Download SOPHowland_01

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Howland_01: Behaviour Tests
University Committee on Animal Care and Supply (UCACS)
Standard Operating Procedure (SOP)
SOP #
Title
Howland_01 Howland Laboratory Behaviour Test Battery in Rats
1. Purpose
This SOP identifies the procedures associated with the standard behaviour tests conducted
in Dr. John Howland’s laboratory.
2. General Information/Responsibility
Individuals performing the procedures outlined in this SOP must be adequately trained. The
principal investigator is responsible to ensure all research personnel performing these
procedures are properly trained and comfortable in completing the procedures.
3. Equipment/Materials
Indicated with each individual procedure below.
4. Procedure
Table of Contents:
A. RECOGNITION MEMORY TESTING ........................................................................................ 1
B. VISUAL-CUE TO RESPONSE SET-SHIFTING AND REVERSAL LEARNING USING AN OPERANT CHAMBER
BASED PROCEDURE .......................................................................................................... 7
C. WATER MAZE TESTING ................................................................................................... 12
D. DELAYED AND TRACE FEAR CONDITIONING ......................................................................... 13
E. LOCOMOTOR ACTIVITY TESTING ....................................................................................... 18
F. STRESS RESPONSE .......................................................................................................... 19
A. RECOGNITION MEMORY TESTING
I. OBJECT RECOGNITION:
(A) ROOM SET-UP
a. General Information:
 Tests are run in the water maze room.
 There are two white object recognition boxes labeled “1” and “2”.
o Generally, odd numbered rats (no tail mark) are run in box 1 and even numbered
rats (black tail mark) are run in box 2
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 Boxes are placed at back of room behind water maze. There are black marks on the
floor for the positioning of the box on test day. For habituations, box 2 can be placed
within these black marks, and box 1 can be placed in front of box 2.
 The numbers on the boxes should face the east wall (towards the preparation room)
b. Set-Up
 Turn 3 lamps on full intensity. Do not turn on the lamp in the West back corner (lamp
nearest the boxes); Then, shut off the main fluorescent lights
 Clean the boxes with 40% EtOH and a damp sponge. Be sure not to touch the inside
walls or floor of the box with hands and fingers
 If it is test day, set up the camera in front of the box and position it so you can see
where the objects will be placed.
o For object recognition, you only need to see the back of the box.
 For objects in space, you need to be able to view the whole bottom of the box; this
requires that you elevate the camera stand off the floor using the empty cages in the
water maze room
 Place “DO NOT ENTER” sign on door and Quiet sign out front.
(B) HABITUATION
a. General Information:
 Total of 3 habituations before first test day
 The last habituation must be completed 48 hours before test day
 Each habituation lasts roughly ten minutes in length
 For the first two habituations, rats can be tested two at a time with one rat in each
box
 For the final habituation, rats must be brought one at a time to the room and
habituated alone.
 The boxes are cleaned with 40% ethanol and a damp sponge between each
habituation.
b. Procedure:
 Remove rat(s) from colony room
o If you have a single rat, you must get a clean cage and transport the rat in this
cage; this cage can be reused for subsequent rats
o If you are running rats two at a time, you may transport them in their home cage
 Transport rat(s) to the water maze room and gently set cage on floor in front of boxes
 Pick up rat (not by tail) and slowly place in bottom of box with nose pointing East
wall; Do the same with other rat if running two at a time
 Leave room and time for approximately ten minutes in box
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 After ten minutes, enter room quietly and take rats out of box
o Try to pick up rat in same way in which they are accustomed to being handled
o If rat is very anxious and running around box, you can pick it up by its tail; make
sure to place on your other arm when you pick it up by its tail. Do not carry it
around only by its tail
 Take rat back to colony room and place in home cage if necessary
(C) TESTING
(a) TRAINING TRIAL
 Set-up room as before
 Clean objects with 40% EtOH and paper towel; Be sure not to touch objects with your
hands or fingers
o Objects must be cleaned for each rat
 Place two of the same object at the back of the box on the Velcro
 Check you can see objects in camera view
 Label white board:
o Date: ex: Sept 29, 2008
o Rat: ex: ORST-51
o Trial: TRAIN
o Experiment: RM + STRESS (ex)
 Double check everything is ready to go
o Check lights, camera, objects, whiteboard
 Get rat from colony room and transport to watermaze room in separate cage
 Place cage down in front of box as before
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 Take white board and place in front of camera so you can read all of the information
on the camera viewer; Press record and record information on white board for ~ 2
secs;
 Keep camera running, pick up rat from cage and place in box in same manner as you
have done in the habituations
 As soon as you place the rat in the box, begin timing;
o If this is the first rat, start two stopwatches
o One stop watch will be used to time the four minute trials and the other will be
kept running throughout the entire period of experimentation – it will be used to
time when you should start each rat in the training trial, when you can begin
stressing the animals, and when you can begin the test trials
 Leave room and allow rat to explore objects for four minutes
o During this time, you can be cleaning and preparing the second box and objects
for the next rat
 As soon as four minutes is up, enter the room and remove rat from the box. Press the
stop button on the camera to stop recording
 Carry rat back to colony room and place in home cage.
