Fear conditioning protocol & startle respond, freezing behavior, Laboras system

Technical Feasibility Study “Freezing & Startle detection with Laboras”

Customer:

University of Münster

Münster, Germany

Prepared by:

Metris b.v., The Netherlands


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


Introduction

The University of Münster 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.


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.


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 6: +30.5 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.


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.


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.


Detection of Startle using LABORAS

Currently, LABORAS doesn’t 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 animal’s 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 20sec 20sec Time

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

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.


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


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

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

Fig. 5b Amplitude Range and Energy variance analysis

1593

2748

1372

338

1CS+ 2CS+ 3CS+ 4CS+

Ener

gy V

aria

nce

Stimulus

Energy variance

24

37

23

11

1CS+ 2CS+ 3CS+ 4CS+

Ampl

itude

Ran

ge

Stimulus

Amplitude Range

Stimulus Time period Sound

Amplitude Range

Energy variance Energy duration(s) Remarks

1CS- 90-100 2,5 kHz - - - - 2CS- 120-130 2,5 kHz 1 37 1 0,2 3CS- 150-160 2,5 kHz - - - - 4CS- 180-190 2,5 kHz - - - - 1CS+ 210-220 10 kHz 24 1593 5 0,2 2CS+ 240-250 10 kHz 37 2748 4,9 0,2 3CS+ 270-280 10 kHz 23 1372 4,4 0,2 4CS+ 300-310 10 kHz 11 338 10 0,2 not clean


Test session 03

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

Fig. 5b Amplitude Range and Energy variance analysis

5360

1011 1149

2895

1CS+ 2CS+ 3CS+ 4CS+

Ener

gy V

aria

nce

Stimulus

Energy variance46

21 22

31

1CS+ 2CS+ 3CS+ 4CS+

Ampl

itude

Ran

ge

Stimulus

Amplitude Range

Stimulus Time period Sound

Amplitude Range

Energy variance Energy duration(s) Remarks

1CS- 70-80(sec) 2,5 kHz - - - - 2CS- 100-110 2,5 kHz - - - - 3CS- 130-140 2,5 kHz - - - - 4CS- 160-170 2,5 kHz - - - - 1CS+ 190-200 10 kHz 46 5360 6,1 0,2 2CS+ 220-230 10 kHz 21 1011 5 0,2 3CS+ 250-260 10 kHz 22 1149 6,1 0,2 4CS+ 280-290 10 kHz 31 2895 4,6 0,2


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 CS- stimuli. 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.


Annex A: Freezing Data

Animal 1700 (test session 01, day 1)

Manual Observer Scores Münster Homemade software Münster

LABORAS immobility detection (current software)

