TSE Multi Conditioning System - Animalabanimalab.eu/.../produkty-dopobrania/tse_multi_conditioning_system.… · 2 ... The TSE Multi Conditioning System ... certain restrictions due

Sophisticated Life Science Research Instrumentation

TSE Multi Conditioning SystemFor mice, rats & other small laboratory animals

TSE MCS Short Description

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Contents

1. Hardware .................................................................................................. 3 1.1. Housing...................................................................................................... 3 1.2. Light barrier frames.................................................................................... 5 1.3. Shock grids ................................................................................................ 6 1.3.1. Mouse ........................................................................................................ 6 1.3.2. Rat ............................................................................................................. 7 1.4. Control unit ................................................................................................ 7 1.4.1. Audio elements .......................................................................................... 7 1.4.2. Light ........................................................................................................... 8 1.4.3. Foot shock ................................................................................................. 8 1.4.4. Additional features ..................................................................................... 8 1.5. Arenas ....................................................................................................... 9 1.6. Optional Components ................................................................................ 9 1.6.1. Video kit for camera observation and synchronized DVD video recording. 9

2. Software.................................................................................................. 12 2.1. Software Package “Fear Conditioning Extended Advanced” ................... 13 2.1.1. Arenas ..................................................................................................... 13 2.1.2. The Fear Conditioning paradigm in the FCS software package............... 20 2.2. Software Package “2-Compartment Paradigms”...................................... 22 2.2.1. “Two-Way Active Avoidance” ................................................................... 22 2.2.1.1. Arenas ..................................................................................................... 23 2.2.1.2. Two-Way Active Avoidance paradigm in the 2-Compartment software ... 24 2.2.2. “Learned Helplessness” ........................................................................... 26 2.2.2.1. Arenas ..................................................................................................... 26 2.2.2.2. Learned Helplessness paradigm in the 2-Compartment software............ 27 2.2.3. “Latent Inhibition” ..................................................................................... 29 2.2.3.1. Arenas ..................................................................................................... 29 2.2.3.2. Latent Inhibition paradigm in the 2-Compartment software...................... 29 2.2.4. “Passive Avoidance” ................................................................................ 30 2.2.4.1. Arenas ..................................................................................................... 30 2.2.4.2. Passive Avoidance paradigm in the 2-Compartment software................. 32 2.2.5. “Light-Dark Exploration” ........................................................................... 33 2.2.5.1. Arenas ..................................................................................................... 34 2.2.5.2. Light Dark Exploration paradigm in the 2-Compartment software............ 34 2.2.6. “Place-Preference”................................................................................... 35 2.2.6.1. Arenas ..................................................................................................... 36 2.2.6.2. Place Preference paradigm in the 2-Compartment software ................... 37 2.3. Software Package “Locomotor Activity & Open Field – ActiMot”.............. 39 2.3.1. Arenas ..................................................................................................... 39 2.3.1.1. Open Field/Activity paradigm in the ActiMot software package............... 40

3. Future extensions .................................................................................. 42



TSE Multi Conditioning System LARGE Short Description of the Main Features

The TSE Multi Conditioning System (MCS) LARGE is suitable for rats and mice. The following text describes the LARGE system only and all components that are used for both species. The MCS system is also available in a smaller version that is only available for mice and that has certain restrictions due to its smaller size concerning the design and variety of the arenas. If you are interested in more information please contact us. 1. Hardware

1.1. Housing



! The experiments are run in a housing that is an integral part of the complete setup. ! The housing is made from expanded PVC with increased sound-attenuating properties. ! Size: approx. 850 x 800 x 900 mm (WxDxH). ! Features fan in side wall, pull-out floor, special cable lead-throughs ! 2-Wing door with 3-dimensional peepholes in each door half. The peep holes can be rotated to

any desired angle so that the animal can be observed independent from its position inside the arena. Peepholes can also be closed to prevent light to shine inside the housing when peepholes are not in use.

Options ! In order to further increase attenuation in the low frequency range – this is especially useful

when experiments are started and stopped in different boxes at different times – special sound-absorbing protective mats are available that can be easily fixed inside the housing. The material has a smooth surface that can be cleaned with isopropanole. When these mats are used the attenuation between boxes is almost complete.

! For very high shock amplitudes (4.5mA) a contact protection device is provided for the housing doors. If the doors are opened shock delivered to the floor grid is deactivated then. For amplitudes below 3.1mA this device is optional

! Centrally positioned loudspeaker for background noise, stimulus sound, stimulus noise. The

loudspeaker is also suitable for carrying out experiments on ultrasound-induced defensive behavior of rats (using 22 kHz frequency)

! The housing roof is equipped with both house lights and stimulus lights. ! The 4 house lights are arranged symmetrically. They are used for continuous background

illumination. The 2-lamp groups at the left and right can be switched on manually via the control unit (left pair only, right pair only, both pairs). The brightness is set manually via the potentiometer on the control unit and applies to all measuring stations – maximum brightness at arena floor approx. 100 lux. The house lights are switched on and off manually via a switch on the housing wall (not software-controlled). These lamps are white; separate red transparent covers permit their use as red house lights.