 Go back to colony room; switch out first box with second box
 Put first box out in hallway so you can clean it during the training trial of the next rat
 Change the information on the white board – ie) switch the rat I.D to I.D. of the rat
you will run next
 If everything is ready to go and the time is right, get the next rat and begin the next
training trial
 WHEN IS THE TIME RIGHT?
o That all depends on how spaced out you are running the trials
o If you are running nine minute trials, you run the first rat at T=0 min, the next rat
at T= 9 min, the next at T=18 min and so on. . . (time this on the stopwatch)
o I find that if I grab the rat at about 1 min before his trial actually should begin,
then I have enough time to grab him, transport him to the colony room, record
the white board and place him in the box before the time his trial should actually
begin; Therefore, if he should start at 9 minutes, I go into the colony room at 8
minutes.
 Run the next rat and each subsequent rat after that in the same manner
(b) TEST TRIAL
a. General Information:
 The test trial for a rat is run 1 hour and 45 minutes after you began the training trial
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 This trial is identical to the training trial except objects are different or are in different
locations
 In object recognition, a copy of the object used in the training trial is used and a novel
object is used. Both are placed at the back of the box (same way as in training trial)
 In object place recognition, the objects are identical to the objects used in the
training trial, but one object is moved to the front of the box.
 Never use the exact objects you used in the training trial in the test trial but use
copies of these objects
 Example:
o Say in object recognition you trained a rat on two cups
 Let’s label these cups C1 and C2 (Cup 1 and Cup 2)
o In the test trial, you will use a cup and a cross.
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 Label these C3 and Cr1 (Cup 3 and Cross 1)
o Therefore, although you will use a cup in the test trial that is identical in form to
the cups used in the training trial, you will not use the exact cup from the
training trial in the test trial. (You will not use C1 or C2 in the test trial.)
Correct: TRAIN :
C1 C2
TEST:
C3 Cr1
Incorrect: TRAIN
C1 C2
TEST
C1 Cr1
 As before, all objects and the boxes are cleaned for each rat
b. Procedure
 Look at watch that is kept running throughout the entire experiment
 When this watch reads 1:45:00, you must start the first rat in the Test trial; be sure
box and objects are ready to go before this time
 Switch white board to read “TEST” trial and label with correct rat I.D.
 Get rat from colony room or off of the stress pole, transport into the water maze
room, record white board, and place rat in box as before
 Run rat in box for four minutes
 At end of four minutes, pick up rat from box and place back in colony room
 Run each subsequent rat in identical manner.
(c) Injections
 Injections must be completed for each rat 60 min before its test trial
 = i.p injections
 For vehicle injections, 25% DMSO is used (2 ml/kg)
 All injections can be completed in the preparation room where the vet techs clean
cages
 Make sure your syringe is ready to go before you bring the rat into the room
i. Procedure:
 At 60 minutes before its test trial, transport the rat in a separate cage into the prep
room.
 Hold rat underneath front limbs or by the scruff of neck
 Both gently on rat’s face if needed and complete injection
 NEVER shake this rat
 Return rat to colony room following injection
(d) Stress
 Stress in conducted ½ hour before the test trial for a rat or 1 h 15 minutes after the
training trial
 On test day, half the rats should be stressed and half not
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 You must work out a schedule so you can stress the required amount of rats at the
correct times
o There are only two poles so a max of two rats can be stressed at any one time
 See Stress Sheet for Procedure
COMMENTS:
 Running these experiments takes much preparation beforehand
o Map out what objects will be used for which rat and how the objects will be
placed in the box; use provided template
o Determine how evenly spaced in time you want to run the rats (ie 6 minute trials,
8 minute trials, etc . . .)
