Customer: University of Mnster Mnster, Germany

Prepared by: Metris b.v., The Netherlands

Technical Feasibility Study Freezing & Startle detection with Laboras

Table of Contents
Introduction........................................................................................................................................3 Objectives ...........................................................................................................................................3 Technical Feasibility using LABORAS....................................................................................................4 Description of data collection sessions ............................................................................................4 Analysis of collected data ................................................................................................................6 Effects of sound stimulus system on LABORAS ............................................................................6 Detection of Freezing using LABORAS..........................................................................................6 Detection of Startle using LABORAS.............................................................................................8 Results analysis startle data....................................................................................................... 12 Conclusions....................................................................................................................................... 12 Annex A: Freezing Data ..................................................................................................................... 13 Annex B: Startle Data ........................................................................................................................ 19 Annex C: The Conditioning Protocol .................................................................................................. 25

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Introduction
The University of Mnster in Germany investigates fear conditioning of different strains of mice using high intensity audible sounds. After the mice have been conditioned they are placed back in a similar environment after a few days and are exposed to the same sounds or combinations of the familiar sounds and new sounds. The response of the animal can be seen in both startles as well as freezing and are also compared to the responses of unconditioned mice. Current technology and products on the market are not able to measure both freezing and startle responses reliable using a single measurement system In addition, Startle response measurement systems require the animal to be in a plastic cylinder increasing stress and limiting the possibilities of measuring subtle behavior responses. Freezing detection systems (on the market and home build) are based on video and lack sensitivity to differentiate between small movements (such as sniffing and chewing) and freezing (no movements at all).

Objectives
The objective is to detect freezing and startle response in a home-cage environment in which the mouse can move freely and unobstructed (non-invasive). The startle response behavior will be measured during and up to 2 seconds after the end of the stimulus. The freezing behavior will be measured during a stimulus and up to 30 seconds after the end of stimulus. The sound stimulus will be synchronized with the measurement system by providing the measurement system a TTL signal (up) when the stimulus begins and a TTL signal (down) when the stimulus ends. The measurement system will have to detect the startle response with a reliability of 80% or better (by comparing video observer scores and measurement system scores). The energy of the measured startle response will be determined and used as a measure of the startle intensity. The measurement system will detect the time periods that the animal is not moving at all (freezing) with a reliability of 80%. The time intervals will be accurate to about 1 second accuracy.

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Technical Feasibility using LABORAS

In this report, we describe the possibilities and technical feasibility of using LABORAS as the measurement system of choice to realize the objectives described in the previous paragraph. Feasibility of LABORAS is based on the analysis of the data that was collected during a two-day visit of Metris representatives at the University of Munster on 16 and 17 September 2010.

Description of data collection sessions

The data consisted of two different experiments performed on respectively day 1 and day 2. At Day 1 (afternoon), the following experiments were performed: Sensitivity of LABORAS system for Sound Pressure Level of the audio stimulus generator (Experiment name: Univ-Munster-demo.LAB) Measurement of mouse startles as a response to two different audio stimuli (sine tones with one frequency audible for humans and mice). Data was collected by LABORAS and the whole experiment was also captured on video (Experiment name: Startle-test.LAB). The LABORAS Control Unit (LCU) display was video-taped to ensure start synchronization between the video equipment and LABORAS later on (test session 1 and 3). Session 2 was skipped.

At Day 2 (morning), the following experiments were performed: The mice were exposed to different sounds (bursts, white noise and tones) to investigate the effects of conditioning mice. All experiments were captured on video and recorded with LABORAS (in a similar way as during the first day). In addition SONOTRACK was used to record all sounds of the equipment and the animals during each experiment Mice were put in cages with saw-dust bedding material (test session 4 and 5) and paper bedding material (test session 6 and 7)

All test sessions on the first day lasted 6 minutes. The test sessions on the second day had durations of 12 minutes. First the video recording was started then the animal was put in the cage and then simultaneous LABORAS, SONOTRACK (only on 2nd day) and the sound system were started.

