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

Neonicotinoids

Controversial class of insecticides Potentially responsible for the decline in Honey

Bees?Among the most effective and prevalent

insecticides in use todayPopularity “stems” from versatility of use

Developed to be structurally similar to nicotinePotent neuronal nicotinic acetylcholine

receptor agonist

ImidaclopridChloronicotinyl 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

Hypothesis and ObjectivesWe 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

0.5mg/kg IMD

Study Overview12 Experienced

CD-1 Female Mice

Treatment A4 Female Mice

Treatment B4 Female Mice

Treatment C4 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

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

Combined Male Female0

1000

2000

3000

4000

5000

6000

Total Distance Travelled

Water

25% DMSO

IMD (0.5mg/kg)

Tot

al D

istan

ce T

rave

lled

(Cm

± S

EM

) ** *

N= 36 51 25 18 20 12 18 31 13 Combined Male Female

0

50

100

150

200

250Zone Data

Water

25% DMSO

IMD (0.5mg/kg)

Tim

e in

Cen

tre

Zon

e (C

m ±

 SE

M)

N= 36 51 25 18 20 12 18 31 13

Combined Male Female0

1000

2000

3000

4000

5000

6000

Total Distance Travelled

25% DMSO

IMD (0.5mg/kg)

Tot

al D

istan

ce T

rave

lled

(cm

± S

EM

)

N= 44 55 21 24 23 31

*

_x0008_Combined

_x0005_ Male

_x0007_ Female

175180185190195200205210215220

Zone Data

25% DMSOIMD (0.5mg/kg)

Tim

e in

Cen

tre

Zon

e (S

ec ±

 SE

M)

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)

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.

Combined Male Female0

50

100

150

200

250

Open Arm Analysis

25% DMSO

IMD (0.5mg/kg)

Tim

e In

Ope

n A

rms (

Sec

± SE

M)

N= 44 54 21 23 23 31

Combined Male Female0

50

100

150

200

250

Open Arm Analysis

Water

25% DMSO

IMD (0.5mg/kg)

Tim

e In

Ope

n A

rms (

Sec

± SE

M)

******

N= 36 55 25 18 22 12 18 33 13

********

Combined Male Female0.00.10.20.30.40.50.60.70.80.9

Water vs IMD Water (6♂,12♀)IMD (0.5mg/kg) (5♂,6♀)

Win

ning

Per

cent

age

(%/1

00)

N(Matchups)= 22 10 12

*** * *

Combined Male Female0

0.10.20.30.40.50.60.70.80.9

Vehicle vs IMD 25% DMSO (6♂,13♀)IMD (0.5mg/kg) (5♂,6♀)

Win

ning

Per

cent

age

(%/1

00) * *

N(Matchups)= 33 15 18

_x0008_Combined

_x0005_ Male

_x0007_ Female

00.10.20.30.40.50.60.70.80.9

Vehicle vs IMD 25% DMSO (21♂, 23♀) IMD (0.5mg/kg) (24♂, 31♀)

Win

ning

Per

cent

age

(%) *** *** ***

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

SummaryPreliminary data suggests that aggression in

male mice may be increased by IMD treatmentSignificantly more wins in the tube testFurther 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

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

AcknowledgmentsHampson 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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