1. A Neurodevelopmental and Behavioural
Study of Mice Following In Utero and
Early Postnatal Exposure to Imidacloprid, A
Neonicotinoid Pesticide
Andrew Patrick Burke
Supervisor: Dr. David R. Hampson

2. Neonicotinoids
 Controversial class of insecticides
 Potentially responsible for the decline in Honey Bees?
 Among the most effective and prevalent insecticides in
use today
 Popularity “stems” from versatility of use
 Developed to be structurally similar to nicotine
 Potent neuronal nicotinic acetylcholine receptor
agonist

3. Imidacloprid
 Chloronicotinyl Nitroguanidine
chemical family
 Structurally similar to Nicotine
 First commercial neonicotinoid
and most widely used insecticide
on planet
 Used to control sucking and
chewing insects
 Currently banned by the European
Union
 Applied to over 140 different
crops, most of which are grown for
human consumption

4. Hypothesis and Objectives
 We hypothesize that chronic prenatal exposure to a low
dose of imidacloprid will induce abnormal behaviours in the
offspring, while also having a lasting effect on the offspring’s
reproductive and immune systems
 Objectives
 Objective 1: To treat pregnant female mice with imidacloprid
from early gestation to postnatal day 21, through subcutaneous
osmotic pump infusion
 Objective 2: To study the behaviour of the adult mouse
offspring using behavioural tests, including tests measuring
locomotor activity, anxiety, aggression, depression,
sensorimotor gating and social dominance
 Objective 3: To perform postmortem examination on the adult
mouse offspring

5. 0.5mg/kg
IMD
Study Overview
12 Experienced
CD-1 Female Mice
Treatment A
4 Female Mice
Treatment B
4 Female Mice
Treatment C
4 Female Mice
4 Pregnant
Female Mice
4 Pregnant
Female Mice
4 Pregnant
Female Mice
25%
DMSO
Water
4 Litters 4 Litters 4 Litters
Behavioural Testing

6. Open Field
Testing
(PND42-46)
Elevated
Plus Maze
(PND47-52)
Tube Test
(PND55-58)
Prepulse
Inhibition
(PND58-61)
Forced
Swim Test
(PND65-67)
Resident
Intruder Test
(PND67-72)
Behavioural Testing
Locomotor Activity
Anxiety
Aggression
Sensorimotor
Gating
Depression
Social Dominance

7. 0
1000
2000
3000
4000
5000
6000
Combined Male Female
TotalDistanceTravelled(Cm±SEM)
Total Distance Travelled
Water
25%
DMSO
IMD
(0.5mg/kg)
**
*
N= 36 51 25 18 20 12 18 31 13
0
50
100
150
200
250
Combined Male Female
TimeinCentreZone(Cm±SEM)
Zone Data
Water
25%
DMSO
IMD
(0.5mg/kg)
N= 36 51 25 18 20 12 18 31 13
0
1000
2000
3000
4000
5000
6000
Combined Male Female
TotalDistanceTravelled(cm±SEM)
Total Distance Travelled
25%
DMSO
IMD
(0.5mg/kg)
N= 44 55 21 24 23 31
*
0
50
100
150
200
250
Combined Male Female
TimeinCentreZone(Sec±SEM)
Zone Data
25%
DMSO
IMD
(0.5mg/kg)
N= 44 55 21 24 23 31
STUDY 1 : PND42-46, 1pm-5pm, 20 minute Trial. Two-way ANOVA + Bonferroni *p<0.05,
**p<0.01
STUDY 2 : PND42-46, 1pm-5pm, 20 minute Trial. Two-tailed Student’s t-test, *p<0.05
OPEN FIELD TEST (Activity and Anxiety)

8. STUDY 1 : PND47-52, 9AM-1pm, 5 minute Trial. Two-way ANOVA + Bonferroni, *p<0.05,
**p<0.01, ***p<0.001, ****p<0.0001
ELEVATED PLUS MAZE (ANXIETY)
STUDY 2 : PND47-54, 9AM-1pm, 5 minute Trial. Two-tailed Student’s t-test, No Significance.
0
50
100
150
200
250
Combined Male Female
TimeInOpenArms(Sec±SEM)
Open Arm Analysis
25% DMSO
IMD
(0.5mg/kg)
N= 44 54 21 23 23 31
0
50
100
150
200
250
Combined Male Female
TimeInOpenArms(Sec±SEM)
Open Arm Analysis
Water
25%
DMSO
IMD
(0.5mg/kg)
***
***
N= 36 55 25 18 22 12 18 33 13
****
****

9. 0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Combined Male Female
WinningPercentage(%/100)
Water vs IMD Water (6♂,12♀)
IMD (0.5mg/kg) (5♂,6♀)
N(Matchups)= 22 10 12
*** * *
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Combined Male Female
WinningPercentage(%/100)
Vehicle vs IMD 25% DMSO (6♂,13♀)
IMD (0.5mg/kg) (5♂,6♀)
* *
N(Matchups)= 33 15 18
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Combined Male Female
WinningPercentage(%)
Vehicle vs IMD 25% DMSO (21♂, 23♀)
IMD (0.5mg/kg) (24♂, 31♀)
*** *** ***
N(Matchups)= 188 85 103
TUBE TEST (Social Dominance and Aggression)
STUDY 1 : PND58-61, 1pm-5pm. Fisher’s Exact Test, *p<0.05, **p<0.01, ***p<0.001
STUDY 2 : PND58-61, 1pm-5pm. Fisher’s Exact Test, *p<0.05, **p<0.01, ***p<0.001

10. Summary
 Preliminary data suggests that aggression in male mice
may be increased by IMD treatment
 Significantly more wins in the tube test
 Further supported by very preliminary resident intruder
data
 In utero and early postnatal exposure to IMD appears
to:
 Have no lasting effect on locomotor activity and anxiety

11. Future Directions
 Complete post-mortem analyses
 Is there a deficit in immune function as a result of
imidacloprid exposure?
 Analyze offspring in 5-choice serial reaction time task
(a test of attention)
 Repeat selected procedures to further validate our
findings

12. Acknowledgments
Hampson Lab Members:
• Dr. Jason Arsenault
• Dr. Shervin Gholizadeh
• Dr. Laura Pacey
• Dr. Sujeenthar Tharmalingham
• David Jiang
• Dr. Yosuke Niibori
• Charlotte Pidgeon
Supervisor and Advisory Committee:
• Dr. David R. Hampson
• Dr. Peter G. Wells
• Dr. Paul J. Fletcher
University and Faculty Members:
• Dr. Jack Uetrecht
• Dr. Marija Milenkovic
• Dionne White
• Joanna Warzyszynska

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