Freezing

Freezing

Freezing Start End

Start End Event

Start End

0:0:23.68 0:0:24.84

00:00:14.000 00:00:14.000 moving

01:56,0 01:57,5

0:3:5.96 0:3:8.0

00:00:14.040 00:00:14.240 immobility

02:23,4 02:24,8 0:4:5.32 0:4:6.56

00:00:14.280 00:00:14.280 moving

04:19,1 04:27,0

0:4:19.20 0:4:31.56

00:00:14.320 00:00:14.320 immobility

04:32,3 04:34,3 0:4:46.76 0:4:47.96

00:00:14.360 00:00:14.480 moving

04:46,2 04:49,5

0:4:55.56 0:4:59.8

00:00:14.520 00:00:14.520 immobility

04:50,7 04:54,0 0:5:16.0 0:5:17.56

00:00:14.560 00:00:14.720 moving

04:55,3 04:58,5

0:5:22.8 0:5:23.64

00:00:14.760 00:00:14.760 immobility

05:17,3 05:23,5

00:00:14.800 00:00:14.920 moving

05:25,6 05:29,0

00:00:14.960 00:00:14.960 immobility

05:48,3 05:49,8

00:00:15.000 00:00:15.120 moving

05:50,8 05:52,5

00:00:15.160 00:00:15.160 immobility

05:55,0 05:56,3

00:00:15.200 00:00:15.320 moving

05:57,6 05:59,8

00:00:15.360 00:00:15.360 immobility

00:00:15.400 00:00:15.520 moving

00:00:15.560 00:00:15.560 immobility ------ rest not displayed ----





Freezing

Freezing

Freezing Start End

Start End Event

Start End

0:3:30.68 0:3:34.0

00:00:02.320 00:00:02.320 sniffing

00:27,8 00:29,0

0:3:35.40 0:3:37.44

00:00:02.360 00:00:02.680 immobility

00:31,5 00:41,7 0:3:37.64 0:3:39.68

00:00:02.720 00:00:02.760 sniffing

01:27,0 01:30,2

0:4:16.8 0:4:17.48

00:00:02.800 00:00:02.800 moving

01:32,5 01:36,2 0:4:18.68 0:4:27.52

00:00:02.840 00:00:15.080 immobility

01:39,3 01:41,3

0:4:32.36 0:4:34.60

00:00:15.120 00:00:17.320 moving

01:42,0 01:54,0 0:4:46.60 0:4:50.8

00:00:17.360 00:00:17.480 immobility

02:03,0 02:04,8

0:4:50.80 0:4:54.16

00:00:17.520 00:00:17.520 sniffing

02:50,8 02:53,0 0:4:54.92 0:4:58.92

00:00:17.560 00:00:17.680 moving

03:08,7 03:09,2

0:5:11.40 0:5:12.60

00:00:17.720 00:00:18.320 immobility

03:51,6 03:56,5 0:5:46.40 0:5:48.28

00:00:18.360 00:00:18.360 sniffing

03:57,8 03:59,2

0:5:48.40 0:5:50.24

00:00:18.400 00:00:19.280 moving

04:00,0 04:03,0 0:5:50.84 0:5:52.44

00:00:19.320 00:00:19.320 sniffing

04:05,3 04:09,0

0:5:55.8 0:5:56.56

00:00:19.360 00:00:19.600 moving

04:19,3 04:31,2 0:5:57.48 0:5:59.12

00:00:19.640 00:00:30.120 immobility

04:52,0 04:59,0

0:6:40.36 0:6:42.16

00:00:30.160 00:00:31.320 moving

05:16,0 05:17,5 0:6:44.36 0:6:45.80

00:00:31.360 00:00:31.360 immobility

05:20,5 05:21,7

00:00:31.400 00:00:31.400 moving

05:22,5 05:23,8

00:00:31.440 00:00:31.440 immobility

05:23,9 05:24,3

00:00:31.480 00:00:31.480 sniffing

05:24,4 05:26,7 ------ rest not displayed ----





Freezing

Freezing

Freezing Start End

Start End Event

Start End

0:5:25.32 0:5:27.56

00:00:00.040 00:00:16.000 immobility

02:40,6 02:42,9

0:5:28.84 0:5:32.16

00:00:16.040 00:00:16.040 moving

02:45,4 02:46,6

00:00:16.080 00:00:16.480 immobility

04:55,7 04:56,8

00:00:16.520 00:00:16.520 moving

05:25,4 05:27,6

00:00:16.560 00:00:16.680 immobility

05:28,4 05:31,8

00:00:16.720 00:00:16.840 moving

05:35,2 05:36,8

00:00:16.880 00:00:16.880 immobility

06:07,4 06:09,1

00:00:16.920 00:00:16.920 moving

06:49,9 06:51,4

00:00:16.960 00:00:17.000 immobility

06:53,1 06:54,4

00:00:17.040 00:00:17.040 sniffing

08:54,7 08:57,3

00:00:17.080 00:00:17.880 immobility

00:00:17.920 00:00:18.040 moving

00:00:18.080 00:00:18.080 immobility

00:00:18.120 00:00:18.120 sniffing

00:00:18.160 00:00:18.360 moving





LABORAS immobility detection Freezing

Freezing

Freezing

Start End

Start End Event

Start End

0:3:9.8 0:3:13.68

00:00:00.040 00:00:06.560 immobility

04:17,2 04:41,3 0:3:15.76 0:3:17.16

00:00:06.600 00:00:06.640 moving

04:50,2 04:52,6

0:4:11.96 0:4:13.76

00:00:06.680 00:00:06.720 immobility

04:54,4 04:55,9 0:4:16.52 0:4:41.48

00:00:06.760 00:00:07.000 moving

08:09,1 08:10,3

0:4:48.8 0:4:52.48

00:00:07.040 00:00:07.080 immobility

08:17,1 08:22,4 0:4:53.76 0:4:55.72

00:00:07.120 00:00:08.400 moving

08:23,1 08:32,1

0:4:57.8 0:4:58.68

00:00:08.440 00:00:08.440 sniffing

08:35,2 08:36,7 0:8:16.40 0:8:18.80

00:00:08.480 00:00:08.960 moving

0:8:22.76 0:8:25.80

00:00:09.000 00:00:09.080 immobility

00:00:09.120 00:00:10.040 moving

00:00:10.080 00:00:10.080 sniffing

00:00:10.120 00:00:10.480 moving