 This time includes the four minutes the rat will spend in the box
o Map out which rats will be stressed and when they must be stressed
 Rats must sit on stress pole for ½ immediately prior to test trial
 Rats can be run in a second test following one week of rest
o For this 2nd test, only one habituation is needed 48 hours before test day; rats
can be habituated in pairs
B. VISUAL-CUE TO RESPONSE SET-SHIFTING AND REVERSAL LEARNING USING AN OPERANT
CHAMBER BASED PROCEDURE
a. Apparatus and Testing Parameters:
i. Background
Behavioural testing will commence utilizing commercially available Plexiglas chamber
(30cm X 24cm X 21cm; Med-Associates, St. Albans, VT, USA) enclosed in sound
attenuating boxes. Boxes are equipped with a fan to provide ventilation and to mask
extraneous noise. Each chamber is fitted with two retractable levers, one located on
each side of a central food receptacle where food reinforcement (45 mg; BioServ,
Frenchtown, NJ) is delivered by a pellet dispenser. A light emitting diode stimulus
light is positioned centrally above each lever and serves as a stimulus for visual-cue
discrimination learning. Each chamber is illuminated by a single 100-mA, 28V house
light located in the top-center of the wall, opposite to the levers. An infrared
photobeam is located in the food receptacle.
ii. Initial Lever Pressing Training
On the day before initial exposure to the operant chamber, rats are given 20 reward
pellets in their home cage. Before the animal is placed in the chamber on the first day
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of training, two to three crushed pellets will be placed in the food cup and on the
active lever. Rats will be trained under a fixed-ratio 1 schedule to a criterion of 50
presses in 30 mins, first for one lever, then the other (counterbalanced left/right
between subjects). On subsequent days, rats will be familiarized with the insertion of
the levers into the chambers and will be trained to press them within 10 s of
insertion. These sessions will consist of 90 training trials and begins with the levers
retracted and the chamber in darkness. Every 20 s, a trial begins with illumination of
the house light and insertions of one of the two levers into the chamber. If the rat
fails to respond on the lever within 10 s, the lever will retract, the chamber darkens
and the trial is scored as an omission. If the rat responds within 10 s, the lever
retracts, a single pellet will be delivered immediately and the house light remains
illuminated for another 4 s. In every pair of trials, the left or right lever will be
presented once, and the order within the pair of trials will be random. Importantly,
the stimulus lights above each of the levers would not be illuminated during these
training sessions. Five daily retractable lever training sessions will be given.
On the last day of training with retractable levers, the side bias for the rat will be
determined. These sessions are similar to pretraining session, except that both levers
are inserted into the chamber. Again, the stimulus lights above the levers are not
illuminated during these trials. On the first trial, a food pellet will be delivered after
responding on either lever. Upon subsequent insertion of the levers, food is only
delivered when the rat responds on the lever opposite to the one chosen initially. If
the rat chooses the same lever as the initial choice, no food will be delivered, and the
house light will extinguish. This continues until the rat chooses the lever opposite to
the one chosen initially. After choosing both levers, a new trial commenced. Thus,
one trial for the side-bias procedure consists of responding on both levers. The lever
(right or left) that a rat responds on first during the initial choice of a trial will be
recorded and counted toward its side bias. If the total number of responses on the
left and right lever is comparable, the lever that a rat chooses initially four or more
times over seven total trials will be considered its side bias. However, if a rat makes a
disproportionate number of responses on one lever over the entire session (i.e.,
greater than a 2:1 ratio), that lever will be considered as its side bias. Once the side
bias is determined, the rat will be returned to its home cage.
