Context-dependent
effects of diazepam
on behaviour in mice
Lawrence Moon
Pat Wallace
Please switch off mobiles!
Goals
1. To critically evaluate design of in vivo experiments
2. To become familiar with practical aspects of in vivo work
3. To apply appropriate statistical methods to in vivo data
4. To experience the effects of neuroactive drugs (!)
Overview
1. Students will be allocated into groups of five
2. Quick exercise in groups
3. Feedback from groups – students will be asked questions!
4. Introduction to experiments
5. Students will be asked questions to check understanding!
6. Demo of behaviours to score
7. Students run first experiment -> two groups of 15
8. Demo of how to scruff and inject mice.
9. Students run second experiment
10.Lunch
11. Statistical analysis
12. Discussion of results
Allocate students into groups
A quick exercise to test prior knowledge.
Discuss in your group of three:
1. What family of drugs does
diazepam (Valium) belong to?
2. What can diazepam be used to
treat / what are the effects of
diazepam in humans?
(I’ll be asking members of each group
to provide replies. But don’t worry if you
don’t know the answers!)
A quick exercise to test prior knowledge.
Discuss in your group:
1. What family of drugs does diazepam (Valium)
belong to?
BENZODIAZEPINES
2. What can diazepam be used to treat?
ANXIETY (e.g. as a sedative / anxiolytic)
INSOMNIA
MUSCLE SPASM
ALCOHOL WITHDRAWAL
SEIZURES
Introduction to experiments
Aim is to show that the same dose of diazepam in
mice can produce behavioural effects that are either
sedative or anxiolytic depending on the testing
environment:
• Boxmaze exploration
• Locomotor activity measurement
First experiment: Boxmaze exploration
PERSPEX
LID GOES
ON TOP
LOW WALLS
MEDIUM WALLS
UNCROSSABLE WALLS
Imagine you are this mouse.
What might you do?
Hypothesis to test: diazepam affects
behaviours in the boxmaze
Experimental design: I’ll ask for class feedback on this!
Three treatments: saline, diazepam and amphetamine
Eighteen mice: six per treatment.
Inject mice, place mouse in centre of boxmaze. Wait one minute.
Student 1 to act as timekeeper and allocate roles.
Student 2 to measure “rearing”
Student 3 to measure “crossing”
Student 4 to measure “dipping”
Student 5 to measure “circling”
Three minutes per mouse. Each group observes nine mice.
More class feedback
What are the outcome measure(s) (what is counted)?
See handout
How will we analyse these (conceptually-speaking)?
We’ll come back to this at the end
What could go wrong? What else might affect the outcome
measures? Any other sources of variability or confound?
Noise
Bad handling
Inaccurate injection
Mouse – to – mouse variability in responsiveness
More elements of experimental design
What order should the mice be injected in? Should we inject and
observe all the diazepam mice first? Or the saline mice first?
Why would this be a problem?
What might happen if I tell you right now which mice will be
receiving diazepam? How could we deal with this?
What’s the difference between single blind and double blind?
We’ll be giving the injections subcutaneously. What happens to
the drug?
How to record your results
Mouse
tail mark
Mouse
number
Tally over three minutes
Rearing
None
1
IIIII
None
2
IIIII
One
3
I
One
4
IIII
Two
5
II
Two
6
I
Crossing
Dipping
Circling
Time for a demo, then your turn
One group of five per boxmaze
One person to act as timekeeper and to make a note of the tail
markings of each mouse. This person to allocate others each to
one behaviour and to distribute hand-counters.
One person to count rearing
One person to count crossing
One person to count dipping
One person to count circling
You need a pen and a lab coat.
Switch mobiles off and don’t talk! Lights will be off.
Locomotor activity test
There are four test regions. One mouse will be placed in each region .
Each person monitors one mouse
Experimental design
• You will each inject a mouse with either saline, amphetamine or
diazepam.
• Place into corner of designated region.
• Score “crossings” for 40 minutes. Staff will keep time.
• Each student will observe and record “crossings” by one mouse.
THIS TIME EACH STUDENT MUST RECORD THE TAIL
MARKING OF HIS/HER MOUSE!
• Record each crossing according to its 5 minute interval. Do this as
shown on next screen..... Staff will call out five minute intervals.
Individual results
MOUSE
TAIL
MARK
Squares entered per 5 minutes at time (mins) after injection
0-5
No bars
5 - 10
10 - 15
15 - 20
20 - 25
25 - 30
30 - 35
II
IIIII
IIIIII
IIIIIII
IIIIIIII IIIIIIII
II
IIIIII
Experimental design
• We want to find out whether there are differences between the
drugs in their effect on locomotion of mice.
• What problems might there be with this experimental design that
we have selected?
• Any other questions?
Now practice scruffing and injecting your mouse......
but DON’T actually inject drug until we say so (so everyone
gets plenty of practice scruffing)
Boxmaze results: on whiteboard
Tail marking
Mean rearing
No bar
One bar
Two bars
Calculate means
Mean crossing
Mean dipping
Mean circling
Statistics for boxmaze results
(Another interactive bit)
• What’s the difference between parametric vs non-parametric tests?
• Name two tests (for testing differences between two groups)
• How do I do a non-parametric test?
• The next slide will show how.....!
Statistics for boxmaze results
Example using mean number of “crossings” per mouse
Mouse number
Control group
Diazepam group
i
15
7
ii
12
6
iii
11
13
iv
9
10
v
14
2
vi
16
4
Observation: D group has lowest mean.
Question: Could this low value have arisen by chance (1/20)?
