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Reprinted from: Brain and Behavior. Raju TR, Kutty BM, Sathyaprabha TN and Shanakranarayana Rao BS (eds.

),
National Institute of Mental Health and Neuro Sciences, Bangalore, India. 2004:145-151.

METHODS OF ASSESSMENT OF LEARNING AND


MEMORY IN RODENTS
Srikumar BN, Bindu B, Priya V, Shankaranarayana Rao BS,
Raju TR and Bindu M Kutty

Among the various functions of the brain, one Currently several of the mechanisms underlying
of the most interesting and puzzling one is the such a magnificent phenomenon as memory have
ability to acquire new information and store them been understood. However, many of them remain to
for future retrieval. Learning is defined as be completely resolved. Animal models are useful in
behavioral modification through experiences or deciphering this complex machinery at molecular,
conditioning and when this persists it is termed cellular and systems level. Several of the CNS
memory. Memory can be broadly classified into disorders are often associated with impairment in
declarative and non-declarative memory (Figure cognitive functions. Alzheimer’s disease and ageing,
1) (Milner et al., 1998). Declarative memory for example have a primary impact on learning and
answers the question ‘what’, i.e. it is possible to memory, and other diseases such as Parkinson’s
verbally declare this type of memory. Memory of disease, bipolar depression, schizophrenia, and many
facts and events are examples of declarative other neurological disorders have been demonstrated
memory. Declarative memory can be further to have a secondary deficit in learning and memory.
classified into episodic and semantic memory. There are drugs available for the treatment of these
Episodic memory is memory of past and personally memory disorders, but clinical improvement is far
encountered events. The knowledge for the from satisfaction. Presently, the pathophysiological
meaning of words and how to use them is phrased mechanisms underlying these disorders have not
as semantic memory. Non-declarative (or been clearly understood and it is imperative that new
procedural) memory answers the question ‘how’ drugs that would be more useful be developed.
and is not stored with respect to time and place. Experimental paradigms of learning and memory are
Skills, habits and priming are examples of non- very enlightening in both elucidation of the
declarative memory. Priming is change in the pathophysiology and development of new
ability to detect or identify objects as the result of medications for the treatment of learning and
recent encounters, for example, Mys in Mysore. memory disorders.

Figure 1. Different mammalian memory systems (Milner et al., 1998)

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Paradigms of Learning and Memory different conditioning paradigms are
Broadly the paradigms can be based on either summarized in Table 1.
avoidance conditioning or maze training. Table 1: Summary of the different types of tasks
Avoidance conditioning can be further classified used to assess learning and memory (Brioni et al.,
into active and passive conditioning. The 1997; Reddy, 1997)

1. Step-down task
2. Step-through task
Passive avoidance
3. Two compartment task
4. Up-hill avoidance

1. Run way avoidance


Active avoidance 2. Shuttle box avoidance
3. Jumping avoidance

1. Classical Pavlovian conditioning


Classical conditioning 2. Rabbit eye blink response conditioning
3. Operant conditioning

1. Y maze
2. T maze
3. Radial arm maze (RAM)
Mazes 4. Water maze (WM)
5. Figure 8 maze
6. Complex maze
7. Stone maze
8. Battig maze

The hippocampus has been shown to be an important structure involved in learning and memory. There have been
many mazes that have been used to test hippocampal function (Table 1). The radial arm maze (RAM) and water maze
(WM) are perhaps the most used among them. A few of the various mazes that are used are depicted in Figure 2.