! The 2 stimulus lamps are arranged symmetrically – they are located to the right and left of the

central loudspeaker. They are used to provide a stimulus. When using the FCS and Shuttle-Box software package the lamps can be switched on independent from each other. In the



ActiMot software package they are always controlled simultaneously. The brightness is set manually via the potentiometer on the control unit and applies to all measuring stations – maximum brightness at arena floor approx. 700 lux. These lamps are white; separate blue transparent covers permit their use as blue stimulus lights. The LEDs are switched on/off via the software; the application of pulsating light is possible to increase effectiveness (with user-defined frequency; depends on paradigm used).

2-Place system

1.2. Light barrier frames

! Square-shaped LARGE – suitable for rats and mice, inner dimensions approx. 500x500mm. ! With stainless steel excrement collection tray, with black coating to avoid reflections what is

especially important for camera observation. ! Special support device for removable foot shock grid. The special design of this support device

allows the shock grid to be used in 2 positions (rotation through 90°) in order to modify the context – a feature exclusively available in the MCS system. The grid is removed from the top.

! Infrared sensors for animal location detection in 3 dimensions: XY-level for horizontal, Z-level for vertical movement (=rearing). 31 sensor pairs in each of the X, Y and Z-axes, each with 14mm distance between sensors (high resolution: 7mm distance between coordinates). XY and Z-levels with stepless height adjustment (Z up to 300mm). Min. distance between light barrier and shock grid is approx. 15mm; this means that all animal movements are reliably recorded.

! Light-beams are evaluated with a maximum sample rate of 100Hz, i.e. a sample is taken every 10ms. This allows ultra-precise movement detection.



Mouse grid

Grid lengthways Grid crosswise ! The Z-frame is made so that it is open to the rear (U-shape); this ensures that the camera has

a free view of the animal from behind 1.3. Shock grids

! Shock grids for mouse and rat are available. ! All shock grids consist internally of 2 independent halves so that shock can be given

independently on both sides in the Shuttle-Box software package (Active Avoidance and Passive Avoidance paradigm); however, the animal cannot recognize the subdivision – the distance between the grid rods is identical over the whole grid, even in the transition area between the 2 halves! If the FCS software is used or if the Learned Helplessness Module of the Shuttle-Box software package is used then both halves of the shock grid will always be switched on at the same time, i.e. a foot shock will be applied throughout the whole arena area.

! PCBs are sealed to effectively prevent damage by urine. ! Shock grids are square, fill the whole light barrier frame. This means that it is possible to use

arenas up to the maximum size of the frame. If a smaller arena is used the grid area accessible from below is defined by the built-in template of the arena.

1.3.1. Mouse

! Stainless steel rods, 4mm rod diameter and 10mm distance between rods (center to center).



Mouse grid Rat Grid 1.3.2. Rat

! Stainless steel rods, 6mm rod diameter and 18mm distance between rods (center to center). 1.4. Control unit

The control unit is available as 1-place, 2-place or 4-place control unit

1.4.1. Audio elements

! 1 manual noise channel per system with built-in amplifier for loudness up to 100dB. This channel is used for background noise. The noise is switched on and off manually on the control unit. Manual amplitude setting on control unit via digital potentiometer for easy reproducibility.

! Noise generator (white noise) for stimulus noise, i.e. application of noise as CS. 1 channel for each measuring station. With built-in amplifier for loudness up to 90dB. Amplitude setting via the software. Noise switch-on/switch off controlled by the software.

! Sound generator (1 channel for each measuring station) for producing sine sound as a stimulus (CS) (frequency: sine 2-22kHz, adjustable by the software in 200Hz steps), (optional: 2-12.5kHz in steps of 100Hz) with built-in amplifier for loudness up to 90dB. Amplitude setting via the software. Sound switch-on/switch off controlled by the software.

! In the FCS software package setting of the frequency (sine) and amplitude (sine, noise) is made for all 4 measuring stations in parallel, i.e. identical stimulus qualities are applied in all 4 measuring stations at the same time. This allows to alter the quality of the stimulus during an experiment (for CS+/CS- studies). In the Shuttle-Box software package there is one common



settings for all boxes; the quality can not be changed in the experiment. In the ActiMot software package only the manually controlle noise is used (no sine sound)

Please note that the maximum intensity for noise and sound that can be achieved at the floor of the arena depends on whether the sound-protecting mats are used or not and varies depending on the arena used. 1.4.2. Light

! Control of housing illumination ("house light"), approx. 150 lux max. in center of arena. Potentiometer on front panel for manually presetting the light intensity. The lamps on the left and right sides of the housing (both channels) can be switched in manually independent of one another.

