1.  Repeated drug exposure leads to brain neural
adaptations.
 It is widely believe that these neural adaptations
mediate the long lasting changes in behavior
associated with substance use disorders (SUD).
 Additional neural adaptations may occur in
response to periods of drug abstinence and
may also contribute to SUD.
 Our research looks for neural adaptations to
cocaine exposure that persist , or develop only,
during long bouts of abstinence in rat models in
order to
 better understand the neural mechanisms
underlying behavioral changes in cocaine use
disorders (CUD).
 Identify neurochemical substrates that might
provide targets for the development of
pharmacological treatments against CUD.
 Identify physiological markers of CUD.
 A database of proteins collected several years ago
using new “shotgun” proteomic methods is used
here.
 In that initial study, protein expression in the brain
of rats exposed to several weeks of repeated, daily
cocaine and either a short (1 day) or long (21 days)
bout of forced abstinence, was compared to a
saline control group (See Sequence of Procedures).
Protein changes in the striatum of the rat brain after short and long periods of forced abstinence from a 21‐day bout of
repeated cocaine exposure
Pearl N. Guerin1,2, Mauricio Suarez2, & Alexis C. Thompson3
1Biological Sciences, 2Psychology, 3Research Institute on Addictions
Introduction
Methods
(Used for original data collection)
Results
Increased on Day 21
(Day 1, Day 21 ‐ % of control levels)
Decrease on Day 21
(Day 1, Day 21 ‐ % of control levels)
Pattern of Change
suggests
No Change on
Day 1
 Carbonic Anhydrase 2 (‐‐, 79%)*
 Gluthione S‐transferase P (‐‐, 73%)*
 Serum Albumin (‐‐. 71%)**
 Stathmin (‐‐, 77%)**
Abstinence‐Induced
Changes
(maybe)
Same Change
as Day 1
 G‐protein signaling modulator (130%, 129%)
 Calretinin (136%, 128%)
 Myelin Basic Protein (72%, 69%)**
Cocaine‐induced
Persistent Changes
(likely)
Opposite
Change to Day
1
 Adenylate Cyclase 5 (136%, 80%)*
 Clathrin Coat Assembly Protein (AP180) (231%, 79%)**
 Cofilin‐1 (140%, 74%)**
 Hemoglobin**
• Subunit Beta 1 (164%, 70%)
• Subunit Beta 2 (128%, 72%)
• Subunit Alpha ½ (136%, 72%)
Cocaine‐induce short
lasting Changes
+
Abstinence‐induced
compensatory
Changes
(likely)
28
15
6
3 4
1 0 0
29
12
7
3
1 0 0
3
0
5
3
0 0 0 0 0 0 0
2
7
4 4
6
2 3 3 4
7
0
5
10
15
20
25
30
35
40
Cell Signaling Cytoskeleton Intracellular
transport
RBC/Plasma Glucose
metabolism
Anti‐Oxidant Axonogenesis
Regulation
Protease inhibitor Unknown
WD1_Up WD1_Down WD21_Up WD21_Down
7
5 5
3 4 3 2 2
15
0 0 0 0
3
1
3 2
9
0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0
6
0
5
10
15
20
25
30
35
40
Chaperones Neurotransmitter
metabolism
Oxidative
phosphorylation
Cell motion Metobolism
Enzyme
Tricarboxylic acid
cycle
Cell adhesion Apoptosis
associated
Other
Functional annotation of the differential proteins identified in WD1 and WD21 (from Jiang et al., 2011 )
Jiang, X‐S, Li, J., Tu, C‐J, Suarez, M., Straubinger, R.M., Thompson, A.C., & Qu, J., Program number
518.15. 2011 Neuroscience Meeting Planner. Washington, DC: Society for Neuroscience., 2011.
Online.
Shichen S. Jiang, X‐S., Li J., Straubinger, R.M., Suarez, M., Tu, C‐J., Duan, X‐T., Thompson, A.C., Qu, J.