METHODOLOGY
I. Operant Conditioning
The operant conditioning apparatus consists
of a rodent test chamber (Skinner’s box), pellet
dispenser, sound attenuating chamber and a
control unit. The test chamber contains two
stainless steel response levers (5 x 4cm each) at a
height of 6cm from the floor, located both on the
left and right side (termed left and right lever
respectively) on one wall of the chamber. A food
hopper (5 x 3cm) is located in between the two
levers at the floor level. Each lever can be pressed
passively by a weight of about 7-8g. The chamber
Figure 2. Different mazes employed to study learning is connected to a solid food dispenser. Food
and memory (Reddy, 1997). pellets (approx. 45mg) are delivered to the
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feeding tray (food hopper) from the pellet Operant conditioning has been extensively used
dispenser through a plastic tube once lever is in our lab as a learning paradigm, to study
pressed. The test chamber and the pellet dispenser neuronal plasticity and as a model to understand
are kept in a sound proof box so as to avoid the the role of different areas in brain’s function.
external disturbances (Bures et al., 1983). Increased dendritic arborisation and spine density
was demonstrated following operant conditioning
Procedure during the brain growth spurt (Mahajan and
Rats are semi starved for 48hrs to motivate them Desiraju, 1988). Operant conditioning was found
towards food reward. On subsequent days 5-8 gm sensitive to detect functional deficits following
of food is provided after the test so as to maintain exposure to several environmental toxins like lead
the body weight at 85% of its initial weight, (Kumar and Desiraju, 1992), mercuric chloride
throughout the experimental period. (Lakshmana et al., 1993), endosulfan (Lakshmana
and Raju, 1994), arsenic (Nagaraja and Desiraju,
Acquisition (learning) test 1994), and metanil yellow (Nagaraja and Desiraju,
The training/shaping period consists of 30min 1993). It has been used as a model to understand
session per day for three days. After three days of the effect of subordination stress on brain
training session, the rats are subjected to 15 min biochemistry (Dhingra et al., 1996; Dhingra et al.,
test session for seven days. The number of lever 1997). The role of subiculum (Govindaiah et al.,
press responses per session is recorded. 1997; Nutan and Meti, 2000), fornix
(Yoganarasimha and Meti, 1999)and substantia
Learning criterion: To assess the acquisition
of the operant learning task, the criterion is set nigra – ventral tegmental area (SN-VTA)
in such a way that the rats should reach 75% of (Yoganarasimha et al., 1998) in brain’s function
the maximum lever press response of normal has been examined using operant conditioning
control rats. paradigm. Furthermore, operant conditioning has
been used to demonstrate the capability of intra
Retention (Memory) test cranial self-stimulation (ICSS) to facilitate learning
(Yoganarasimha et al., 1998) and bring about
Retention test can be carried out, 48h, 72h or
functional reversal following fornix lesion
7 days after the acquisition phase of the operant
(Yoganarasimha and Meti, 1999). Thus, operant
conditioning. Here the rats are subjected to one
conditioning is a powerful test to evaluate learning
trial and the number of pedal presses is
calculated. and memory.