Method:
Designate group with lowest mean “L” and the other one “H”
Rank the scores (mixing two groups) and label as “L” or “H”
Take the lowest L and count how many Hs precede it.
Repeat for all Ls and calculate the total
Statistics for boxmaze results
Example using mean number of “crossings” per mouse
Mouse number
Control group
Diazepam group
i
15
7
RANK
4 6
7
9
10 11
12 13 14 15 16
GROUP D D D
D
C
D
C
NUMBER
OF Cs
BELOW
THIS D
2
0
0 0
0
ii
12
6
1
iii
11
13
C
iv
9
10
D
v
14
2
C
vi
16
4
C
C
3
• Observed U statistic for lowest group = total = 4.
• Could this have arisen by chance (1 in 20) with n1=6 and n2=6 ?
• From tables, theoretical (critical) value of U is 5 (at 1/20)
• Because Uobserved <= Utheoretical, difference is significant (at 1/20)
Statistics for boxmaze results
• Now each group calculates stats for one given comparison.
• Then we break the codes (end of blinding).
Statistics for boxmaze results
• If you had only two groups, you would run one Mann Whitney for
each outcome measure (“rearing”, “crossing”, etc).
• But we have three groups.
• Some people would use separate Mann Whitney tests to compare
all four outcome measures for
• Diazepam vs control, and
• Amphetamine vs control
• But this would be dodgy – who knows why?
Statistics for boxmaze results
• To avoid “multiple testing errors”, what tests could be used to
determine whether there is a difference between multiple groups in
any one particular outcome measure. (e.g. “crossing”)?
• Analysis of variance (ANOVA). This is a parametric test.
• Kruskal Wallis. This is the nonparametric equivalent.
• If the test provided evidence against the null hypothesis, then
pairwise tests should be conducted to determine which group
differs from the other.
• This is beyond the scope of this class. (Covered by Dr. Spina?)
Discussion of boxmaze
• Is it a good test environment for detecting sedative or anxiolytic
effects of diazepam?
• What about amphetamine?
• How might you improve the assay?
• How do we know the drugs worked? (Need for positive control)
Take a break
Results from second experiment
Tail marking
Mean squares entered per 5 minutes at time (mins) after injection
0-5
5 - 10
10 - 15
15 - 20
20 - 25
25 - 30
30 - 35
35 - 40
No bar
One bar
Two bars
Fill in your individual totals on the whiteboard. When all scores
are present, calculate and write the means onto your handout.
Analysis of second experiment
Students to plot out the group means against time, then we break
codes
Discussion of results:
Which line do you think is which drug?
Should we have included an uninjected mouse? If so, why?
Does Diazepam look like an anxiolytic or sedative?
What is variability of this data like? In future would you need more
mice?
Knowing the time course of activity, how could you redesign the
first experiment?
Statistical analysis of locomotor data
• Observations of the same outcome measure were recorded for
each mouse repeatedly => REPEATED MEASURES DESIGN
• REPEATED MEASURES ANALYSIS OF VARIANCE will tell
us whether there were
• i) differences between groups in mean locomotion overall, and
• ii) differences in amount of locomotion over time
• Needs a computer!
Discussion: validity of boxmaze
Behavioural tests that are designed to identify novel anxiolytic
drugs should have good predictive validity.
In an unfamiliar environment, diazepam boosts activity (reduces
anxiety). In a familiar environment, it does not.
Did the data show this?
However, amphetamine can also boost activity in an unfamiliar
environment.
Did the data show this?
Amphetamine is a locomotor stimulant and definitely not an
anxiolytic.
Is the boxmaze therefore a good assay for discovering anxiolytics?
Discussion: mutating receptors
• Neurons have ion channels.
• Some ion channels have both GABA receptors and
benzodiazepine receptors of varying subtypes.
• You can mutate each subtype and then give mice diazepam.
• This way, you can figure out the mechanism of action.
• You might also develop better sedatives / anxiolytics.
Conclusions
• Be exceptional!
• Choose your experimental designs carefully.
• Positive controls
• Negative controls
• How many animals? What effect size do you expect?
• Know which statistical methods you will use in advance.
• Get a statistician to look at your plans beforehand!
• Then –
• Perform your studies blind!
• Randomise!
• Analyse blind!
• Good luck!
End of practical - thank you!
Using your feedback forms anonymously,
please name one thing you did not
understand or name one thing could be
done to improve this practical.
Simplistic overview of diazepam
1. Diazepam binds to receptors including the GABAA receptor and some voltage-dependent sodium
channels.
2. Diazepam enhances the activity of GABA-A
receptors, allowing more chloride ions to enter the
neuron, depolarizing the membrane and boosting
neuronal inhibition.
3. Hence diazepam reduces spasticity and seizures.
Diazepam pharmacology
1. Can be taken orally, IV or IM.
2. Fast absorption and fast onset of action.
3. Crosses BBB.
4. Long lasting: has half-life of 20 to 50 hours.
5. Side effects: sleep, addiction, dizziness, amnesia
Previous boxmaze data (PTO for
interpretation)
MOUSE
DRUG
TREATMENT
Mean squares entered per 5 minutes at time (mins) after injection
0-5
5 - 10
10 - 15
15 - 20
20 - 25
25 - 30
30 - 35
35 -40
Diazepam
32 0 0
030
11 0 0
0 0 56
0 27 0
000
000
0 71 0
Amphetamine
81 109 161
261
66 114 117
295
78 123 73
344
1 135 55
469
0 116 35
405
0 120 24
430
0 120 33
396
0 112 18
361
Saline
24 118 182
10 120 167
14 76 158
20 79 127
006
100
000
000
Previous boxmaze data (interpretation)