Materials and Reagents

Animals
Male C57BL/6 mice (Envigo, Jerusalem) weighing 20-25 g and approximately 12 weeks old were used in this study. This protocol can also be used to study the function of the hippocampus in other strains and different age groups of mice (Shoji et al., 2016; Sharma et al., 2018). The mice were housed individually, on a 12/12 h light/dark cycle, and provided with water and standard rodent chow ad libitum. Animals were handled according to approved protocols and animal welfare regulations of the University of Haifa Institutional Ethics Committee.
70% ethanol (Fisher Scientific, catalog number: BP82011 )

Equipment

TFC chambers (Coulbourn Instruments, model: H10-11M-TC )
Place TFC chambers measuring 25 x 25 x 25 cm internally inside a larger, insulated plastic cabinet that excludes external light and noise (Panlab, Harvard Apparatus, model: LE116 76-0280 ).
Visual (CCD) and infrared camera (Sensor Technologies America, model: STC-CMB4MPOE ) along with an infrared illuminator (Bosch, model: EX12LED-3BD-8W ).

Software

FreezeFrame 3.0 and FreezeView software (Coulbourn Instruments)
Note: Both software components can be downloaded from the Actimetrics website.
FreezeView manual
Note: The manual can be downloaded from the Coulbourn webpage.

Procedure

See Video 1 for the procedure to perform the experiment described in this protocol.

Video 1. Trace Fear Conditioning Protocol. This video describes the trace fear conditioning protocol to assess hippocampal function in mice (Animals were handled according to approved protocols and animal welfare regulations of the University of Haifa Institutional Ethics Committee).

Acquisition of TFC
1. Start the FreezeFrame 3.0 software (Coulbourn Instruments) and select the protocol for TFC See Figure 1 for protocol settings in FreezeFrame 3.0.
  
  Figure 1. The screenshots from FreezeFrame 3.0 software showing settings used in the TFC protocol. A. Conditioning trial; B. Contextual test; C. Tone and trace test.
2. Turn on the light and fan of the conditioning box and calibrate the shock levels, light levels, and sound intensity levels for the testing chamber.
3. For the TFC protocol, place mice in a chamber (with a 20 W bulb and a 16-bar metal grid floor) for 120 sec. Apply a 2.9 kHz tone for 20 sec at 80 dB (conditioned stimulus, CS) and a 0.5 mA foot shock for 2 sec (unconditioned stimulus, US) at the end of the 20 sec trace interval.
4. Repeat the previous step four times and separate each trial by a 120 sec inter trial interval (ITI).
5. After administration of the last shock, keep the animals in the chamber for 120 sec before taking them back to the home cage in order to maintain a constant ITI during the conditioning procedure. Do not house the mice in the same room as the testing room.
6. Clean the chambers with 10% ethanol between successive sets of mice.
Contextual memory testing
1. Place mice in the conditioning chamber 24 h after conditioning and record freezing for 300 sec (without tone or foot shock) using a visual camera. Analyze the data with FreezeView software (Coulbourn Instruments).
2. Clean chambers with 10% ethanol between successive sets of mice.
Tone and Trace memory testing
1. For the tone-trace test, place animals in the dark chambers 48 h after conditioning, but hide the grid floor with black plastic to create another context.
2. Present the animals with the TFC protocol as on the conditioning day, but without shock.
3. Record animal behavior using an infrared camera.
4. Clean the chambers with water between successive sets of mice.
Extinction of fear memory
Extinction of TFC refers to a reduction in the freezing response after the repeated presentation of a CS in the absence of the previously paired US.
1. Measure extinction of tone-trace fear response 24 h and 48 h after Tone and Trace testing.
2. Place mice in the dark chambers and hide the grid floor with black plastic to create another context.
3. Present the animals with the TFC protocol as on the conditioning day, but without shock.
4. Record animal behavior using an infrared camera.
5. Clean the chambers with water between successive mice. Do not use 10% ethanol, since the mice may associate the smell with the conditioned context. In this case, water constitutes a different context, allowing to test auditory fear response.

Data analysis

The indication for fear memory is the percentage of time spent freezing during the context, tone, and trace event. See Figure 2 for an overview of the experiment and representative results.

Figure 2. Mice tested in the TFC protocol show evidence of contextual fear and freezing during tone and trace interval. A. Experimental design; B. TFC protocol used for the conditioning; C. Freezing response of mice during the conditioning, Base (% freezing during first 120 sec), Tone (average % freezing during the tones), Trace (average % freezing during the traces) and ITI (average % freezing during the ITI); D. Contextual memory test (% freezing during 300 sec in context); E. Tone and trace fear response, Base (% freezing during first 120 sec), Tone (average % freezing during the tones), Trace (average % freezing during the traces) and ITI (average % freezing during the ITI); F-G. The mice show normal extinction of fear response during tone and trace interval.
The software reports freezing behavior to the tone and trace interval as percent freezing across all five trials.
Shapiro–Wilk test was used as a numerical means of assessing normality. Independent-samples t-test was used as a parametric test and the Mann-Whitney U test was used for the nonparametric equivalent.

Notes

To establish a contextual memory, it is important that the mice be kept in the chamber for 120 sec prior to the stimuli. In addition, a 20 sec trace interval ensures a proper association between the conditional and unconditional stimuli. Five repetitions of this pairing induces a strong response to the trace interval.

Acknowledgments

Our laboratory used this protocol to assess TFC in mice in a recent publication (Sharma et al., 2018). That work was supported by the Israeli Ministry of Science, Technology, and Space (MOST 3-12080 to K.R.); the Israel Science Foundation (ISF 1003/12, ISF-IDRC 2395/2015 to K.R.); the Wolfson Charitable Trust (K.R.); and the Israeli Planning and Budgeting Committee Program Fellowships for Outstanding Post-Doctoral Fellows from China and India (V.S.) and the Ministry of Science and Technology (Eshkol post-doctoral fellowship) and the Tauber foundation fellowship to HO-S. We have modified the protocol in an attempt to make it more efficient to evaluate hippocampus function (Lugo et al., 2014).

Competing interests

The authors have no conflicts of interest or competing interests to declare.

Ethics

Animals were handled according to approved protocols and animal welfare regulations of the University of Haifa Institutional Ethics Committee.

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