Variants of the task II. Evaluation of spatial Learning and memory


in T-maze
1. Continuous reinforcement schedule (CRF): In
CRF, each response is followed by reinforcement The T-maze consists of a start box (12x12cm),
after a brief interval (i.e. for each pedal press, stem (35x12cm), a choice area (15x12cm) and two
there is one food pellet delivered). arms (35x12cm); each arm has a goal area
(15x12cm) containing a food-well. The sidewalls
2. Fixed ratio schedule (FR): In FR the
are 40cm high. The stem and start box are separated
reinforcement is scheduled after a fixed number
of responses or when a fixed minimum time is by a sliding door, and a cloth curtain separated
elapsed. the arm and goal areas so that the food well from
the choice area is not visible to the rat. 16W bulbs
3. Variable ratio or variable interval schedule (VR
illuminated the start box, choice and goal areas.
or VI): Here the response reward ratio or the
The T-maze is kept in a dimly lit, sound attenuated
inter reward time is randomly varied around
room (Bures et al., 1983).
some average value.
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Procedure memory. Laxmi et al showed ventral subicular
Rewarded alternation test lesion results in spatial memory impairment in T-
maze (Laxmi et al., 1999). ICSS-induced facilitation
This is analogous to non-matching to sample
of spatial tasks and stress-induced impairment has
task, where the rat is rewarded only if the current
been shown using T-maze (Sunanda et al., 2000;
choice does not match the previous one.
Yoganarasimha et al., 1998).
Behavioural testing is carried out in two phases;
a. Orientation and training session and
III. Evaluation of working and reference memory
b. Learning performance test (acquisition test) in Radial arm maze task (RAM)
The radial arm maze was introduced in 1976 to
Orientation and training session study hippocampal dependent learning and
Rats are semi starved for 48h and allowed to memory (Olton and Samuelson, 1976) .The eight
explore the T-maze for 30 min. After the 30 min arm radial maze is a computer monitored plexiform
orientation session, the animals are returned to maze (Columbus Instruments, USA). It consists of
their home cage. Rats are then trained for eight equally spaced arms radiating from an
alternation food reward. In these sessions rats are octagonal central platform. Each arm is 56.2cm
subjected to 10 trials / session / day. In each trial, long by 7.9cm wide. The entire maze is elevated
the rat is placed in the start box, then the sliding 80cm above the floor.
door is released slowly and the rat is forced to move
into one arm that leads to the goal area having the Acquisition of spatial task
food pellet by blocking the other arm that does not
The rats are maintained on a restricted feeding
have the food pellet. In the consecutive trials (10
at 85%of their free feeding body weight. These rats
trials), rats are again forced to enter a particular
are allowed to familiarize themselves with the
arm for food reward.
radial maze. Prior to each trial, all the eight arms
are baited with food pellets. The rat is placed in
Acquisition (learning) test the center of the maze and allowed to freely explore
The rats are subjected to 10 trials / session (inter- the maze. The rats are required to take the food
trial interval of 30s) until they reach the criteria of pellets from each arm without making a re-entry
7-8 correct choices out of 10 trials. Acquisition test into the arm already visited. The trial is terminated
is conducted for eight days consisting of one session when the animal takes the food reward from all
per day. Percentage of correct responses and errors the eight arms or after 10 min if all the eight arms
are calculated for each rat. In each trial, the rat is are not visited. The average criteria for acquisition
placed in the start box, the sliding door released are attaining 7.5/8 correct choices. Re-entry into
slowly and the rat is allowed to move into either an already visited arm is considered as an error.
left or right of the goal area for food reward. In Each animal is given two trials daily; retention test
each session of 10 trials, the number of errors, i.e., is carried out ten days following acquisition. The
entry into the non-rewarded arm is recorded. performance of the animal is scored by calculating
the percentage of correct responses (a correct entry
is when the animal has not previously entered the
Retention (Memory) test
arm) divided by the total number of entries made
Seven days after the last day of acquisition of by the animal.
the task, the rats are subjected to a retention test.
In this, rats are given one session of 30 trials. The
Variants of Radial arm maze tasks
performance is evaluated by calculating the
number of errors committed during the session. Minor variants
Studies from our lab have documented that T- 1. Increase or decrease in the number of arms,
maze is a useful tool to evaluate learning and thereby changing memory load
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2. Water deprivation with water reward can be etc.). An external cue is provided on the wall
used rather than food deprivation with solid opposite to the target quadrant. Diffused
reward. illumination is provided by indirect lighting from
3. Impose increased retention intervals between four 200W flood lamp fixtures aimed towards
arm choices. the ceiling. The rats are marked with black colour
on their foreheads to facilitate tracking.
Major variants For the place navigation task, the escape
1. Partially baited RAM task: In this, four of the platform is placed in the center of one of the four
eight arms are baited and the rats are trained imaginary quadrants of the pool and kept in this
to choose only the baited arms. This task permits location throughout training (Figure 3). All trials
discerning of reference memory and working in each experiment are performed at the same
memory components of spatial memory. An time of the day (± 2 hrs) during the animals’ light
entry into an unbaited arm is regarded as a phase. All rats are given two trials a day with
reference memory error and any re-entry (either an inter-trial interval of 20 min for 9 days (9
to a baited or unbaited arm is considered as a sessions) (Note: the number of trials taken to
working memory error. learn the task varies with the number of trials
2. Non-spatial working memory: Here, a cue per day). For each trial, the rat is placed into the
(colour or surface texture) differentiates baited pool from one of the two start positions located
arms from the unbaited arms and allow cue around the rim of the pool and is then given a
and place memory to be tested. maximum of 90s to find the escape platform. If
the animal found the platform, it is allowed to
The radial arm maze has been widely used in rest on it for 15s before being removed from the
behavioural neuroscience and behavioural pool. If the rat does not locate the platform
pharmacology. Thus, RAM tests are useful in within 90s, the rat is hand guided to the
evaluating the effect of drugs, stress and various platform. After the experiment, the rats are dried
other environmental factors on learning and with a towel and placed under warming lamps.
memory (Devi et al., 2003). On the 10 th training day, the rats are given a
probe trial without the platform and rats allowed
IV. Assessment of Spatial learning in Water to swim for 60s. It is generally believed that rats
maze task with an intact hippocampus spend more time in
This task is called “Morris water maze task”, the target quadrant. Retention is tested after 10
named after RGM Morris, who developed it in days. The animals’ movements are tracked with
1981. The maze used for rats is a circular pool of a camera attached to the ceiling. Data are
water, with a diameter of 1.5m, height 57cm, and analysed using an automated tracking program
depth of water 29cm. The pool is a metal cylinder (Columbus Instruments). Specialized software
painted white on the inner surface and the provided measures such as latency, path length,
escape platform is also made of metal cylinder swim speed and the amount of time spent in
with flat metallic top having a surface diameter different quadrants of the pool.
of 10cm and is 1–1.5cm below the water surface
during water maze training. The pool is filled
with water (23±2.0°C) and made opaque with
1.5 litre of milk in order to obscure the platform
and allow efficient tracking of the rats swim
paths. The pool is situated in a 3.6 x 3.3 m2 room
with black curtains hanging from the ceiling and Figure 3. Platforms used in different water maze tasks
covering other cues (doors, electrical fixtures, (adapted from (Reddy, 1997).
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Variants procedural learning. Mazes differ in the extent to
1. Short-term spatial memory: In this procedure, which they permit the use of diverse source of
the hidden escape platform is put in a different spatial information. They remain indispensable in
location in the pool each day and the focus is behavioral neuroscience and drug discovery
upon the savings in escape performance research.
between the first and subsequent trials. The
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