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The synchronization times between the video recordings and the other systems are as follows: Test session 1: + 35.3 sec Test session 2: not used Test session 3: + 23.0 sec Test session 4: + 52.1 sec Test session 5: + 15.1 sec Test session 6: +30.5 sec Test session 7: + 22.2 sec

The experimental setup with the LABORAS platform is shown in the picture below:

Figure 1 Laboras /Sonotrack setup & startle speakers In the middle the camera, directed towards the long-side of the cage. The camera was tilted about 15 degrees in elevation to improve visibility of the different body parts of the mice. Behind the LABORAS platform, the LABORAS Control Unit can be seen which was also recorded on video too. On the left, the Sonotrack system components can be seen that are connected to the Ultrasound Microphone above the cage.

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Analysis of collected data

Effects of sound stimulus system on LABORAS

From the sound tests (without animals) it can be concluded that the effect of the sounds (air movement caused by the sound pressure) on the LABORAS system can be neglected. The sounds produced during the first day were visible but led to only very small signal changes in LABORAS. The sounds produced during the second day were not visible in the LABORAS signals. However, it would be advisable to use speakers that are horizontally orientated and not vertical, because this would eliminate any effect on the LABORAS system, because LABORAS measures only vertical force changes.

Detection of Freezing using LABORAS

LABORAS is presently already able to detect immobility. This behavior is in LABORAS defined as sleeping (inactive period of the animal) and periods without movements (during the active period of the animal). The LABORAS immobility algorithm is designed to allow small short movements during a period of motionless behavior of animal. This was done to limit the number of behavior changes in the LABORAS detection. In the analysis LABORAS immobility detection was compared to freezing -scores from video using precise human observations and automated scores from home-made software. The analysis shows the following: The immobility score of the home-made system is not correlated with the human observer scores LABORAS score about 50% of the time intervals of the human observer more or less the same. LABORAS scores in general more intervals (false positives) than the human observer (this is because the LABORAS immobility algorithm is designed to allow small movements during periods of motionless behavior). By informing the LABORAS system when the sounds are generated, more than 50% of the false positives that are in unrelated timeframes can be easily eliminated. In some cases the human observer scores can be debated. Some freezing intervals are missing and also the start and end times are not always corrected. In Test session 3 no correlation between the LABORAS scores and the human scores could be found. It is assumed that the time synchronization is incorrect.

Detailed data, including all manual scores, LABORAS scores and the manual scores can be found in Annex A.

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A screenshot showing the LABORAS signals during freezing is provided below:

Figure 2 Example of Laboras score during freezing Detection of freezing behavior with LABORAS is technically feasible and a performance for automated freezing detection of 80% or more seems possible, assuming the following improvements: A proper definition of freezing behavior will be provided to Metris. Current manual scores show that the consistency of the freezing score can be improved. Allowance of small movements in the LABORAS immobility detection algorithm should be changed or removed by changing the interpolation algorithm that is used in the algorithm. Sensitivity of the LABORAS algorithm might need some changes to correctly pick-up all freezing behavior The LABORAS algorithm will be informed about the timestamps of the sound system, such that it will only look at those time periods during and up to 30 seconds directly after the sound stimuli. Proper functioning of a freezing algorithm will require a quiet environment in terms of mechanical vibrations, similar to the environment that was used during the Metris visit. Although no problems were observed during our visit, it would be advisable to ask any personal to leave the test room during freezing experiments as part of the experimental protocol.

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Detection of Startle using LABORAS

Currently, LABORAS doesnt have a startle response detection algorithm. However, the signals can be analyzed at the times during and directly after the sound stimuli to investigate if LABORAS is sensitive enough to pick up the startle of the mice. During the visit it was clear that not all animals responded during the second day of our visit as expected. The customer notified us about 3 weeks later that changes made to the protocol and the sound stimulus equipment resulted in more distinctive startle responses of the mice. For the feasibility of startle detection we have therefore limited ourselves to the data of the first day. The animals were trained using the fear conditioning protocol described in Annex-C. After this the animals were used in the Laboras setup using the following protocol: Step1: After 60 sec. adaptation phase the animal receives a CS- voice signal of 2.5 kHz with 10 sec. duration and 85dB power, then 20 sec. pause, after this again 10 s CS- signal and 20 sec. pause. This process was repeated 4 times. Step2: The animal receives a CS+ voice signal of 10 KHz with 10 sec. duration and 85dB power, then 20 sec. pause. The sequence is done 4 times. The goal of this experiment is to analyze the animals response (behavioral changes) after receiving different type of calls (2.5 KHz, 85dB and 10 kHz, 85dB), after the fear conditioning training.

