Lab 5 - The ANATOMY of BRAIN FUNCTION

Question 1

What regions of the brain are involved in establishing a spatial memory?

Answer 1

The hippocampus is involved in the formation of spatial memories

Question 2

A classical test for assessing spatial memory is the

Answer 2

Morris water maze (MWM)

Question 3

Hypothesis about during the learning of a spacial task

Answer 3

e.g. the reference memory version of the Morris Water Maze (MWM), there should be an increase in activity in hippocampal neurons and in neurons in other specific regions of the brain that are used during the watermaze procedure.

Question 4

Aim of this laboratory session

Answer 4

To use a cellular marker of brain activity, to determine in which brain regions cells are activated during a spatial memory task.

Question 5

c-Fos define

Answer 5

c-Fos, an immediate early gene is activated in many cells in response to increased activity of the cell and thus can be used to indicate which cells are active during spatial learning by comparing c-Fos staining in trained and non-trained brains.

Question 6

Morris Water Maze

Answer 6

This Morris Water Maze (MWM) test is based on the ability of a rat to associate a specific location with external visual cues. In the MWM, the specific location is the location of a submerged and thus not visible escape platform, in a water filled pool. Rats are natural swimmers so swimming does not distress them but they do not like water and so are motivated to learn the task to get out of the water.

In the reference memory version of the MWM they will initially find the platform by chance. The rat is allowed to stand on the platform for a short time, normally 10 seconds, and view the surroundings. During this time they will make a spatial map using cues external to the water filled pool, the maze, which allows them to find the platform from any position in the pool.

MWM is one of the most widely used tasks and is fantastic for studying memory processes and spatial learning memory

Question 7

Procedure of MWM summarised

Answer 7

water maze is circular pool with an escape platform of clear perspex submerged beneath the surface of the water such that it is not visible to a swimming rat

maze in centre of room, Distinct visual cues, large black and white patterns on card, are placed on the walls in addition to the standard equipment found in the room and these act as the external visual cues. Internal light sources are kept to a minimum to avoid platform detection and inducing stress in the animals.

allowed to swim for 60s, allowed to remain on platform for 10 seconds, then lifted out

divided into 4 equal quadrants representing an arbitrary location with resect to true compass north

if the animal does not reach the platform within 60 seconds it is guided to the platform and remains on the platform for 10s

for training, the target is kept in one quadrant got the duration of the training so it begins to learn where the platform is located

Question 8

Parameters determined from the MWM procedure

Answer 8

path-length (the distance travelled) and latency (time) to find the platform
distance travelled as it gives a good idea of the way that the animal searched for the platform

Question 9

Learning the MWM task requires

Answer 9

..the activation of NMDA receptors in the hippocampus

Question 10

Rat exposures used for this experiment

Answer 10

three alcohol treated and one control
alcohol exposed animals were given specific does of alcohol, this is an animal model of binge alcohol exposure during the 3rd trimester equivalent of pregnancy
6,5.25 or 4.5g/kg of ethanol

Question 11

MWM training for the studied rats

Answer 11

trained on 4 trials per day with an inter-trial interval of 8 minutes, over 4 consecutive days

Question 12

The quicker they learn, ….

Answer 12

the shorter the mean distance traveled and shorter latency time (time to get on the platform)

Question 13

Alcohol regime results in

Answer 13

permanent cell loss in the CA1 region of the hippocampus that is dose dependent. All alcohol- exposed animals has significantly less CA1 neurons than controls with the 6g/kg animals have only 50% of the control number of neurons. (This data is from; Paul Shoemack, BSc Hons thesis, Univ. of Otago, 2007.)

Question 14

How did each group perform over time?

Answer 14

All groups learned where the platform was, as indicated by the decrease in the distance travelled to get to the platform.

shows they are learning the area spatially and then consolidating it each day

Question 15

How did the different treatment groups differ? was this difference (treatment effect) different on the different days?

Answer 15

Controls started with the 2nd lowest distance travelled and by day 4 had the lowest distance travelled out of the 4 groups, improvement from day 1 to day 2 is the largest out of all groups (learnt faster)

by the end of the trainings, a dose dependent relationship is shown between the amount of ethanol exposure and path length.
the 6g/Kg group having the longest path length on day 4 likely due to ethanol’s influence on the hippocampus

Learning was significantly slowed by ethanol treatment. Discuss the variation in learning) i.e path length across the treatment groups. Discuss the profound effects of 6 g/kg ethanol on path length and subsequent learning compared to other groups. (path lengths by day 4 for other groups was comparable).

Question 16

What would you say about the performance of an animal that has lost 50% of its CA1 neurons?