iii. Visual-Cue Discrimination
On the next day, after pretraining, rats will be trained on the visual-cue
discrimination. Here, the rat is required to respond on the lever that has a visual-cue
stimulus light illuminated above it. A session begins with both levers retracted and
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the chamber in darkness (the intertrial state). Every 20 s, a trial begins with
illumination of one of the stimulus lights above one of the levers. Three seconds
later, the house light illuminates and both levers are inserted into the chamber. A
response on the lever with the stimulus light illuminated above it (a correct response)
results in the retraction of both levers and delivery of one food pellet. After food
delivery, the house light will remain on for another 4 s, after which the chamber
returns to the intertrial state. If the rat responds on the other lever (incorrect
response), both levers retracts immediately and the chamber reverts to the
intertribal state. Failure to respond on either lever within 10 s results in the
retraction of both levers, extinguishing of the house light, and the trial will be
recorded as an omission. In every pair of trials, the left or right stimulus light will be
illuminated once, and the order within the pair of trials will be random. For each trial,
the lever that the animal chooses and the location of the stimulus light will be
recorded. Trials will continue until either (1) a rat has received a minimum of 30 trials
and achieved criterion performance of 10 consecutive correct responses or (2) after
150 trials, whichever comes first. Omission trials are not included in the trials to
criterion measure. Upon completion of a training session, the rat will be removed
from the chamber and placed back into its home cage. If a rat did not achieve
criterion on the first day of training, on the following day, the rat will receive a
second, identical session of the visual-cue discrimination training. During these and
all subsequent sessions, the latency to respond on a lever after insertion into the
chamber will also be recorded.
iv. Set-Shift to Response Discrimination
The acquisition of this discrimination will require the animal to cease the use of the
visual-cue discrimination strategy, and instead use an egocentric spatial response
strategy to obtain food reward. Here, a correct response entails responding on the
lever opposite of its side bias (left or right), regardless of the location of the stimulus
light illuminated above one of the levers. As with the initial visual-cue discrimination,
in every pair of trials, the left or right stimulus light will be illuminated once, and the
order within the pair of trials will be random. Trials will be given in a manner identical
to initial visual-cue discrimination trials, and again, for each trial, the lever that the
animal chooses and the location of the stimulus light will be recorded. Trials continue
until either (1) a rat achieves criterion performance of 10 consecutive correct
responses or (2) after 150 trials.
For the set-shift, we will analyze the errors into 3 subtypes: 1) perseverative errors
are scored when a rat responds on a lever with the stimulus light illuminated above it
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on trials where it requires the rat to press the opposite lever. Eight out of every
sixteen consecutive trials requires the rat to respond in this manner. These types of
trials will be separated into consecutive blocks of eight trials each. Perseverative
errors will be counted when a rat presses the incorrect lever on six or more trials per
block of eight trials that requires a press on the lever that did not have the stimulus
light illuminated above it. Once a rat makes five or fewer perseverative errors in a
block for the first time, all subsequent errors of this type are no longer counted as
perseverative errors, because at this point the rat is using the original strategy less
than 75% of the time. Instead, these errors will be scored as 2) regressive errors. 3)
Never-reinforced errors are scored when a rat presses the incorrect lever on trials
where the visual-cue light is illuminated above the same lever that the rat is required
to press during the set-shift.
v. Reversal Learning (Response Reversal)
On the day after the set-shift, rats will be required to learn a reversal of the response
rule, whereby rats have to press the lever opposite to the one they were trained
during the set-shift on the preceding day. Thus, if a rat is required to press the left
lever during the set-shift, during the reversal phase, it has to press the right lever,
regardless of the position of the visual-cue. Otherwise, this training will be identical
to the response discrimination-training day. One of the visual-cue lights will be
illuminated prior to the start of each trial, same as before, and serves as distracters.
Errors will be scored as presses of the lever that is rewarded during the set-shift and
are subdivided into preservative and regressive errors, using a procedure similar to
that used for analyzing these types of errors during the set-shift. Perseverative errors
are scored when a rat makes 10 or more errors within a block of 16 trials, excluding
the first block. Once a rat makes fewer than 10 errors within a block of 16 trials, all
subsequent errors will be scored as regressive errors.