! Control of stimulus LEDs (approx. 700 lux in center of arena), for application of light as CS in FCS, LI and AA trials. Potentiometer on front panel for manually presetting the light intensity. The lamps on the left and right sides of the housing (both channels) can be switched in independent of one another when the FCS software package is used (software-control). In the Passive Avoidance and Light-Dark paradigm and in the ActiMot software package the light can be used to increase aversiveness of the test area.

1.4.3. Foot shock

! Shocker module, 2 channels per measuring station. The shocker module allows the application of foot shocks with a max. amplitude of either 1.55mA (in steps of 0.05mA), 3.1mA (in 0.1mA steps) or 4.65mA (in steps of 0.15mA). Please define the required range with your order.

! With integrated current flow recognition and switching electronics for scrambled shock; suitable for very short shock times. The selected current strength is achieved independent of the transition resistance (i.e. the voltage is changed to achieve a preset current strength in Amp).

! Constant current or pulsating current (the frequency can be set in the software). ! Galvanic separation of the 2 shocker channels per shock grid; this means that both shock grid

halves can be operated at the same time (FCS software package; LHN or LI paradigm in the Shuttle-Box software package) or independent from each other in Active Avoidance or Passive Avoidance trials.

1.4.4. Additional features

! With 1xTTL output (BNC socket) per measuring station for controlling external systems, e.g. TSE VideoKit or customer-specific data recording systems. Provides TTL signal at start (high) and end (low) of the trial.

! Including PCI interface for building into PC



! Observation LED Unit: This small signal unit can be connected to the MCS housing. If the FCS software is used (use only possible with this software!) the software switches on the built-in LED at user-defined intervals for user-defined durations. It supports the user in the manual recording of freezing events, by showing the time at which the status of the animal (freezing or not) can be assessed by the observer and entered in the system via the keyboard. These manually recorded events are shown in the results table.

! The Shock Status LED Unit permits observation of the current shock status of the shock grid.

It is connected to the housing roof. Each half of the shock grid is shown separately 1.5. Arenas

The multi conditioning hardware permits operation with various specialized software packages. - Fear Conditioning Extended Advanced (called “FCS” software package in the text) - Software package “2-compartment paradigms” (called “Shuttle-Box” software package in the text) - ActiMot software package for OpenField and Activity analysis Some paradigms – such as Place Preference run with the Shuttle-Box software package – require their own set of arenas. Some of the arenas can be used for several paradigms or can even be shared between software package (all FCS arenas can be used in the ActiMot software package and vice versa). Please read the chapters below for details. 1.6. Optional Components

1.6.1. Video kit for camera observation and synchronized DVD video recording

! 1 kit per measuring station is required - each measuring station has its own camera and own DVD recorder

! A mini CCD camera is used that can be mounted either centrally above the arena or at the rear wall of the housing depending on the image that is preferred by the user

! All arenas are suitable for camera observation; black acrylic arenas have a transparent rear panel.

! The light-sensitive CCD camera is capable of recording an image in darkness or in red light. An additional IR illumination is provided for this purpose. The IR lamps do not interfere with the functioning of the light barriers

! Each measuring station is provided with a DVD recorder for recording the trial on a hard disk or DVD. The recorder is modified to that its recording function can be triggered by the TSE MCS system. At the start of the experiment the DVD recorder is automatically switched to the



recording mode by the MCS control unit; recording is automatically stopped at the end of the experiment.

Camera mounted above the arena Camera mounted behind the arena

Fear Conditioning Mouse arenas - View from behind

! If more than 1 measuring place is provided the recorders are connected to a so-called quad-

splitter, which shows up to 4 images on one monitor. Each image can be enlarged to full screen as required. This does not affect recording the whole image on DVD.

! Offline video evaluation: Trial videos can be evaluated offline at any time. This function is reserved for the Fear Conditioning software package. This is done by starting the FCS software in the Demo mode and starting a pseudo-experiment. During this “experiment” manual event keys are available, with which freezing can be documented manually by the evaluator (1 key per measuring station; can be used in parallel for all 4 measuring stations) and/or other types of behavior can be recorded (only measuring station 1; 10 event keys). Manually recorded freezing events appear in the results table, the other behavior markers in a separate table. The manual markers of the offline evaluation are available in special export files for further statistical evaluation.



Setup shown with DVD recorders (1 recorder for each housing), a multiplexer and a separate

monitor to display up to 4 camera images in parallel

IR Lamp



2. Software

The MCS system can be controlled using 3 different software packages. All 3 packages can be provided separately. An existing system can be retrofitted with any package any time later.



- Fear Conditioning Extended Advanced (called “FCS” software package in the text) - Software package “2-compartment paradigms” (called “Shuttle-Box” software package in the text) - ActiMot software package for OpenField and Activity analysis All packages are suited for operating systems from Windows XP. Windows 7 will follow soon. Only some of the software features are described below as examples. Please also refer to the brochures available separately for the FCS and ActiMot software for details. 2.1. Software Package “Fear Conditioning Extended Advanced”

2.1.1. Arenas

Example: Round mouse arena placed inside the frame with foot shock grid

(shown with optional sound-protection mats) ! As a minimum 1 conditioning arena and 1 arena for testing the animal in a new context

are provided. Please select the arenas that you need from the list below. Additional arenas can be added at a later date any time.