1CS-

2CS-

3CS-

4CS-

1CS+ 2CS+

3CS+ 4CS+

20sec 60sec Adaptation 10sec 2.5 kHz, 85dB Signal

20sec

20sec 10sec 10 kHz, 85dB Signal

Time

Fig. 3 Schematic representation of fear conditioning experiment with Laboras

Laboras data was analyzed in a time window of a few seconds during the onset of the CS- and CS+ tones. In figure below the results are shown and clearly show the startles related to the CS+ tones.

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Fig. 4 Startle test with Laboras. Experiment name is Startle-test, session01 and session03; 4 times CS+ signal in the Laboras.

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For both test session 01 and test session 03, the following signals features were derived from the Laboras signals for time periods directly following the CS- and CS+ stimuli: - Amplitude Range, - Energy Variance - Energy

Test session 01

Stimulus 1CS2CS3CS4CS1CS+ 2CS+ 3CS+ 4CS+

Time period 90-100 120-130 150-160 180-190 210-220 240-250 270-280 300-310

Sound 2,5 kHz 2,5 kHz 2,5 kHz 2,5 kHz 10 kHz 10 kHz 10 kHz 10 kHz

Amplitude Range 1 24 37 23 11

Energy variance 37 1593 2748 1372 338

Energy 1 5 4,9 4,4 10

duration(s) Remarks 0,2 0,2 0,2 0,2 0,2 not clean

Fig. 5a Values of LABORAS sensor signal parameters following CS- and CS+ stimuli (test session 01)

Energy variance
2748
Energy Variance Amplitude Range

Amplitude Range
37 24 23 11

1593

1372 338

1CS+

2CS+

3CS+

4CS+

1CS+

Stimulus

2CS+ 3CS+ Stimulus

4CS+

Fig. 5b Amplitude Range and Energy variance analysis

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Test session 03 Time period 70-80(sec) 100-110 130-140 160-170 190-200 220-230 250-260 280-290 Amplitude Range 46 21 22 31 Energy variance 5360 1011 1149 2895

Stimulus 1CS2CS3CS4CS1CS+ 2CS+ 3CS+ 4CS+

Sound 2,5 kHz 2,5 kHz 2,5 kHz 2,5 kHz 10 kHz 10 kHz 10 kHz 10 kHz

Energy 6,1 5 6,1 4,6

duration(s) Remarks 0,2 0,2 0,2 0,2

Fig. 6a Values of LABORAS sensor signal parameters following CS- and CS+ stimuli (test session 03)

Energy variance
5360
Energy Variance Amplitude Range

Amplitude Range
46 31 21 22

2895 1011 1149

1CS+

2CS+ 3CS+ Stimulus

4CS+

1CS+

2CS+ 3CS+ Stimulus

4CS+

Fig. 5b Amplitude Range and Energy variance analysis

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Results analysis startle data

Average duration of the Startle-Respond behavior is about 0,25sec. This is very short with high energy levels in the power spectrum of the Laboras data. The main parameters are Amplitude Range and Energy Variance. Analysis of the Laboras signals in the time intervals directly following the onset of the CS+ stimuli shows similar changes in the Laboras signals that can be used for recognition. These Laboras signals are significantly different from Laboras signals related to normal animal behaviors. In case of the Laboras signals following the onset of the CS- stimuli, no significant or obvious changes can be found in the Laboras signals. The correlation between the Laboras signals following the 4 CS+ stimuli is very high (For details refer to Annex B).

Amplitude range (spread of amplitude) directly after the CS+ stimuli is very high and also Energy Variance is high. This is contrast to the neglect able values of these two parameters after the CSstimuli. This means that amplitude range and energy variance are useful d parameters to use as a start point for the Startle Response detection algorithm.

Conclusions
It is concluded that it is feasible to detect startle response using the Laboras technology. The startle response leads to Laboras signals that are easy to differentiate from the other Laboras signals that are related to normal animal behaviors. Several potential signal features (parameters) were investigated that could be the base for a detection algorithm. Reliable freezing detection might be possible by making minor changes to the immobility algorithm in Laboras. For reliable freezing detection the environment should be quiet and free from building and equipment vibrations. Development activities have been initiated directly following this feasibility study. To do the full scale development Metris will need more and accurately annotated data from the customer. It is expected that the customer will provide further data around the 15th of January. Development is expected to be finished during the second half of February, assuming that proper data is received by January 15th.