The above specific training protocol is adapted from Floresco et al., 2009 (Floresco
SB, Zhang Y, Enomoto T (2009) Neural circuits subserving behavioral flexibility and
their relevance to schizophrenia. Behav Brain Res doi:10.1016/j.bbr.2008.12.001)
a. Procedure:
1. Food Restriction Procedure:
 control and maternally infected rats will be paired housed until one week
before set-shifting commences after which they will be individually housed
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 during the week before testing, rats will be carefully handled each day for 3-5
min
 on the day they are switched to individual cages, rats will be weighed and their
food removed
 over the next 3-4 days, rats will be fed 20-30 g of food per day until they reach
85% of their free feeding weight
 rats will then be maintained at 85% of their free feeding body weight through
daily weight measurements and feeding (previous experiments suggest that 2030 g of food per day is necessary for maintaining the body weight of a 300-400
g rat)
 once the set shifting protocol has been completed, rats will be returned to free
feeding
 it is important to note that throughout the experiment, all rats will be weighed
daily and fed daily by the experimenter. They will continuously have free
access to water throughout the experiment.
2. Daily Testing Procedure:
 at the start of each training day, Med-Associate boxes are turned on and a
check of levers, hopper and lights will be done through the FR1Test program
and ensure that it is clean and ready to use
 once operant chambers are functioning in a normal manner, the training
program required on that day will be turned on and ready to use
 four animals will be removed from the colony and moved into the operant
chamber room using a cart
 place animals into chambers and close the door of the chambers
 run/start the training program
 once the program closes (time varies depending on type of training session
required; ranges from 30-45 mins), animals will be taken out of the chambers
and return to the colony room
 clean trays of the operant chambers
 prepare testing procedure for the next batch of rats
 repeat procedure
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C. WATER MAZE TESTING
a. Procedure:
1. Pre-test set-up:
 Check lighting [60W (13W CFL) bulbs used in lamps]
 Place spatial cues
 Fill pool, and darken water using Tempora Paint- Dust
Free Powder Paint™ (Use 1 can=454grams)
 Warm Pool to 25°C (Empty water approximately 6cm,
and refill to appropriate temperature)
 Place Platform in the North West Quadrant:
 30cm radius from the edge of the pool to the
edge of the platform (edges set parallel to one
another)
 Top of platform 3cm below water surface
2. Training Procedure for a Given Training Day:
1) Obtain a clean empty plastic cage (the test cage)
2) Place two rats in the test cage and bring into Morris Water Maze Testing Room
3) Start Video recording
4) Indicate the experiment, date, trial number and the two rat numbers using a white
board and show on Video recording
5) Gently place Rat-1 in the pool at predetermined location (either North, West, South,
or East)
6) Walk directly to the Television (Experimenter Cue Position-ECP) and stand there for
the remainder of the trial
7) Record the amount of time it takes for the Rat to locate the platform—(If the rat
doesn’t locate the platform after 60 seconds, then guide the rat to the platform, and
immediately return to the ECP)
8) Allow the rat 10-15 seconds to sit on the platform
*If the rat jumps off the platform, place him back on the platform before
removing from pool*
9) Remove rat from the platform, gently rub it for 10 to 15 s with a towel, and place
back in cage [If the rat’s total time in the pool was less than 75 seconds, then wait
until 75 seconds in total has expired before placing the next rat in the pool. (ex: Rat-1
trial 1= 75s total, no break Rat-2 trial 1= 40s total, 35s break, Rat-1 trial 2=55s, 20s
break, Rat-2 trial 2=75s…]
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10)
11)
12)
13)
*Clear pool of any excess debris*
Repeat steps 4-9 for trial 2, trial 3, and trial 4.
Place rats in home cage
Wash out the empty test cage
Proceed to next testing trials with the next two rats (rat-3 and rat-4)…
3. Testing Procedures:
1) Place first two rats into the test cage, and bring into Morris Water Maze Testing
Room
2) Remove the platform from the pool
3) Place rat in pool at a new arbitrary position (ex: SW)-consistent for all rats
4) Let rat swim for 60s
5) Remove rat from pool
6) Repeat steps 1-5 for next two rats
7) Score Videos for time spent in each quadrant
D. DELAYED AND TRACE FEAR CONDITIONING
a. Brief Overview of Conditioned Fear and its Association with Clinical Disorders and
Psychiatric Illnesses
Both delayed conditioning to an auditory cue and trace conditioning to an auditory cue
are used to measure associative learning in rodents. One of the ways to examine
associative fear learning in rodents and higher order mammals is to use well established
Pavlovian fear conditioning techniques. During Pavlovian fear conditioning a harmless
sensory specific cue called a conditioned stimulus (CS: e.g. tone or light) is repeatedly
paired with an aversive event such as a mild foot shock (the unconditioned stimulus:
UCS). Presentation of the UCS (i.e., foot shock) typically generates a host of physiological
and behavioral responses that are indicative of a central fear-state (e.g. defensive
freezing, elevated startle and cardiovascular activity, increased respiration and hypervigilance) and these are referred to individually as an unconditioned response (UCR).