! The arenas do not have a fixed floor so that they can be used together with the floor grid, with the grid cover plate or with the litter/sand tray.

! The black arena that is normally used for training is provided with a lid to prevent the animal from jumping out

! The arenas have a built-in PVC positioning template for placing the arena centrally; this prevents it from lying on the grid. The color of this template is black. Other colors can be provided on request.

! Perfect functioning of the infrared light barriers - and therefore automatic detection of freezing - is guaranteed for all arenas, irrespective of their shape, size, material or floor properties.

! A video recording of the experiment for later manual evaluation is possible for all types of arena and floor versions.

The following arenas are available to date. If you need arena with a different size/height/shape please let us know.



Please note that you can also use arenas with rat size for mice, if you want to offer the mice a very large exploratory area. Although originally labeled as “rat arenas” they can be used for mice as well. Shape Picture Comment 1. SQUARE MOUSE Square, black 200x200mm Height 400mm

! The IR arena rear side is the

clear wall that allows for camera observation. The rear wall can also be provided in black on request.

! The arena can be closed with a lid if desired in order to prevent the animal from jumping out during the foot shock (included)

! Please note that this arena has a reduced exploratory area in order to facilitate the freezing response. Larger arenas such as 300x300mm area are also available.

MOUSE Square, black 300x300mm Height 400mm

! The rear side can be clear or

black ! The arena can be closed with

a lid if desired in order to prevent the animal from jumping out during the foot shock (included)

Size Comparison



MOUSE Square, clear 200x200mm Height 400mm

! Provided without lid. The lid of

the black arena can be used.

RAT Square, black 460x460mm Height 400mm

! The IR arena rear side is the

clear wall that allows for camera observation

! The arena can be closed with a clear lid if desired in order to prevent the animal from jumping out during the foot shock (included)

RAT Square, clear 460x460mm Height 400mm

! Provided without lid. The lid of the black arena can be used (shown in this picture)



2. ROUND ! Material clear acrylic

! The arena is supplied without lid. If necessary, an optional lid is available

MOUSE Round, clear 200mm diam. Height 400mm

RAT Round, clear 400 mm diam. Height 400mm

3. TRIANGLE ! Material clear acrylic ! The triangle can be used in

both orientations, i.e. with the flat side pointing to the front or to the rear (the latter is better for camera observation)

! The arena is supplied without lid. If necessary, an optional lid is available (not included in the quote).



MOUSE Triangle Height 400mm

RAT Triangle Height 400mm

Picture N/A

! One pair of grid cover plates is supplied per light-beam frame: light gray and black. The plates

can be placed on the grid so that the animal no longer moves on the grid itself (context variation).

Grid cover plates used to modify the context



Selection of FCS arenas

4. Homecage

! Home cages can also be used as a test arena. Home cages are to be provided by the customer.

! A template is provided that allows to center the home-cage inside the frame.

! The cage is to be used in horizontal position only

MOUSE Template for Homecages



RAT Template for Homecages

5. Use of Sand/Litter

Sand & Litter Tray

RAT: Tray

RAT: Tray in Frame

MOUSE: Template / Template in Tray

MOUSE: Tray plus Template in Frame

! These trays for litter/sand are made from PVC and stainless steel.

! They consist of a lower part, the tray proper, and the upper part, the template

! The tray is positioned in the shock grid mounting. If the trays are used then the shock grid is removed.

! The tray is suitable for rats & mice

! After the arena has been placed on the tray the arena is filled with litter/sand.

! The tray is designed for a filling level of approx. 1cm litter/sand. The height of the tray base has been determined so that after filling the top edge of the filling material corresponds approximately to the top edge of the shock grid, so that the height of the light barriers does not have to be adjusted between trials

RAT ! Only the lower part is used MOUSE ! Both tray and template are

used



2.1.2. The Fear Conditioning paradigm in the FCS software package

! All connected boxes are always controlled simultaneously with this software so that there is no interference between boxes

! All features provided in the standard TSE Fear Conditioning software package described in the dedicated FCS brochure (available separately) are provided. Please refer to the FCS brochure for details.

! The control file editor allows to define trace and delay fear conditioning experiments for fear learning, retention and extinction trials. The sequence of stimuli is defined by the user in control files that can be stored for future use.

! Timing of stimuli can be done in steps of 50ms allowing very short stimulus durations ! Data acquisition can be started at a defined time set by the user. The measuring stations are

prepared and loaded with animals. The experiment is then started without any user intervention required. This function is primarily intended for use with home cages as “new context” arenas.