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Annex A: Freezing Data

Animal 1700 (test session 01, day 1) LABORAS immobility detection (current software) Freezing Start End 01:56,0 02:23,4 04:19,1 04:32,3 04:46,2 04:50,7 04:55,3 05:17,3 05:25,6 05:48,3 05:50,8 05:55,0 05:57,6 01:57,5 02:24,8 04:27,0 04:34,3 04:49,5 04:54,0 04:58,5 05:23,5 05:29,0 05:49,8 05:52,5 05:56,3 05:59,8

Manual Observer Scores Mnster Freezing Start End 0:0:23.68 0:3:5.96 0:4:5.32 0:4:19.20 0:4:46.76 0:4:55.56 0:5:16.0 0:5:22.8 0:0:24.84 0:3:8.0 0:4:6.56 0:4:31.56 0:4:47.96 0:4:59.8 0:5:17.56 0:5:23.64

Homemade software Mnster Freezing Start End Event 00:00:14.000 00:00:14.040 00:00:14.280 00:00:14.320 00:00:14.360 00:00:14.520 00:00:14.560 00:00:14.760 00:00:14.800 00:00:14.960 00:00:15.000 00:00:15.160 00:00:15.200 00:00:15.360 00:00:15.400 00:00:15.560 ------ rest not 00:00:14.000 00:00:14.240 00:00:14.280 00:00:14.320 00:00:14.480 00:00:14.520 00:00:14.720 00:00:14.760 00:00:14.920 00:00:14.960 00:00:15.120 00:00:15.160 00:00:15.320 00:00:15.360 00:00:15.520 00:00:15.560 displayed ---moving immobility moving immobility moving immobility moving immobility moving immobility moving immobility moving immobility moving immobility

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Animal 1699 (test session 03, day 1) LABORAS immobility detection (current software) Freezing Start End 00:27,8 00:31,5 01:27,0 01:32,5 01:39,3 01:42,0 02:03,0 02:50,8 03:08,7 03:51,6 03:57,8 04:00,0 04:05,3 04:19,3 04:52,0 05:16,0 05:20,5 05:22,5 05:23,9 05:24,4 00:29,0 00:41,7 01:30,2 01:36,2 01:41,3 01:54,0 02:04,8 02:53,0 03:09,2 03:56,5 03:59,2 04:03,0 04:09,0 04:31,2 04:59,0 05:17,5 05:21,7 05:23,8 05:24,3 05:26,7

Manual Observer Scores Mnster Freezing Start End 0:3:30.68 0:3:35.40 0:3:37.64 0:4:16.8 0:4:18.68 0:4:32.36 0:4:46.60 0:4:50.80 0:4:54.92 0:5:11.40 0:5:46.40 0:5:48.40 0:5:50.84 0:5:55.8 0:5:57.48 0:6:40.36 0:6:44.36 0:3:34.0 0:3:37.44 0:3:39.68 0:4:17.48 0:4:27.52 0:4:34.60 0:4:50.8 0:4:54.16 0:4:58.92 0:5:12.60 0:5:48.28 0:5:50.24 0:5:52.44 0:5:56.56 0:5:59.12 0:6:42.16 0:6:45.80

Homemade software Mnster Freezing Start End Event 00:00:02.320 00:00:02.360 00:00:02.720 00:00:02.800 00:00:02.840 00:00:15.120 00:00:17.360 00:00:17.520 00:00:17.560 00:00:17.720 00:00:18.360 00:00:18.400 00:00:19.320 00:00:19.360 00:00:19.640 00:00:30.160 00:00:31.360 00:00:31.400 00:00:31.440 00:00:31.480 ------ rest not 00:00:02.320 00:00:02.680 00:00:02.760 00:00:02.800 00:00:15.080 00:00:17.320 00:00:17.480 00:00:17.520 00:00:17.680 00:00:18.320 00:00:18.360 00:00:19.280 00:00:19.320 00:00:19.600 00:00:30.120 00:00:31.320 00:00:31.360 00:00:31.400 00:00:31.440 00:00:31.480 displayed ---sniffing immobility sniffing moving immobility moving immobility sniffing moving immobility sniffing moving sniffing moving immobility moving immobility moving immobility sniffing

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Animal 1682 (test session 04, day 2)

