1. INVOLVEMENT OF INCREASED CB1R ACTIVATION IN ALTERED
SOCIAL PLAY INDUCED BY DEVELOPMENTAL CHLORPYRIFOS EXPOSURE
Nicole E. Rowbotham, Carole A. Nail, Jenna A. Mosier, Aubrey M. Lewis, and Russell L. Carr
Center for Environmental Health Sciences, Department of Basic Sciences, College of Veterinary Medicine,
Mississippi State University, Mississippi State, MS
Introduction
1. Chlorpyrifos (CPF) is the most commonly used organophosphorus (OP)
insecticide in the US. Although the usage of CPF in households was
eliminated in 2000, CPF is still heavily used in agriculture creating a risk
for exposure of children living in agricultural communities.
2. It has been well established that OP insecticide toxicity results from
acetylcholinesterase inhibition. However, neurochemical and behavioral
effects result at levels below the no observed effect level (NOEL) for
inhibition of cholinesterase, indicating toxic effects from a currently
unknown “noncholinergic mechanism of action”.
3. One potential candidate for the unknown mechanism is the
endocannabinoid (EC) system, due to its role in brain development and
modulation of emotionality and anxiety. It has been reported that
alterations in EC signaling during development results in negative
long-term neurologic effects.
4. Anandamide (AEA) AND 2-arachidonoylglycerol (2-AG) are the most
well study endocannabinoids and are primarily hydrolyzed by fatty acid
amide hydrolase (FAAH) and monoacyglcerol lipase (MAGL),
respectively.
5. We have previously reported that low-level developmental CPF
exposure inhibits FAAH and MAGL, resulting in the accumulation of AEA
and 2-AG in the brain (Carr et al., 2013) and leads to increased social
play behavior once adolescence is reached (Figure 1).
6. During social play, there is an increased phosphorylation of the
cannabinoid 1 receptor (CB1R) (Trezza et al, 2012). The CB1R, a G
protein-coupled receptor, is the primary EC receptor in the brain and the
location for AEA and 2-AG binding. Phosphorylation of the CB1R is an
indicator of EC system activation. This suggests either activation of the
EC system increases play or play activates the EC system.
7. The objective of this research is to compare the phosphorylation of the
CB1R (pCB1R) in brain regions of control and treated rats immediately
following behavioral testing. The regions assessed (hippocampus,
amygdala, prelimbic cortex, agranular insular cortex, and nucleus
accumbens) are associated with social play behavior. The goal was to
determine if greater EC activation is occurring in the CPF treated rats
compared to controls.
Methods
1. Chemicals: Chlorpyrifos (>99%) was a generous gift from DowElanco
Chemical Company (Indianapolis, IN). PF-04457845 was purchased
from MedChem Express (Monmouth Junction, NJ). All primary
antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). The secondary antibody was purchased from Sigma Aldrich (St.
Louis, MO).
2. Animals: Adult male and female Sprague Dawley rats [CD IGS] were
bred to obtain rat pups. The day of birth was designated as postnatal day
0 (PND0). All procedures were approved by the MSU-IACUC.
3. Exposure: Beginning on PND10, rats were exposed daily for 7 days to
0.5 mg/kg CPF, 0.75 mg/kg CPF, 1.0 mg/kg CPF or 0.02 mg/kg
PF-04457845 by oral gavage at a volume of 0.5 ml/kg. PF-04457845
yielded slightly higher FAAH inhibition as the highest CPF dosage. CPF
and PF were dissolved in corn oil and delivered to the back of the throat
using a 50-ul tuberculin syringe equipped with a 1-inch 24-gauge straight
intubation needle (Popper and Sons, Inc., New Hyde Park, NY).
4. Behavioral Testing: The behavioral arena was a clear empty litter-cage
with bright light (600 lux). Each test session was filmed and recorded
using a remote operated Canon EOS Rebel camera. Testing was
performed on PND35-36. Following a 24 hour isolation period, two rats
of the same treatment, sex, age and size but from different litters were
placed into different corners of the behavioral apparatus. The rats
remained in the arena together for 600s. Two blind observers viewed the
video and recorded: time to first interaction, grooming, chasing, body
sniffing, anogenital sniffing, nape attack, crawling over/under, play fights,
pinning, and time spent playing. After each test, the cage was emptied,
cleaned with 70% ethanol, dried, and refilled with fresh litter.
5. Sample Preparation: Immediately following behavioral testing, brains
were extracted and frozen on dry ice. Frozen brains were sliced at 500
micron increments, and the brain regions of interest were collected using
punches. Samples placed into working lysis buffer, sonicated for 15-20s
twice, and then underwent centrifugation (14,000 x g at 4°C for 30 min).
Supernatant was collected and the pellet was resuspended in lysis buffer
and reprocessed. The final supernatant was collected an added to the
first fraction. Total protein concentration was measured using the
Bradford protein assay.