Control file editor

! The following add-on is available exclusively in the MCS FCS software package: Both stimulus

lamps can be used independent from each other. It is therefore possible to use white stimulus light and blue stimulus light alternatively in the same experiment

! All analysis functions of the FCS software are available. This includes:



New features in the MCS system are: ! A freezing list that shows all freezing episodes in chronological order ! An interval table allowing to evaluate all parameters in user-defined intervals

! A special export file has been implemented for freezing analysis. The user defines an interval

(1 second or 10 seconds, etc.) – each phase is then divided into these intervals. In the file the total freezing duration per interval is given in milliseconds. Freezings that overlap interval limits are also determined. A check is made whether the freezing as a whole exceeds the minimum duration. If this is the case then the durations are separated and assigned to the particular intervals

! In all tables that output freezing those freezing episodes are defined by the user entering a minimum duration. The lower limit for this duration is 100ms. The maximum pause (also defined by the user in ms) also applies for summarizing briefly interrupted freezing events

! All analyses can be exported as Excel-compatible files.



A variety of parameters are available to adjust the analysis to the special requirements of the user 2.2. Software Package “2-Compartment Paradigms”

- TWO-WAY ACTIVE AVOIDANCE - LEARNED HELPLESSNESS - LATENT INHIBITION - PASSIVE AVOIDANCE - LIGHT-DARK-TEST - PLACE PREFERENCE ! All the paradigms of this group are carried out with the additional “Shuttle Box” software

package, for which special trial modules are available. The software package allows actions within the box (light, sound, noise as well as shock applications) to be controlled according to preset user instructions depending on the animal’s behavior (sojourn to left or right of light barrier frame).

! Both halves of the shock grid can be addressed independently of one another, so that simultaneous or alternative shock application on both sides is possible. This function is an essential precondition for carrying out most of the test procedures described below.

! The trials in the 4 measuring stations can be started and stopped independently of one another.

! It is also possible to carry out different trials in different boxes (precondition: identical stimulus qualities).

2.2.1. “Two-Way Active Avoidance”

In this paradigm the animal is to learn in several runs on successive days that a not-aversive stimulus (CS) is followed by an aversive stimulus (US) and that it is possible to avoid this aversive stimulus by moving to the other compartment as soon as the CS occurs (associative learning). The number of correct avoidance reactions), i.e. transfer to the "safe" compartment after the



occurrence of the signal stimulus and before the aversive stimulus is a measure of the learning success. 2.2.1.1. Arenas

! Special 2-compartment arenas which are made from transparent and black acrylic. The arenas have no floors and are always used with the shock grid.

! The arenas are divided into two equally sized sections by a central separating black wall. The separating wall has a central opening in species-specific size.

! The arena is provided with a built-in holder for positioning the arena centrally. The distance between shock grid and arena has been minimized in order to able to reliably record all the animal’s movements.

! The shock grid can be used in 2 positions (rotation through 90°). Shape Picture Comment ! The arena has 2 clear and 2

black walls. The clear walls allow for camera observation or observation through the peep hole of the housing

! A central groove allows the use of a divider with a gate (this divider has to be used, operation without divider is not possible)

! The arena can be closed with a lid if desired in order to prevent the animal from jumping out during the foot shock (included)

MOUSE Square 300x300mm Height 250mm



RAT Square 450x450 mm Height 400 mm

Optional: ! Separate LED lid modules for selective compartment illumination. It could be necessary to

shine light selectively into the compartment. A special LED lid module is available as an accessory that is placed on the arena. If this LED module is used then the stimulus LEDs in the housing roof are inactive. The LED module can be switched on and off via the software so that the animal has to jump towards the light or away from it in Active Avoidance trials The housing is already prepared for the connection of this module so that an existing system can be retrofitted with this option any time later.

2.2.1.2. Two-Way Active Avoidance paradigm in the 2-Compartment software

! Both light and sound or noise can be used as a conditioning stimulus (CS). Light and an audio

signal can also be applied at the same time. The light can also be applied as a pulsating light at a frequency set by the user; available colors are white or blue.

! The setting of the frequency (sine sound) and loudness (sine sound, noise) is made via the software. The setting applies to all 4 measuring stations.

! Foot shock is available as the US. It can also be applied in pulsating form at a frequency set by the user. Slower pulsations can increase the effectiveness of the shock.

! The AA paradigm allows the definition of complex user-defined program sequences. New features only available in the MCS system are:

• The animal has to show more than one transfers during the CS in order to escape the shock • The animal has to endure a minimum shock time in order to escape the shock • The CS can be given in a randomized order instead of constant sine sound or regularly

pulsating sound (“unpredictable sound”).



Trial Parameters

Selecting data records for analysis

! Various tables are available for the analysis of the learning behavior of the animal; these include

a run table and a special results table, from which the learning success can be read off directly (number of avoidance reactions).



Run table Results table ! Group tables permit a direct comparison between the animals.