After just a few fear conditioning trials the CS becomes firmly associated with the foot
shock (UCS) and a CS-UCS association or fear-memory-trace are formed in discrete
regions of the brain. Thus, after fear conditioning has been completed, presentation of
the CS-alone can now produce a constellation of fear-like behaviors and responses that
are indicative of a central fear-state. Individually these behavioral, autonomic and
reflexive responses are called a conditioned response (CR) or a conditioned fear response.
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In addition, an entire environment, experimental context, or testing apparatus can also be
paired with an aversive event (foot shock) when fear conditioning laboratory animals.
Repeated pairings of a context with an aversive UCS (foot shock) leads to what is called
contextual conditioned fear because the fear that develops is not associated with a
specific sensory cue or CS. Rather, the expression of contextually-based conditioned fear
occurs when a research animal is exposed to the environment or context where the
aversive event (e.g. foot shock) was administered during training. After a few training
sessions animals pickup on the various sensory cues of an environment and associate
them with the UCS (foot shock). Thus, animals re-exposed to this conditioned context will
now exhibit fear-like behaviors and responses.
Since conditioned fear can be easily measured and quantified in research animals and is
also observed to occur in humans, neuroscientists can employ several novel research
approaches to unravel the mechanisms and neurobiological processes that contribute to
learning and memory in humans. One of the main ways this is accomplished is to use well
established fear conditioning paradigms and animal models of fear that closely mimic the
types of fear responses that occur in humans. A particular effective behavioral method
that has been developed to study the neural substrates underpinning conditioned fear is
the “defensive freezing paradigm”. This paradigm takes advantage of the fact that both
humans and laboratory rats become completely immobile (i.e. defensively freeze)
immediately after encountering a fear-eliciting stimulus or dangerous situation in the
environment. This type of defensive response is highly adaptive from an evolutionary
standpoint as it reduces the chances of being detected by a predator, and it also allows
the fearful animal to initiate other defensive reactions that are designed to ensure
survival (e.g., increased startle reflexive responses for jumping away from danger and
fleeing).
b. Rationale for Examining the Impact of Prenatal Infections on Associative Learning:
The “defensive freezing paradigm” is a valid and reliable method that can be used to
examine associative learning and memory functions in rodents. In order to adequately
examine the effects of prenatal infection on associative learning, both delayed and trace
fear conditioning techniques will be employed using defensive freezing behavior as a
dependent measure and behavioral indicator of associative learning. In the current series
of experiments, both delayed and trace fear conditioning will be employed to evaluate
any cognitive impairment that occurs in the offspring of infected pregnant female rats
(i.e. dams). The basic rationale for conducting such experimental work is based on the
fact that alterations in implicit forms of associative learning often exist in persons who
suffer from psychiatric disorders (e.g., schizophrenia and autism spectrum disorder) and it
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is believed that these deficits in learning and cognition are at least partly due to exposure
to infections during critical prenatal periods of neurological development. The use of
effective animal models may enable the mechanisms through which early neurobiological
development causes cognitive impairments later in life to be understood.
1. Procedure:
Young adult offspring of pregnant dams treated with saline or poly(I:C) will be used in
these experiments. One or two rats from each litter will be used in a specified behavioral
test in order to exclude the possibility of confounds that may arise due to specific litter
effects.
2. Apparatus and Testing Parameters:
Associative fear conditioning (delayed and trace) and testing will take place in four
identical Med-Associates sound-attenuating test chambers (30 cm X 24 cm X 21 cm: MedAssociates, St. Albans, VT, USA). The test chambers will be closed during all testing and
training procedures. Each test chambers is equipped with a fan to provide ventilation and
to mask any extraneous noise or lingering odors. Fear conditioning and testing using the
defensive freezing paradigm will adopt methods and techniques that are currently in
practice in reputable neuroscience research labs that adhere to ethical animal use
practices. With this in mind, the experimental protocols employed in our research will
endeavor to minimize the level of pain, discomfort or distress experienced by the
laboratory animals.
