合确定的冻结回合,如黄色框。非冻结期不着色。下面的下一个矩形为协议提供了视觉效果,并显示了协议期间所有刺激的开始/偏移(蓝色矩形:气味开始,黄色矩形:真空偏移,绿色矩形:清洁空气偏移)。运动指数直方图应该用于设置阈值。可以设置回合长度以满足特定需求;在这种情况下,2秒。直方图右侧的视频显示了整个测试过程中鼠标的行为,其中有一个由上面的绿灯指示的冻结(通过用户定义的阈值和回合长度确定)。在这个例子中,鼠标在1 st 60秒间隔(基线冻结)的0%和在2 nd 60秒间隔期间74.28%冻结条件刺激。

  3. 查看左下角的运动索引图。使用光标将图形内的蓝色垂直线拖动到黄线的第一个槽中。这将设置你的冻结阈值。
    注意:在7.5 Hz采样时,我们手中的阈值通常出现在2到5之间的运动指数值上,并且不高于10(视频1)。
  4. 手动将回合设置为2秒(视频1)。
    注意:这表示运动指数必须低于设定的阈值,总计2秒才算作冻结。
  5. 导航到“分析”选项卡,并在每个鼠标的测试协议期间以60秒的间隔记录%冻结。
    注意:您可以选择为所有鼠标设置相同的阈值,并将所有鼠标的所有分析导出到单个电子表格中。
  6. 从分析中排除在测试方案的第一分钟过度冻结的任何小鼠。此外,小鼠应该在气味提示期间冻结,但应该减少冰冻行为,因为在下一个提示之前气味从腔室中排出(图2C)。
    注意:根据我们实验室收集的数据,我们排除了在第一分钟内冻结超过20%的任何鼠标,但这可以根据个人用户的特定需求进行调整。第一分钟的高度冷冻表明高度广泛的焦虑,并且很难确定气味呈现期间的冷冻是否与气味提示有关。
  7. 对于每只鼠标,在E5演示期间平均显示%冻结值(即,如果您在测试过程中将E5两次从60秒提升到120秒再从300秒提高到360秒,平均两个对应为了获得每只老鼠的平均冻结百分比值,我们将这些值相加。
    注意:如果在测试过程中每次两次出现两种不同的气味,将1 st 气味的两个值平均值和2 nd 气味的两个值一起平均分开使每只小鼠的每种气味平均冻结%;如果您每次只出现一次多种气味,请不要平均。
  8. 将所有小鼠的E5%冷冻值平均在一起以产生组平均值。
  9. 将所有小鼠一起测试的第一分钟内的冻结%平均,以产生基线冷冻的组平均值。
  10. 使用配对样本 t -test将E5诱导的冷冻与基线冷冻进行比较。显着的结果表明由于对E5的恐惧条件而产生了学习恐惧(图2D)。
    注意:如果您对比较不同实验组之间的获得性冷冻感兴趣,您可以使用适当的测试来统计比较各组之间E5诱导的冷冻。此外,如果您测试冷冻到多种气味,您可以使用适当的测试来将冷冻与一种气味与其他气味进行比较。


    图2.培训和测试原理图以及代表性的测试分析。 A.训练范例的示意图,说明每种刺激的时间安排。 B.测试范例的示意图,说明每个刺激的时间。 C.代表性测试数据,证明在整个测试范例中60秒箱内B6小鼠(n = 10)的冻结行为%。彩色条代表60秒的箱,其间E5是填充室。未着色的条形代表60秒的箱子,在此期间空气和真空开启且气味不是填充室。冷冻在E5的第一次和第二次呈现之间没有显着差异。 D.代表性测试数据,证明在基线1 st 分钟和60秒E5呈现(n = 10)期间的平均冷冻行为。在E5呈现期间,小鼠比在基线分钟期间明显冻结更多,表明对条件刺激的获得性恐惧。误差棒表示SEM,* P &lt; 0.05。

笔记

  1. 很少,对照小鼠意外地表现出对条件刺激的非常低的冷冻;因此,我们不包括分析中的对照小鼠,其不会使条件刺激的第一次呈现冻结至少25%。这些老鼠被认为是非学习者。
  2. 老年小鼠通常冻结较少,但我们观察到对照小鼠中长达8个月的条件刺激持续至少50%的冷冻。

致谢

该出版物得到了NIDCD奖项R01DC013779对MLF和F31DC016485对JMR的支持。 Ross和Fletcher(2018)使用了这种用于测试恐惧泛化的协议的改编形式,其利用了多种强度匹配的气味(通过光电离检测器测量的~200ppm,Rae Systems,目录号:059-4020-000)。测试(Sigma-Aldrich,目录号:E15701,148962,418099,537683,00790)。

利益争夺

作者声明他们没有利益冲突。

伦理

所有实验方案均由田纳西大学机构动物护理和使用委员会批准。

参考

  1. Blanchard,R。J.和Blanchard,D。C.(1969)。 蹲伏作为恐惧指数。 J Comp Physiol Psychol > 67(3):370-375。
  2. Fanselow,M。S.(1980)。 震后冻结的有条件和无条件组成部分。 Pavlov J Biol Sci 15(4):177-182。
  3. LeDoux,J。(2003)。 情绪化的大脑,恐惧和杏仁核。 Cell Mol Neurobiol 23(4-5):727-738。
  4. Pavesi,E.,Gooch,A.,Lee,E。和Fletcher,M。L.(2012)。 获取嗅觉恐惧条件期间的胆碱能调节改变了小鼠的学习和刺激概括。 学习内存 20(1):6-10。
  5. Ross,J。M.和Fletcher,M。L.(2018)。 学习依赖性和独立性增强二尖瓣/簇状细胞肾小球气味反应后清醒嗅觉恐惧条件小鼠。 J Neurosci 38(20):4623-4640。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ross, J. M. and Fletcher, M. L. (2018). Assessing Classical Olfactory Fear Conditioning by Behavioral Freezing in Mice. Bio-protocol 8(18): e3013. DOI: 10.21769/BioProtoc.3013.
  2. Ross, J. M. and Fletcher, M. L. (2018). Learning-dependent and -independent enhancement of mitral/tufted cell glomerular odor responses following olfactory fear conditioning in awake mice. J Neurosci 38(20): 4623-4640.
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