Group table ! Number of rearing events and number of jumps permit the detailed observation of the

exploratory behavior of the animals as well as an assessment of their individual shock response. The output of the number of freezing events and their duration within the framework of this experiment is a feature that has become possible for the first time by having high-resolution light barriers at 3 levels and allows a completely new quality in the assessment of learning behavior.

! In the results tables the distance travelled of the animal during the different experimental phases is given as well

2.2.2. “Learned Helplessness”

Learned helplessness is an established paradigm for carrying out tests on animal models for research into depression (“behavioral despair”). If the animal is repeatedly given a shock that it cannot avoid then it alters its ability to demonstrate normal learning behavior in subsequent learning trials. 2.2.2.1. Arenas

! For this test the arenas used in the standard active avoidance test are used, as the

animals then have identical surroundings to those in the shock pre-exposure session (context) to those in the following learning test.



2.2.2.2. Learned Helplessness paradigm in the 2-Compartment software

! LHN tests are carried out in 2 steps. 1. In the first the animal is trained in the test module “shock pre-exposure”, in which the animal

is given a shock that it cannot avoid. 2. In the second step the altered learning behavior of the animal that this causes is tested in the

“active avoidance” test module – where shock avoidance is possible by moving to the other compartment. During pre-exposure no CS (= stimulus light, sound or noise) is used. Foot shock is available as the US. It can also be applied in pulsating form at a frequency set by the user. Slower pulsations can increase the effectiveness of the shock.

! UNIQUE FEATURE: It is possible to read in test sequences of active avoidance experiments

(US-only trials) as a template (so-called “yoked” experiments), in order to copy the foot shock pattern between animals 1:1. This is a completely new test approach, and is used to observe more closely the influence of the controllability of the test events on the behavior of the animals (the total shock length is the same – the only difference is the controllability vs. the non-controllability of the situation)

Trial parameters LHN pre-exposure

Left: Standard Right: Yoked



Run Table

Results Table ! In addition to run tables for showing the transfer activity of the animal, a results table is also

generated, which also provides information about the animal’s exploratory behavior (rearings), shock response (jumps) and freezing behavior.

! All analytical functions of the “active avoidance” test module are available for the analysis of learning behavior.



2.2.3. “Latent Inhibition”

Latent inhibition (LI) describes a lessening in the associative learning when a stimulus is applied several times in a preliminary test (pre-exposure) without any consequences whatsoever, before it is paired with a conditioned stimulus (CS) and an unconditioned stimulus (US) in a learning phase (acquisition). 2.2.3.1. Arenas

! For this test the arenas used in the standard active avoidance test are used, as the

animals then have identical surroundings to those in the CS pre-exposure session (context) to those in the following learning test.

2.2.3.2. Latent Inhibition paradigm in the 2-Compartment software

Trial Parameters ! LI tests are carried out in 2 steps. 1. In the first step the animal undergoes training in the “Latent Inhibition - Pre-Exposure” test

module. No shock is given here. The animal can be either pre-exposed to the context or to a stimulus. If sine sound is used frequency and loudness of the sine sound are set via the software. The setting applies for all 4 measuring stations

2. In the second step the test is carried out in the “active avoidance” test module to determine whether the associative learning of the animal has been altered by the pre-exposure. All analytical functions of the “active avoidance” test module are available for the analysis of learning behavior.



Example: ! Habituation time ! Followed by switching between sine sound and pause. The duration of the sound and pause

can be preset by the user (random durations are also possible). ! End after the number of trials defined by the user. ! Delay period after the end of the last sound. ! The module can also be operated in a “no pre-exposure” mode. In this case no sound is

applied. The “no pre-exposure” animals are placed in the arena for a time that is identical with that of the pre-exposed animals.

! Data evaluation produces tables with numerical parameters for the exploratory behavior of the

animals and number of compartment transfers during pre-exposure, extended by the presentation of the run pattern.

2.2.4. “Passive Avoidance”

The Passive Avoidance paradigm is an established paradigm for studying associative learning. The animal learns to not carry out a particular behavior (to avoid it) – in this case to transfer to the dark compartment of a 2-part test arena. This is done by applying a punishing electrical stimulus (unconditioned stimulus = US) the first time that the compartment is entered. Successful associative learning is demonstrated on renewed confrontation with the test environment by a missing or delayed transfer to the dark compartment (latency >0). 2.2.4.1. Arenas

! Special passive avoidance arenas are used, these consist of 2 compartments. These arenas

are divided 50:50 into light and dark – one half of the arena is made from clear acrylic, the other from black acrylic. The arenas have no floors and are always used together with the shock grid.

! Mouse arenas are available in extra-large size that allow more explorative behavior ! An automatic door (black acrylic) is used as the separator and is inserted between the 2

halves. The door can be opened and closed by the software. ! The distance between shock grid and arena has been minimized in order to able to reliably

record all the animal’s movements. ! The bright side of the arena can be closed with a lid if desired in order to prevent the animal

from jumping out ! The dark compartment is closed with a dark lid and is characterized by its very low residual

light.