Manual Observer Scores Mnster Freezing Start End 0:5:25.32 0:5:28.84 0:5:27.56 0:5:32.16

Homemade software Mnster Freezing Start End Event 00:00:00.040 00:00:16.040 00:00:16.080 00:00:16.520 00:00:16.560 00:00:16.720 00:00:16.880 00:00:16.920 00:00:16.960 00:00:17.040 00:00:17.080 00:00:17.920 00:00:18.080 00:00:18.120 00:00:18.160 00:00:18.400 ------ rest not 00:00:16.000 00:00:16.040 00:00:16.480 00:00:16.520 00:00:16.680 00:00:16.840 00:00:16.880 00:00:16.920 00:00:17.000 00:00:17.040 00:00:17.880 00:00:18.040 00:00:18.080 00:00:18.120 00:00:18.360 00:00:18.680 displayed ---immobility moving immobility moving immobility moving immobility moving immobility sniffing immobility moving immobility sniffing moving immobility

LABORAS immobility detection (current software) Freezing Start End 02:40,6 02:45,4 04:55,7 05:25,4 05:28,4 05:35,2 06:07,4 06:49,9 06:53,1 08:54,7 02:42,9 02:46,6 04:56,8 05:27,6 05:31,8 05:36,8 06:09,1 06:51,4 06:54,4 08:57,3

Animal 1664 (test session 05, day 2)

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Manual Observer Scores Mnster Freezing Start End 0:3:9.8 0:3:15.76 0:4:11.96 0:4:16.52 0:4:48.8 0:4:53.76 0:4:57.8 0:8:16.40 0:8:22.76 0:3:13.68 0:3:17.16 0:4:13.76 0:4:41.48 0:4:52.48 0:4:55.72 0:4:58.68 0:8:18.80 0:8:25.80

Homemade software Mnster Freezing Start End Event 00:00:00.040 00:00:06.600 00:00:06.680 00:00:06.760 00:00:07.040 00:00:07.120 00:00:08.440 00:00:08.480 00:00:09.000 00:00:09.120 00:00:10.080 00:00:10.120 00:00:10.520 00:00:10.680 00:00:10.800 00:00:10.840 ------ rest not 00:00:06.560 00:00:06.640 00:00:06.720 00:00:07.000 00:00:07.080 00:00:08.400 00:00:08.440 00:00:08.960 00:00:09.080 00:00:10.040 00:00:10.080 00:00:10.480 00:00:10.640 00:00:10.760 00:00:10.800 00:00:11.000 displayed ---immobility moving immobility moving immobility moving sniffing moving immobility moving sniffing moving immobility moving immobility moving

LABORAS immobility detection Freezing Start End 04:17,2 04:50,2 04:54,4 08:09,1 08:17,1 08:23,1 08:35,2 04:41,3 04:52,6 04:55,9 08:10,3 08:22,4 08:32,1 08:36,7

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Animal 1665 (test session 06, day 2) LABORAS immobility detection (current software) Freezing Start End 01:20,8 01:51,5 02:00,7 02:06,0 04:36,0 04:39,3 04:58,1 05:00,5 05:06,5 05:09,7 05:10,4 07:30,5 07:33,5 07:40,0 08:50,5 01:22,0 01:59,3 02:02,8 02:08,1 04:37,9 04:53,5 04:59,0 05:03,0 05:07,3 05:10,4 05:10,5 07:32,8 07:35,3 07:42,7 08:52,0

Manual Observer Scores Mnster Freezing Start End 0:4:35.96 0:4:39.4 0:4:51.0 0:7:30.36 0:7:33.48 0:7:39.76 0:7:56.48 0:8:29.24 0:4:38.36 0:4:50.88 0:4:53.68 0:7:33.12 0:7:35.56 0:7:42.76 0:7:57.64 0:8:30.44

Homemade software Mnster Freezing Start End Event 00:00:00.040 00:00:00.200 00:00:00.240 00:00:00.600 00:00:00.680 00:00:00.760 00:00:00.880 00:00:01.120 00:00:01.440 00:00:01.520 00:00:01.600 00:00:01.800 00:00:01.840 00:00:02.360 00:00:02.520 00:00:02.760 ------ rest not 00:00:00.160 00:00:00.200 00:00:00.560 00:00:00.640 00:00:00.720 00:00:00.840 00:00:01.080 00:00:01.400 00:00:01.480 00:00:01.560 00:00:01.760 00:00:01.800 00:00:02.320 00:00:02.480 00:00:02.720 00:00:03.160 displayed ---immobility moving immobility moving immobility sniffing immobility moving sniffing immobility moving sniffing moving immobility moving immobility

