Fear conditioning (A1, A2 and A3)

(NS Intern experiment procedure Fear Conditioning, see appendix 3.35.)

Three fear conditioning experiments were executed with the three different drugs:

alprazolam (experiment A1), α5IA-II (experiment A2) and NS.A (experiment A3).

Alprazolam was tested with the use of 40 animals which randomly were classified into five groups: Vehicle (cremophor 5 %) no electric shock, vehicle (cremophor 5 %)

with electric shock, 0.4 mg/kg alprazolam with electric shock, 1.33 mg/kg alprazolam with electric shock, and 4 mg/kg alprazolam with electric shock. The terms no/with electric shock refers to the conditioning experiment at day 3 and these terms will be used through out the report. Prior to the conditioning experiment the rats were injected with either vehicle (cremophor 5 %) or 0.44 mg/kg, 1.33 mg/kg or 4 mg/kg of alprazolam according to the group they belonged to (see appendix 3.19.).

α5IA-II was tested with the use of 64 rats in a full factorial design with eight different groups: Vehicle (tween 5 %) with electric shock, vehicle (tween 5 %) no electric shock, 1 mg/kg α5IA-II with electric shock, 1 mg/kg α5IA-II no electric shock, 3 mg/kg α5IA-II with electric shock, 3 mg/kg α5IA-II no electric shock, 10 mg/kg α5IA-II with electric shock, and 10 mg/kg α5IA-II no electric shock. The rats were randomly classified into these eight different groups and injected prior to the conditioning experiment with either vehicle (tween 5 %) or 1 mg/kg, 3 mg/kg or 10 mg/kg of α5IA-II according to the group they belonged to (see appendix 3.20.).

NS.A was tested with the use of 40 animals which randomly were classified into five groups: Vehicle (tween 5 %) no electric shock, vehicle (tween 5 %) with electric shock, 0.3 mg/kg with electric shock, 1 mg/kg with electric shock, and 3 mg/kg with electric shock. Prior to the conditioning experiment the rats were injected with either vehicle (tween 5 %) or 0.3 mg/kg, 1 mg/kg or 3 mg/kg of NS.A according to the groups they belonged to. (see appendix 3.21.).

Equipment

8 TSE StartleResponse Box (TSE Technical & Scientific Equipment GmbH, Germany).

The 8 TSE startle boxes were separated in two systems named system 1 and system 2 8 rat cages (Aluminium, plexi glass, integrated floor grid) (270 X 100 X 125 mm) (TSE Technical & Scientific Equipment GmbH, Germany)

3.3.1. Day 1: Handling of animals and calibration

Program

Trial (system 1): Soren test, 110dB Trial (system 2): Soren test

The sampling interval was 2 ms

Procedure

The rats were handled in at least one minute in order to get familiar with the operators.

When the rats became calm they were weighed and marked, see experimental protocols (see appendix 3.19., 3.20. and 3.21.).

The transducers were calibrated to 150 (±2) Maximum g values with the use of an artificial rat and the programs: ‘Soren test, 110dB’ and ‘Soren test’ in system 1 and system 2, respectively. The programs included the same trials, but were named differentially in system 1 and system 2.

3.3.2. Day 2: Habituation

Program

Trial: FC Habituation 45dB KBT

Habituation: 180sec (with background noise, 45dB) Baseline: 30sec (with background noise, 45dB) The sampling interval was 4 ms

The rats were placed in the startle boxes and exposed to the background noise (white noise at 45 dB). See appendix 3.37. for details regarding the program

Procedure

The computer program was started and the startle boxes were tested to ensure that the noise stimulation was functional. Hereafter it was entered into the program which rat was allocated to which startle box. See appendix 3.35. for details about the upstart of the computer program.

The rats were placed in the rat cages and inserted into the startle boxes whereupon the habituation program was started. Eight animals were habituated simultaneously in system 1 and system 2.

Throughout the rest of the experiment the individual animal was placed in the same startle box in every experiment to ensure that the animals were presented for the exact same noise and tones during the different stages of the experiment.

3.3.3. Day 3: Conditioning experiment

Program

Trial: FC conditioning 2006-04

Habituation: 5sec (with background noise, 45dB) Baseline: 30sec (with background noise, 45dB) The sampling interval was 4 ms.

In the conditioning experiment, the rats were exposed to the background noise (45 dB) for 2 x 30 seconds, separated by an Inter-Trial-Interval at 60 seconds, where after they were presented to a tone at 5 kHz and 80 dB for a period of 10 seconds. In the last second of the period the rat simultaneously received an electric foot shock at 0.6 mA through the steel grid, which makes up the floor in the startle cages. This was followed by a period of 180 seconds with background noise. See appendix 3.37. for details regarding the program

Procedure

The rats were prior to the conditioning experiment injected in agreement with the dose groups they belonged to.

The computer program was started and the startle boxes were tested in regard to ensure that the noise stimulation and the electric stimulation were functional. Hereafter it was entered into the program which rat was put in which startle box. See appendix 3.35. for details about the upstart of the computer program.

The rats were 30 minutes prior to the conditioning test injected i.p.

Four animals were injected simultaneously. After the injections the rats were placed in their cages again and maintained there for approximately 30 minutes before they were placed in the rat cages and inserted into the startle boxes. The rat cages were connected to electricity through sockets placed inside the startle boxes, if the tested rat belonged to a ‘with electric shock’ group. Hereafter, the conditioning program was started. Four animals were tested at the same time, alternately in system 1 and system 2.

The same drug dose was not tested in the same box as the experiment was repeated; see experimental protocols (appendix 3.19., 3.20. and 3.21.)

3.3.4. Day 4: Testing the animals

Program

Trial: FC Test 2005-12

Habituation: 5sec (with background noise, 45dB) Baseline: 30sec (with background noise, 45dB) The sampling interval was 4 ms.

The test experiment was carried out after approximately 24 hours. The rats were presented to the same tone as they were at day 3, but without the additional foot shock.

See appendix 3.37. for details regarding the program

Procedure

The computer program was started and the startle boxes were tested in regard to ensure that the noise stimulation was functional. Hereafter it was entered into the program which rat was assigned to which startle box. See appendix 3.35. for details about the upstart of the computer program.

The rats were placed in the rat cages and inserted into the startle boxes whereupon the test program was started. Four animals were tested at the same time, alternately in system 1 and system 2.

3.3.5. Day 10: Re-testing the animals

Program

Trial: FC Test 2005-12

Habituation: 5sec (with background noise, 45dB) Baseline: 30sec (with background noise, 45dB) The sampling interval was 4 ms.

The experiment was a repetition of the day 4 test experiment. At the end of the experiment the rats were sacrificed by suffocation with CO2. The fear conditioning experiments were executed by Merete Hansen and Line Sørensen.