Picture MOUSE Rectangular 450x300mm Height 250mm

RAT Rectangular 450x450 mm Height 300 mm

Left: Automatic door

Right: With additional IR lamps even the dark side (seen to the left) can be monitored with the camera!



2.2.4.2. Passive Avoidance paradigm in the 2-Compartment software

The “Passive Avoidance” test module supports the following experiments: ! Pre-exposure / Training / Retention test / Reversal as well as 2 small conditioning programs. This means that a wide range of test variations can be imagined. The LED above the light compartments can be switched in via the software, which means that the aversiveness level of the light compartment can be defined by the user (up to 700 lux).

Training Test ! The run pattern of the animal is available for evaluation – this opens up a new quality for

studying the exploratory behavior of the animal – this is of crucial importance, particularly for determining the baseline activity. Phase-related results parameters, which can be calculated separately for both compartments, allow detailed conclusions to be made about the learning and exploratory behavior of the animal and also provide information about the shock response of the animal – in this form this was never previously possible in any standard passive avoidance measuring system.

Results table



Training – part-presentation (shock) Test – complete test

Compartment-specific results table for studying exploratory behavior

2.2.5. “Light-Dark Exploration”

The Light-Dark test is a quick and uncomplicated test of the non-conditioned anxiety behavior of the animal (unconditioned anxiety). It does not require prior training of the animal. In the light-dark test the animal can transfer between a protected dark area and a new aversive illuminated area (conflict between staying in a safe compartment and the exploration of the new compartment). The latency to entering the light area and the number of visits to the light area provide information about the “anxiety” of the animal. Anxiolytic compounds decrease the total duration of time spent in the dark compartment and the number of transfers between compartments while anxiogenic compounds work in the opposite way



2.2.5.1. Arenas

! For this test the Passive Avoidance arenas are used (see section 2.2.4.1). This 2-

compartment arena has a dark compartment that comprises 50% of the arena area and it is equipped with a central tunnel gate to let the animal pass. An automated door allows to open and close the tunnel gate via software.

! The floor grid is usually covered by a plastic cover plate (no shock is given here). ! The high-intensity stimulus LEDs can be used to increase aversive conditions (up to 700 lux can

be created on the bright side of the arena) 2.2.5.2. Light Dark Exploration paradigm in the 2-Compartment software

Trial Parameters ! The “Light-dark test” test module supports the following sequence:

• The animal is inserted in one of the two compartments with the door closed. • Habituation period. • Then automatic door opening. • End after a user-defined time.



The automatic doors with which these arenas are equipped can be opened by the software to start the experiment after a habituation period has elapsed, i.e. without requiring any action by the user ! In the analysis in addition to the evaluation of the transfer behavior of the animal – in a similar

way to Passive Avoidance – numerous locomotor parameters can also be calculated, including activity and hyperactivity times.

! Due to the high-resolution infrared sensors a detailed look into the locomotor behavior of the animal throughout the task is possible in parallel. An example is the pattern of movement that can be generated with a keystroke.

Pattern of Movement

2.2.6. “Place-Preference”

Conditioned place preference is a widely used technique to assess the rewarding properties of a psychotropic drug. Treatment with a specific preparation is repeatedly paired with a distinct en-vironment while a control treatment is paired with a different environment. When the animal has access to both environments, preference for the drug-paired cues, i.e. increased time of stay in the drug-paired compartment, will indicate the rewarding effects of the test drug. Learning processes like those observed in conditioned place preference are believed to be involved in the development of addiction.



2.2.6.1. Arenas

Place Preference Arena seen through the camera

mounted to the rear wall of the housing ! The Place Preference arena has 2-compartments providing visual cues through differently

patterened walls (standard: vertical pattern of black/white stripes vs. transparent acrylic plastic, other configurations on request)

! Both compartments are divided by an automated door made of black acrylic to restrict access of the animal to one compartment during conditioning. For the test this door is opened by the software. The tunnel gate is provided in animal-specific size.

! 2 clear lids are included. Picture MOUSE Rectangular 450x300mm Height 250mm



RAT Rectangular 450x450 mm Height 300 mm

! 3 pairs of differently structured floor inserts to facilitate tactile discrimination what is especially

useful for mice are included. They can be combined with each other as is required.

Example of tactile floor inserts

2.2.6.2. Place Preference paradigm in the 2-Compartment software

! The Place Preference function module available for the TSE Multi Conditioning System allows

to select between training and test sessions.



Control Parameters

Evaluation of the test trial includes:

• number of visits to each compartment



• latency first transfer to the other compartment • time of stay: absolute & percentage for each compartment • distance travelled: absolute & percentage for each compartment • number of rearings for each compartments • average speed

! Due to the high-resolution locomotory information and the ability of the system to measure

rearing behavior much more data is available about potential sedative effects of a specific treatment – not available in standard systems that rely on transfer detection only – another advantage of the TSE Multi Conditioning System.