The dependent measure in these particular experiments is “conditioned freezing”, which
is operationally defined as the complete cessation of all movements except for
respiration. Conditioned defensive freezing behavior will be assessed with a
commercially available software package called “Video-Freeze (Med-Associates). The test
chamber will be configured and specifically altered to create two different experimental
contexts. This will be accomplished by varying the lighting (lights on vs. lights off), the
shape (rectangular vs. triangular) the flooring texture (steel grids for training vs. plastic
covering) and the odor (70% alcohol vs. Windex) of the chamber. For example; {Context
#1 = lights on + rectangular shape + steel grid + 70% alcohol}, {Context #2 = lights off +
triangle shape + plastic floor + Windex}. In fact several combinations and permutations of
contextual stimuli can be used to create a diverse range of contextual environments for
training and testing. However, once specified contextual environments are selected for a
particular experiment they should remain the same or be held constant until the
completion of the study.
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In all defensive freezing experiments (delayed or trace conditioning), the unconditioned
stimulus (UCS) will be a 1 s footshock (0.8 mA current intensity). For studies employing
delayed conditioning, the conditioned stimulus (CS) will be an auditory tone (4 kHz, 80 dB,
20 s in duration). In delayed conditioning the auditory CS will be presented for a period of
20 s which co-terminates with a 1 s footshock (UCS). This sequence is a single delayed
fear conditioning trial. Approximately 10 such delayed fear conditioning trials are
typically required to produce robust conditioned fear learning (i.e., defensive freezing
behavior) in laboratory rats. With trace-fear conditioning the CS is also and auditory tone
(4kHz, 80 dB) but the duration of the tone (CS) is only 10 s in duration and it terminates
10 s before the UCS (footshock).
A) Fear Acquisition to Contextual Cues and to a Specific CS
Animals will be subjected to a conditioning day (Day 1) and a test day (Day 2). On Day 1, rats
will be administered 10 CS-UCS fear conditioning trials over a period of 20 min. A 24 hour
consolidation period will be given and then on Day 2, the test phase will commence. During the
Day 2 test phase, rats will be exposed to the experimental context where fear conditioning took
place (no specific CS; Tone) and to a novel chamber that is different to chamber where the
specific CS (tone) was previously paired with footshocks. The rats will be exposed to both
experimental environments in a counterbalance order. This is done so that each animal is
assessed for fear acquisition to the context and to the specific CS (Tone). Contextual
conditioned fear learning will be evaluated by simply exposing rats to the same context where
fear conditioning took place for a period of 10 min. No auditory CS will be presented at this
time instead rats will be evaluated for amount of freezing behavior they exhibit to context.
Cue-specific conditioned fear learning (CS-induced freezing) will be assessed in a novel chamber
that is different to the chamber where CS-UCS fear conditioning occurred. This technique is
used because it assesses the degree of fear generated only by the specific CS (tone). This
method essentially separates out CS-specific fear from contextual fear. Cue fear conditioning
(CS specific) will be evaluated in the novel chamber by first exposing rats to a pre-tone baseline
phase for a period of 2 min. Animal movement during this pre-tone baseline period will be
recorded and assessed by the Video-Freeze software. This procedure is then followed by a 6
min period of continuous tone presentation where defensive freezing behavior to the CS is
recorded and evaluated by the Video-Freeze software package. Finally, a post-tone phase is
administered for a period of 4 min and freezing behavior is recorded.
B) Fear Extinction to a Specific Sensory Cue
In this experimental design, conditioning and testing will take place over a period of three
consecutive days. On Day 1 rats will be fear conditioned using the delayed and trace
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conditioning procedures as described in section (A) list above. On Day 2, fear-extinction
training will be administered to the rats. This consists of presenting rats with 20 presentations
of the CS-only over a period of 60 min. This research protocol produces robust extinction when
a delayed fear conditioning method is used to train rats. On Day 3 a retention test is
administered to assess memory for the extinction training session. This memory/retention test
will involve subjecting rats to an additional session of CS (tone) presentation that is 6 min in
duration. Thus, rats will get the 2 min pre-CS phase (pre-tone), followed immediately by two 6
min test blocks run back to back (2 x 6 min = 12 min total testing time). This extinction test
session will then be followed by the customary 4 min post-tone period where no CS is
administered. During each of the test phases freezing behavior data will be collected and
assessed by the Video-Freeze software package. During the collection and assessment of this
data special care will be taken to ensure that the freezing-behavior of the rats is congruent with
the operational definition of complete immobility described above. Also, it will be necessary to
ensure that the freezing behavior of the research animals is indicative of normal waking
behavior and motor activity and not sleep.