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Animal 1690 (test session 07, day 2) LABORAS immobility detection (current software) Freezing Start End 01:10,3 01:35,7 04:42,2 05:24,7 05:39,4 07:08,5 08:36,2 01:12,9 01:36,4 04:45,2 05:28,0 05:42,7 07:09,6 08:37,7

Manual Observer Scores Mnster Freezing Start End 0:1:10.28 0:4:25.48 0:4:29.52 0:4:40.52 0:5:24.68 0:5:53.40 0:1:13.36 0:4:26.72 0:4:31.12 0:4:45.20 0:5:28.68 0:5:54.60

Homemade software Mnster Freezing Start End Event 00:00:00.040 00:00:10.600 00:00:10.680 00:00:11.240 00:00:11.280 00:00:11.600 00:00:11.640 00:00:13.480 00:00:13.600 00:00:13.760 00:00:15.080 00:00:15.120 00:00:16.040 00:00:16.080 00:00:23.400 00:00:23.440 ------ rest not 00:00:10.560 00:00:10.640 00:00:11.200 00:00:11.240 00:00:11.560 00:00:11.600 00:00:13.440 00:00:13.560 00:00:13.720 00:00:15.040 00:00:15.080 00:00:16.000 00:00:16.040 00:00:23.360 00:00:23.400 00:00:23.480 displayed ---immobility moving immobility moving immobility moving immobility moving immobility moving immobility moving immobility moving sniffing moving

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Annex B: Startle Data

Etholog / Observation data; Experiment name is startle-test; session01; Date 16-9-2010; Laboras delay =35,26 sec Laboras data strtle session01 Start end 21480 21530 +_0,2 sec 24480 25530 +_0,2 sec 27480 30480 27530 +_0,2 sec 30530 +_0,2 sec Video data Session 01 start duration min 4 4 5 5 sec 10 40 10 40 end min 4 4 5 5 sec 11 41 11 41

Etholog/ Observation data; Experiment name is startle-test; session03; Date 16-9-2010; Laboras delay =23 sec Laboras data strtle session03 start end 19930 19980 22930 22980 25930 25980 28930 Video data Session 03 start end min sec min sec 3 42 3 4 12 4 4 42 4 5 12 5

Duration
+_0,2 sec +_0,2 sec +_0,2 sec

43 13 43 13

28980 +_0,2 sec

Analysis of Session 01

Figure1. 1CS - signal (2.5 kHz/85db/10 sec); session01

After the 1CS- signal, there are some changes but they are small and can be related to other behaviors. The video shows some sniffing with head movement, locomotion and immobility behavior around the 1CS- onset.

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Fig. 2a. 2CS (2.5Khz/85db/10 sec); session01

Fig. 2b 2CS- signal1 2,5Khz

During the 2CS- signal, there are some changes in the Laboras signal but they are small and related to some non-startle behaviors. Video analysis reveals that there are short chewing periods with vertical head movement in the same time period. These behaviors are normal behaviors.

Fig. 3 3CS- (2,5 kHz/10 sec); session01

Also the 3CS- signal, doesnt lead to obvious changes in the Laboras signals. The video recording shows normal behavior. Detailed analysis of the video indicates sniffing, locomotion and immobility behavior in this interval.

Fig. 4 4CS- (2,5 kHz/10 sec); session01 Technical Feasibility Study Freezing & Startle detection with Laboras Page 20

The 4CS- signal shows only minor movements in the Laboras signal. Video analysis shows that there is sniffing, freezing and locomotion behavior in this time period. Sometimes, there are also combinations of two behaviors (locomotion with sniffing).

Fig. 5a. 1CS+ (10kHz/10 sec); session01 Laboras signal in the 10sec time period

Fig. 5b 1CS+ (10kHz/10 sec); Startle Duration 0,2sec

After the 1CS+ the Laboras signal changes strongly in terms of high amplitude range, high energy in a very short time.

Fig.6a. 2CS+ (10kHz/10sec); session01 Laboras signal in the 10sec time period

Fig. 6b. 2CS+ (10kHz/10 sec); Startle Duration 0,2sec

Similar strong changes are observed in the Laboras signals after the 2CS+ signal.