2.3. Software Package “Locomotor Activity & Open Field – ActiMot”

In the Open Field paradigm the exploratory behavior of the animal (conflict between exploratory drive and fear) is analyzed in an environment that is unknown to the animal and which is normally aversive (illuminated). The animal usually avoids free, open spaces (the center of the open field). Depending on the test duration, habituation effects can also be studied; repeated testing provides information about the ability of the animals to remember. 2.3.1. Arenas

! The Open Field arenas are made from transparent acrylic and have a permanently mounted

floor made from light gray PVC. In order to use these arenas the shock grid is removed. With clear Plexiglas cover. Operation without a cover is also possible when camera observation from above is required.

! Dimensions : approx. 450x450mm outer dimensions (maximum possible size), height 400mm. The standard arena is identical for rats and mice thus offering mice an extra-large area.



Optional ! The arenas can be provided with a groove into which a separating wall can be slid (transparent

or black). The separating walls have no openings and are only used to reduce the available movement area – in the experiment the animal is inserted into one of the two compartments.

! Smaller arenas can be manufactured if required. In this case this test can also be used for

measuring pure locomotor activity (spontaneous activity). The difference between “open field” and “locomotor activity” applications lies solely in the choice of test arena and the selection of suitable evaluation parameters for the application.

! It is also possible to use all arenas provided for FCS experiments – including the round and triangular arenas. The use of foot shock is not intended, i.e. if arenas without floor are used the grid is solely used as run grid. In floorless arenas the grid cover plates can also be used as the floors.

! Homecages can also be used – the same templates available for the FCS software package are available then.

Homecage Mouse in template 2.3.1.1. Open Field/Activity paradigm in the ActiMot software package

! The multi conditioning hardware permits operation with various specialized software packages. In order to run these experiments we provide our proven ActiMot software package that has been specifically adjusted to work together with the MCS hardware (for Windows XP and Vista operating systems). All the results parameters that are provided in this comprehensive software package for open field and locomotor activity tests are available for the evaluation, so that maximum flexibility is achieved (except hole-board test and “2-in-1” feature – these are not available in the MCS system). Please read the ActiMot brochure for all details.

! The trials in the 4 measuring stations can be started and stopped independently of one another.

! Manually switching in background noise or house light (red or white) is possible (with identical quality for all measuring stations).

! “Aversion” software extension: The software package has been extended so that software-controlled switching of the stimulus lamps is possible. Both lamps are switched on at the same time. As these lamps produce a very high level of brightness on the arena floor this means that the level of aversity of the test surroundings can be increased. Before the test the intensity is set manually by the user on the control unit; switch-on then takes place before the animal is inserted into the box.



! “Start at a particular time” software extension. The software package has been extended so that the test can be started at a particular time without the user having to take any action. This extension is primarily intended for measuring locomotor activity with home cages. The starting time can be set independently for each measuring station.

! Numerous different analyses are possible: Graphic analyses such as run pattern, histograms and location graphs together with various tables for output as numerical parameters. More than 100 different parameters are available.

Histogram

Pattern of Movement Spacial Graph

Selection of results parameters



Results table / Interval analysis ! Interval analyses permit the observation of a user-specific time window. 3. Future extensions

! If light-dark trials are to be carried out with the dark compartment occupying 33% of the space then special black inserts are available for the open field arenas. In this case the trials are carried out using the ActiMot software package. It is not possible to use an automatic door in this case; the tunnel can only be opened using an optionally available manual slider.

! Alternatively special 2-part light-dark arenas with a 33% dark compartment can be supplied. The arena (rat or mouse size) consists of a light and a dark compartment made from clear and black acrylic (infrared-permeable) with floor. The dark compartment occupies 33% of the arena. The black separating wall (infrared-permeable) has a central opening and is permanently mounted. With two 2 covers (clear and black).

! It could be necessary to shine light selectively into the arena compartments for Active Avoidance or Place Preference experiments. A special LED lid module is available as an accessory. The LED lid module is placed on the arena. If this LED module is used then the stimulus LEDs in the housing roof are inactive. The LED module can be switched on and off via the software. The housing is already prepared for the connection of this module.

! Air-puff module as an alternative to foot shock (FCS software and Shuttle software) – in each case coupled with a necessary software extension.

! 3-Compartment place preference boxes for use with ActiMot or FCS software package (no automatic doors are possible here)

TSE_MCS_SHORT4.doc

Sophisticated Life Science Research Instrumentation

TSE Systems is a leading supplier of sophisticated research instrumentation in the

global life science market. Our focus is on providing the total customer solution, with

modular designs of integrated hardware and software platforms for neuroscience,

metabolic and behavioral phenotyping, drug screening and toxicology. NewBehavior as part of the TSE Systems International Group is specialized in state-of-the-art

technology for testing animal behavior within the social context.

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