6. Western Blot Analysis: Western blotting was performed using a 6-20%
gradient gel. Samples were loaded at 175 µg/ml per lane. Proteins were
transferred to PVDF membranes at 4°C overnight at 50V. The
membranes were then blocked in either 3% BSA or 5% Blotto for 2 hrs
and then incubated overnight in the following dilutions of rabbit-anti-rat
antibodies in TBS-T (CB1 1:1000; pCB1 1:300; CB2 1:300; or α-tubulin
1:300). After washing, membranes were incubated overnight in
goat-anti-rabbit antibody (1:500 dilution in TBS-T). Blot membranes were
developed using 4-CN and DAB in conjunction and imaged using
ChemiDoc™ XRS+ System with Image Lab™ Software (Bio-Rad). The
bands were then quantified using ImageJ software (National Institutes of
Health).
7. Statistical Analysis: Statistical analysis was by analysis of variance
(ANOVA) using the Mixed procedure (Littell et al., 1996) followed by
mean separation by Least Significant Difference (LSD). The criterion for
significance was set at p <0.05. Initial analysis indicated no significant
sex differences so male and females were combined for further analysis.
Control
0.5 mg/kg CPF
0.75 mg/kg CPF
1.0 mg/kg CPF
0.02 mg/kg PF-04457845
D. Nucleus Accumbens
NormalizedProteinRatio
0.5
1.0
1.5
Figure 2. CB1 Receptors
A. Agranular Insular Cortex
NormalizedProteinRatio
1
2
3
4
B. Amygdala
NormalizedProteinRatio
0.5
1.0
1.5
2.0
2.5
3.0
3.5
C. Hippocampus
NormalizedProteinRatio
1
2
3
D. Prelimbic Cortex
NormalizedProteinRatio
0.5
1.0
1.5
2.0
2.5
3.0
Control 0.5 mg/kg CPF 0.75 mg/kg CPF
1.0 mg/kg CPF 0.02 mg/kg PF-04457845
FrequencyofChasing
2
4
6
8
10
TimeSpentPlayFighting
0
20
40
60
80
100
120
FrequencyofCrawling
OverandUnder
2
4
6
8 B
* *
D
FrequencyofPlayFighting
0
2
4
6
8
10
A
C
**
*
*
**
****
*
*
** **
Figure 1. Social Behaviors
Control
0.5 mg/kg CPF
0.75 mg/kg CPF
1.0 mg/kg CPF
0.02 mg/kg PF-04457845
E. Nucleus Accumbens
NormalizedProteinRatio
0.0
0.5
1.0
1.5
Figure 3. pCB1 Receptors
A. Agranular Insular Cortex
NormalizedProteinRatio
0.1
0.2
0.3
0.4
0.5
0.6
B. Amygdala
NormalizedProteinRatio
0.2
0.4
0.6
0.8
C. Hippocampus
NormalizedProteinRatio
0.2
0.4
0.6
0.8
1.0
1.2
D. Prelimbic Cortex
NormalizedProteinRatio
0.0
0.2
0.4
0.6
0.8
Non-Behavioral Behavioral
Non-Behavioral Behavioral
Results & Discussion
1. Endocannabinoid signaling plays a role in the modulation of social
behaviors. Developmental exposure to exogenous cannabinoids (such
as THC) leads to long-term neurologic effects as demonstrated by
multiple studies involving deficits in children of exposure pregnant
mothers and deficits in adults following adolescent exposure.
2. Our data demonstrate that inappropriate elevation of endogenous
cannabinoids during development via inhibition of FAAH can result in
altered emotional and social behavior at adolescent ages. Specifically,
the rats exposed to CPF and to PF-04457845, a specific inhibitor of
FAAH, exhibited higher levels of chasing (Figure 1A) and crawling over
and under (Figure1B), which are both play solicitation behaviors. In
addition, these treated rats had higher number of episodes of play
(Figure 1C) and spent more time playing (Figure 1D).
3. With respect to the level of the CB1R protein, there were no significant
effects of treatment in any of the five brain regions (Figures 2A-2D).
4. As expected, rats that had engaged in social play had higher levels of
CB1R phosphorylation in all five brain regions as compared to the
nonbehavioral rats (Figures 3A-3D). However, developmental CPF
exposure did not effect the level of CB1R phosphorylation.
5. If CPF exposure increased steady state EC tone, we would have
expected to see increased phosphorylation in the treated rats who had
not undergone behavioral testing. Increased phosphorylation in rats who
underwent social play testing would have indicated that CPF effected the
responsiveness of the system.
6. Since all groups subjected to behavioral testing had similar levels of
CB1R phosphorylation regardless of treatment, these data suggest that
differences in the level of phosphorylation of the CB1R is not related to
the increased social play observed in CPF treated rats.
7. Despite the lack of differences in phosphorylation of the CB1R, tt is
possible that dysfunction of the EC system plays a role in the altered
social play behavior observed in the treated rats. However, it could be
that the alteration of EC signaling that occurred during the
developmental CPF exposure period induced a functional alteration in a
different neurotransmitter systems that is closely tied to social play (i.e.,
dopaminergic and opioid).
This summer research experience was funded by NIH T35OD010432. The
research was funded NIH R15ES023162.
ENVIRONMENTAL
TOXICOLOGY