00:00:10.520 00:00:10.640 immobility

00:00:10.680 00:00:10.760 moving

00:00:10.800 00:00:10.800 immobility

00:00:10.840 00:00:11.000 moving

------ rest not displayed ----





Freezing

Freezing

Freezing Start End

Start End Event

Start End

0:4:35.96 0:4:38.36

00:00:00.040 00:00:00.160 immobility

01:20,8 01:22,0

0:4:39.4 0:4:50.88

00:00:00.200 00:00:00.200 moving

01:51,5 01:59,3 0:4:51.0 0:4:53.68

00:00:00.240 00:00:00.560 immobility

02:00,7 02:02,8

0:7:30.36 0:7:33.12

00:00:00.600 00:00:00.640 moving

02:06,0 02:08,1 0:7:33.48 0:7:35.56

00:00:00.680 00:00:00.720 immobility

04:36,0 04:37,9

0:7:39.76 0:7:42.76

00:00:00.760 00:00:00.840 sniffing

04:39,3 04:53,5 0:7:56.48 0:7:57.64

00:00:00.880 00:00:01.080 immobility

04:58,1 04:59,0

0:8:29.24 0:8:30.44

00:00:01.120 00:00:01.400 moving

05:00,5 05:03,0

00:00:01.440 00:00:01.480 sniffing

05:06,5 05:07,3

00:00:01.520 00:00:01.560 immobility

05:09,7 05:10,4

00:00:01.600 00:00:01.760 moving

05:10,4 05:10,5

00:00:01.800 00:00:01.800 sniffing

07:30,5 07:32,8

00:00:01.840 00:00:02.320 moving

07:33,5 07:35,3

00:00:02.360 00:00:02.480 immobility

07:40,0 07:42,7

00:00:02.520 00:00:02.720 moving

08:50,5 08:52,0






Freezing

Freezing

Freezing Start End

Start End Event

Start End

0:1:10.28 0:1:13.36

00:00:00.040 00:00:10.560 immobility

01:10,3 01:12,9

0:4:25.48 0:4:26.72

00:00:10.600 00:00:10.640 moving

01:35,7 01:36,4 0:4:29.52 0:4:31.12

00:00:10.680 00:00:11.200 immobility

04:42,2 04:45,2

0:4:40.52 0:4:45.20

00:00:11.240 00:00:11.240 moving

05:24,7 05:28,0 0:5:24.68 0:5:28.68

00:00:11.280 00:00:11.560 immobility

05:39,4 05:42,7

0:5:53.40 0:5:54.60

00:00:11.600 00:00:11.600 moving

07:08,5 07:09,6

00:00:11.640 00:00:13.440 immobility

08:36,2 08:37,7

00:00:13.480 00:00:13.560 moving

00:00:13.600 00:00:13.720 immobility

00:00:13.760 00:00:15.040 moving

00:00:15.080 00:00:15.080 immobility

00:00:15.120 00:00:16.000 moving

00:00:16.040 00:00:16.040 immobility

00:00:16.080 00:00:23.360 moving

00:00:23.400 00:00:23.400 sniffing

00:00:23.440 00:00:23.480 moving ------ rest not displayed ----


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 Video data Session 01 session01 start end Start end duration min sec min sec

21480 21530 +_0,2 sec 4 10 4 11 24480 25530 +_0,2 sec 4 40 4 41 27480 27530 +_0,2 sec 5 10 5 11 30480 30530 +_0,2 sec 5 40 5 41

Etholog/ Observation data; Experiment name is startle-test; session03; Date 16-9-2010; Laboras delay =23 sec Laboras data strtle Video data Session 03 session03 start end start end Duration min sec min sec

19930 19980 +_0,2 sec 3 42 3 43 22930 22980 +_0,2 sec 4 12 4 13 25930 25980 +_0,2 sec 4 42 4 43 28930 28980 +_0,2 sec 5 12 5 13

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.


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, doesn’t 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


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 Fig. 5b 1CS+ (10kHz/10 sec); Laboras signal in the 10sec time period 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 Fig. 6b. 2CS+ (10kHz/10 sec); Laboras signal in the 10sec time period Startle Duration 0,2sec

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


Fig. 7a. 3CS+ (10Khz/10sec); session01 Fig. 7b. 3CS+ (10Khz/10sec); Laboras signal in the 10sec time period 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 Fig. 8b. 4CS+ (10Khz; 10sec) Laboras signal in the 10sec time period 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).


Fig 9 1CS– (2.5kHz/85dB/10 sec); Fig. 10. 2CS– (2,5kHz/85dB/10 sec); Session 03 Laboras signal session 03 Laboras signal

Fig 11 3CS– (2.5kHz/85dB/10 sec); Fig. 12. 4CS– (2,5kHz/85dB/10 sec); Session 03 Laboras signal session 03 Laboras signal

Fig. 13a. 1CS + (10kHz/85db/10 sec); Fig. 13b. 1CS + (10kHz/85dB/10sec); Session 03 Laboras signal Startle Duration 0,2sec


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, inter-stimulus (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 (R1–R6) 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.

Fear conditioning protocol & startle respond, freezing behavior, Laboras system

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