Answer 16

They would not be learning as quickly as the controls, CA1 neurons are in the hippocampus which is involved in spatial memory formation therefore having less of the neurones will impact on spatial memory formation therefore will take longer for them to find the platform

Question 17

What could you do to improve the performance of the animals in the 6g/kg ethanol group? (There is not correct answer so any ideas will be considered – we will discuss this in groups.)

Answer 17

Cognitive enrichment and enhanced physical activity.
Addition of more distal visual cues.
Repetition of trials.
Lengthen the period the rat remains on the platform to extend the period of learning of the spatial cues (> than 10 seconds on the platform).
More days of training
Decreases intertrial interval

Question 18

c-Fos is a marker of

Answer 18

cellular activity

Question 19

c-Fos is a

Answer 19

transcription factor

Question 20

c-Fos as a marker of cellular activity

Answer 20

Neuroscientists commonly want to obtain morphological information that is related directly to a physiological outcome. In this laboratory class we want to obtain information on the brain regions that are activated during the process of spatial learning.
c-Fos is a cellular proto-oncogene that is a member of the immediate early gene (IEG) family of transcription factors. Many extracellular signals e.g. growth factors, result in an increase in the transcription of c-Fos. c-Fos along with other IEGs acts to increase the transcription of a diverse range of other genes involved in a huge range of cellular activities e.g. proliferation and differentiation, learning, response to noxious stimuli and defense against invasion and cell damage.
The protein product of c-Fos, c-Fos protein, can be identified by immunohistochemical techniques and thus the extent of c-Fos expression can be detected.
c-Fos is expressed within some neurons following depolarization and therefore the presence of c-Fos protein in a neuron can be used as a marker for neuronal activity. c- Fos protein can be visualized using immunohistochemical techniques and thus the regions of brain activity can be determined although this does not necessarily indicate the order of activation of brain regions along the functional pathway.
Generally one would expect to see an increase in c-Fos protein, following a relevant stimulus, to levels greater than found in control animals that have not received the stimulus.

Question 21

Staining used to detect the protein protect of the immediate early gene c-Fos

Answer 21

the nuclei will be stained brown, dark to light, and there may also be a light brown staining of the background tissue
the slide with the most labelled nuclei is tissue from the trained animal

Question 22

List the steps you would take in order to label the protein c-Fos in a brain section.

Answer 22

Make or buy an antibody to c-Fos
Apply solution with antibody to c-Fos to section and incubate for a number of hours Wash off antibody
Apply another antibody, secondary antibody, that is an antibody to the species in which the primary antibody was made (in this case goat).
The secondary antibody has a molecule of HRP (horse radish peroxidase) attached to it that can be visualized using the reaction with DAB to make it turn into a brown coloured precipitate.

Question 23

general IHC method

Answer 23

Protein of interest known as the antigen - bind primary antibody to the antigen - secondary antibody to the primary antibody - then add fluent molecule to bind to secondary antibody to allow for visualisation (flurophore)

Question 24

how has the protein product of c-Fos has been detected in the tissue section….

Answer 24

labeled cells have a brown precipitate. The primary antibody is to c-Fos protein which is then recognized with a secondary antibody that has an HRP (horse-radish peroxidase) molecule attached. The HRP is then reacted with DAB (diaminobenzidine) to form a brown precipitate that is visible down the microscope. Thus the only brown should be where the c-Fos protein occurs.

Question 25

Labelled cells in what areas (c-Fos)

Answer 25

Piriform cortex is primary olfactory cortex in rat and send projections to orbital frontal cortex, may thus see activation here. Piriform cortex has multiple subdivisions, thought to have distinct functions that are used in parallel to process olfactory information. Why are these being used in the MWM? rat used olfactory information whenever it is doing anything. There may be olfactory stimuli is can use that we are not aware of. Does it leave traces on platform for next trial, should the water be well stirred

field of CA1, CA2, CA3 of the hippocampus
pyramidal cell layer of the hippocampus
piriform cortex layer 1

Question 26

Regions identified as activated…. indicate how they why they may relate to a spatial learning task like MWM …

Answer 26

Spatial learning- Hippocampus
Fear/Anxiety – the labeling in amygdala – the MWM is a stressful task, could you see any correlation between the degree of anxiety, lack of looking around on platform and the amount of amygdala staining and is there a negative correlation here with learning.
Visual cortex
Motor cortex for swimming

Question 27

Similar and different structures in the rat and human

Answer 27

Human brain cortex highly folded with sulci and gyri whereas rat brain cortex is smooth
both have corpus callosum and anterior commissures
rat ventricle sizes are larger than humans relative to brain size
hippocampus at different locations in rat and human (in rat more superior)
both have internal capsules, thalamus, hypothalamus, amygdala