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Fig. 7a. 3CS+ (10Khz/10sec); session01 Laboras signal in the 10sec time period

Fig. 7b. 3CS+ (10Khz/10sec); Startle Duration 0,2sec

In this case we see in the Laboras signal CS+ stimulus changing with very high amplitude range, high energy and short signal.

Fig. 8. 4CS+ (10kHz, 10sec); session01 Laboras signal in the 10sec time period

Fig. 8b. 4CS+ (10Khz; 10sec) Startle Duration 0,2sec

In this case, some changes can be observed in Laboras signal directly following the 4CS+ signal onset, but not a very high amplitude range and also not a high energy. This signal change is also very short. The amplitude pattern is not as obvious as the other three CS+ signals. In the video the startle is hardly visible either and is weak and combined with some other behavior.

Session03 Analysis:
In the test session 03, the CS- stimuli do not lead to strong significant changes in the Laboras signals or signals with high amplitudes or energy. From video analysis, it is concluded that the animal in the intervals around the stimuli onsets is performing sniffing, rearing, immobility/freezing and locomotion behavior. Sometimes, combinations of two behaviors can be observed (example: locomotion with sniffing, see figures 9, 10, 11, 12). Directly after the CS+ stimuli, strong changes in the Laboras signals are found. These changes occur with very high amplitudes, high energy and in a short time period (see figure 13a, 13b, 14a, 14b, 15a, 15b, 16a, 16b).

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Fig 9 1CS (2.5kHz/85dB/10 sec); Session 03 Laboras signal

Fig. 10. 2CS (2,5kHz/85dB/10 sec); session 03 Laboras signal

Fig 11 3CS (2.5kHz/85dB/10 sec); Session 03 Laboras signal

Fig. 12. 4CS (2,5kHz/85dB/10 sec); session 03 Laboras signal

Fig. 13a. 1CS + (10kHz/85db/10 sec); Session 03 Laboras signal

Fig. 13b. 1CS + (10kHz/85dB/10sec); Startle Duration 0,2sec

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Fig. 14a. 2CS + (10kHz/85db/10 sec); Session 03 Laboras signal

Fig. 14b. 2CS + (10kHz/85dB/10sec); Startle Duration 0,2sec

Fig. 15a. 3CS + (10kHz/85db/10 sec); Session 03 Laboras signal

Fig. 15b. 3CS + (10kHz/85dB/10sec); Startle Duration 0,2sec

Fig. 16a. 4CS + (10kHz/85db/10 sec); Session 03 Laboras signal

Fig. 16b. 4CS + (10kHz/85dB/10sec); Startle Duration 0,2sec

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Annex C: The Conditioning Protocol

On day 1: Animals were adapted through two presentations of six CS-(2.5 kHz tone, 85 dB, stimulus duration 10 s, interstimulus (pause) interval 20 s; inter-trial interval 6 hour). On day 2: Fear conditioning was performed through two exposures of three randomly presented CS+ (10 kHz tone, 85 dB, stimulus duration 10s,randomized inter-stimulus interval 20 s; inter-trial interval 6 hour), each of which was co terminated with a US (scrambled foot shock of 0.4 mA, duration 1 s). On day 3: Single animals were transferred to the retrieval environment (novel context) and habituated over a period of 30 min, before being exposed to six retrieval sessions (R1R6) for extinction training (intertribal interval 30 min), each consisting of a set of four CS-and (40 s later) a set of four CS+ (stimulus duration 10 s, inter-stimulus interval 20 s). On day 4: Recall of extinction was tested by exposing the animal to one set of 4 CS- and 40 s later to a set of 4 CS+ (stimulus duration 10 s, inter stimulus interval 20 s). Extinction recall was tested twice (E1, E2; interval 30 min). For renewal of extinct fear (RN), mice were returned to the initial shock context and received a set of 4 CS- and 40 s later a set of 4 CS+.

Figure a. Schematic representation of fear conditioning paradigm.

Adaptation (day1), fear conditioning training (day 2), retrieval, extinction (day 3; R1 to R6) and recall of extinction (day 4; E1, E2) of fear memory as well as renewal (day 4; RN) of extinct fear memory. CS-, neutral stimulus, CS+, conditioned stimulus.

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