If prenatal poly (I.C:) exposure does indeed cause extinction-learning deficits in the current
experiment, then it is quite likely that regions of the medial prefrontal cortex which normally
acts to inhibit the limbic regions that are responsible for fear expression may have been
neurologically altered.
a. Procedure:
1. Daily Testing Procedure:
 at the start of each training day, Med-Associate boxes are turned on and a check of
cameras, speakers, lights, and shockers will be done through the FR1Test program and
ensure that it is clean and ready to use
 once operant chambers are functioning in a normal manner, the training program
required on that day will be turned on and ready to use
 four animals will be removed from the colony and moved into the operant chamber
room using a cart
 place animals into chambers and close the door of the chambers
 run/start the training program
 once the program closes (time varies depending on type of training session required;
ranges from 20-45 min), animals will be taken out of the chambers and return to the
colony room
 clean trays of the operant chambers
 prepare testing procedure for the next batch of rats
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 repeat procedure
E. LOCOMOTOR ACTIVITY TESTING
a. Apparatus and Testing Parameters:
Rats will be testing in commercially available locomotor activity testing boxes (Med
Associates Test Chambers ENV-008; 30.5 cm X 24.1cm X height 21.0 cm fitted with 4 pairs of
infrared photocells 3.5 cm from the floor evenly spaced on the long walls. The chambers
have metal grid floors and two operant levers (which are retracted during locomotor activity
testing). Each chamber is contained within a Med Associates sound attenuating cubicle with
a house light illuminated during testing.
b. Procedure:
 at the start of each day, initiate the software program for recording locomotor activity
on the PC computer connected to the locomotor activity chambers
 enter the relevant details related to the rats to be tested that day
 ensure the locomotor activity boxes are cleaned and ready
 remove the animals from the colony using a clean cage, weigh all animals, and deliver
immediately to the apparatus
 close doors to sound attenuating chambers
 start computer controlled locomotor activity monitoring sequence
o while drug-free locomotor activity is being monitored, mix up the appropriate
volume of drug for the rats in the testing apparatus, prepare needles for
injections
 after 60 min (computer program will automatically pause), remove each animal from
the locomotor activity testing chamber and inject (i.p.) with the appropriate volume of
drug
 re-initiate locomotor activity monitoring sequence
 after 90 min, remove all animals from locomotor activity monitoring boxes and return to
the colony room
 clean apparatus with sponge and water
 if necessary, repeat procedure for additional rats
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F. STRESS PROCEDURE
a. General Information:
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Our protocol is ½ hour on the elevated platform
This ½ hour is immediately prior to the TEST trial for behavioural experiments
There are two stress poles, so two rats can be run in the room at any one time
The room typically used for stress is the maze room and poles are placed in the centre
of the room (there are black marks on the floor for each pole)
 Make sure room is cleaned up after last rat is stressed
b. Procedure:
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Turn on main fluorescent lights
Turn on lamp in corner
Turn on second set of overhead lights (switch next to main light switch)
Place stress poles in centre of room within marked areas
Put “DO NOT ENTER” sign on door
Transport rat from colony room to maze room and place on pole
Stand in corner of room and watch for around 1- 2 min to make sure rat remains on pole
o if rat falls off, pick him up and put him back on.
 Rat must be stressed for ½ hour. At the end of this half hour, start it on the behavioural
task.
5. Safety
What precautions should be taken to complete this procedure safely?
Who should be contacted if something goes wrong?
6. Potential Complications and Troubleshooting
What are some common problems that arise and what steps can be taken to fix them?
7. References
List the references used to generate the SOP.
8. Revision History
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List the changes made during the most recent revision, as well as the reasons for the
changes.
Date Created: February 11, 2015
Written by: John Howland
SOP Review and Revision History
Revision Number
Review/Revision Date
Reviewer
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