(In press, April 2016). Large‐Scale, Ion‐Current‐Based Proteomic Investigation of the Rat Striatal
Proteome in a Model of Short‐ and Long‐Term Cocaine Withdrawal. Journal of Proteomics .
Brady, S., Siegel, G., Albers, R. W., & Price, D. (Eds.). (2011). Basic neurochemistry: principles of
molecular, cellular, and medical neurobiology. Academic Press.
My project focuses on those proteins that were found on both Day 1 and Day 21 and altered, at least, on Day 21
There were fewer…
* Meets criterion 1 and 2 for significance. Risk of Type 1 error likely greater than 0.05.
** Meets criterion 1 ,2 and 3 for significance.
Using Criterion 1 and 2, the original study showed that cocaine induced a large number (223) of change in proteins in the rat striatum
1. Review literature on function of these target proteins.
2. Identify one protein to study further, based on function
and interest to the lab.
3. Use Western Blot technique and/or
Immunohistochemistry to verify and more carefully
evaluate the change in function.
4. Generate a hypothesis and test how it might work to
mediate CUD.
 40 male rats were used; 60 days old at the start of
the study.
 Procedures: see Sequence of Procedures
 On Day 1 or Day 21 the brain of each rat was
harvested and the striatum (dorsal and ventral) was
removed and rapidly frozen for later analysis.
 Proteomic analysis of the striatal tissue was
conducted by our collaborators Drs. Jun Qu and
Robert Straubinger in Pharmaceutical Sciences
using Comprehensive and Reproducible Ion‐
Current‐Based Proteomic Expression Profiling
Strategy (Shichen et al., in press).
 Determination of significant differences between
experimental and controls groups for the
concentration of each identified protein used
either 2 or 3 criteria:
 Criterion 1: Quantitative differences between
the experimental and control groups on each
day were only consider if the experimental
group protein levels were below 80.1% and
above 124.9% of control levels. The range (80.1‐
124.9%) was defined by a series of control
studies as the 99% confidence interval over
which control values do vary.
 Criterion 2: A p < 0.05 value in the t‐test
comparison between the experimental and
control group.
 Criterion 3: A p < 0.00001 allowing for the
Bonferroni correction for the multiple t‐tests.
SEQUENCE OF PROCEDURES
5mg/kg 10mg/kg 15mg/kg 20mg/kg 30mg/kg
HISTORY (SALINE or COCAINE, 21 days)
24h
FORCED ABSTINENCE (1 day or 21 days)
Protein Class Function [Brady et al., 2011]
Carbonic Anydrase 2 Anti‐Oxidant
• Role in oxidative stress
• Anti‐oxidant enzyme
Glutathione S‐tranferase P Anti‐Oxidant
• Role in oxidative stress
• Anti‐oxidant enzyme
Serum Albumin RBC/ Plasma
• Important for BBB oncotic pressure
• Regulation of blood plasma
Stathmin Cytoskeleton
• Role in cell cytoarchitechture
• Regulatory protein of microtubule
dynamics
Calretinin Cell Signaling
• Calcium‐binding protein
• Role in second messenger pathways
G‐protein signaling
modulator
Cell Signaling
• Facilitates G protein‐coupled
receptors function
• Helps initiate second messengers
pathways
Myelin Basic Protein Cell structure
• Integral component of myelin
• Facilitation of action potential
propagation
Adenylate Cyclase 5 Cell Signaling
• Role in cAMP second messenger
pathway
Clathrin Coat Assembly
Protein
Cell Signaling
• Role in Endocytosis
• Plasma membrane protein trafficking
• Role in recycling neurotransmitter
receptors
Cofilin‐1 Cytoskeleton
• Actin‐binding protein
• Depolymerizes actin filaments
• Role in plasticity
Hemoglobin Plasma
• Role in oxygen transport
• Made in select dopamine neurons
Outcome from Initial Analysis
Work in Progress
2 2
3
4
0
1
2
3
4
5
Antioxidant RBC/Plasma Cell Structure Cell Signalling
No. of Proteins/Class
Class of Proteins
References