1. • Male rats self-administered (SA) cocaine
(0.5 mg/kg/.2 mL inf) 14 x 2hrs/days on FR4.
• Following SA, rats underwent extinction
training wherein cocaine was replaced with
saline. Reinstatement tests were
administered the day after rats reached
extinction criteria (< 15 lever presses).
• For stress-potentiated reinstatement tests, a
stressor, 15-min intermittent uncontrollable
electric footshock stress (EFS; 3 x 0.5 mA,
200 ms duration, mean intershock interval
40s, range 10-70s), was administered prior to
a low-dose cocaine injection (2.5 mg/kg, i.p.)
or either stimuli were given alone.
The Role of the Cortico-Accumbens Pathway in Corticosterone Potentiated
Reinstatement
Alexandra Gabor1, Jayme R. McReynolds1, Paul Jacob Gottshall1, Colten P. Wolf1, Oliver Vranjkovic1, Evan N. Graf1, Cecilia
J. Hillard2, John R. Mantsch1
1Biomedical Sciences, Marquette University, Milwaukee, WI, 2Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI
Self-administration
apparatus
Fig. 1 Stress-potentiated reinstatement is
corticosterone-dependent
Fig. 2 Intra-prelimbic cortex administration of
corticosterone potentiates reinstatement
Fig. 5 Experimental Timeline
Fig. 4 Examination of the glutamatergic PL-NAc core
pathway following reinstatement
Fig. 6 Retrograde tracer injection into the nucleus
accumbens core
Fig. 7 Glutamatergic projections from the PL
to the NAc core
Fig. 8 Example double
immunohistochemistry for CTb and c-Fos
Stress affects everyone in daily life; the impact of
stress on individuals could be detrimental. In
previous studies conducted in our lab, we have
seen that stress can potentiate reinstatement
under certain conditions. Electric footshock stress
given along with a subthreshold dose of cocaine
was able to induce reinstatement whereas either
stimuli given alone did not. We have been able to
mimic the effects of stress-potentiated
reinstatement by using corticosterone in place of
the stressor indicating that corticosterone
mediates the ability of stress to potentiate
reinstatement. We have also been able to isolate
this corticosterone effect on reinstatement to the
prelimbic cortex. The pathway between the
prelimbic cortex and the nucleus accumbens core
(the cortico- accumbens pathway) is critical for
reinstatement and drives drug craving and drug
seeking behavior. This study examines cortico-
accumbens pathway activation following
corticosterone-potentiated reinstatement.
We hypothesize that corticosterone potentiates
reinstatement through increased activation of
this cortico- accumbens pathway.
Acknowledgements
Fig. 9 Double immunohistochemistry for CTb and c-Fos
• Stress-potentiated reinstatement is corticosterone-dependent and corticosterone can act in the
prelimbic cortex to potentiate reinstatement.
• Corticosterone likely potentiates reinstatement through increased activation of the cortico- accumbens
pathway.
• Corticosterone or cocaine given alone do not appear to greatly activate the cortico- accumbens
pathway and will continue to be processed for CTb and fos expression in the prelimbic cortex
following reinstatement conditions.
• We believe that the reinstatement following both corticosterone and a sub- threshold dose of cocaine
will produce the most activation of the cortico- accumbens pathway, as assessed by highest number
of CTb and c- Fos positive neurons.
• Understanding the neurobiology mechanisms and neurocircuitry behind relapse can hopefully lead to
effective treatments to rehabilitate addicts.
Electric Footshock stress (EFS)-induced potentiation of reinstatement is observed in SHAM-
operated (A) but not adrenalectomized (B) rats and was reproduced by systemic administration
of corticosterone (C; CORT, 2.0 mg/kg, i.p.) 40 min prior to the cocaine (COC; 2.5 mg/kg, i.p.)
injection. (Graf et al, 2013; *p<.05, compared to extinction).
Intra-prelimbic cortex (PL) administration of corticosterone (CORT; 0.05 μg/0.3 μL) 15 min
before systemic administration of a subthreshold dose of cocaine (2.5 mg/kg, i.p.) produced
significant reinstatement. However, either stimuli given alone did not produce significant
reinstatement. (*p<.05, compared to extinction)
The cortico- accumbens pathway is the pathway between the prelimbic cortex and the
nucleus accumbens core. This is a glutamatergic pathway that is critical for
reinstatement and drives drug craving and drug seeking behavior.
Introduction
Materials and Methods
• For corticosterone (CORT)-potentiated reinstatement tests, rats were given
a systemic injection of CORT (2 mg/kg, i.p.) 40 min prior to the low-dose
cocaine injection or either stimuli were given alone.
• To test the involvement of the prelimbic cortex (PL) in potentiated
reinstatement, rats were given an intra-PL infusion of corticosterone (0.05
µg/.03 µL) instead of EFS 15 min prior to low dose cocaine.
• All drug treatments and reinstatement tests were given in a randomized
order.
• To examine activation of glutamatergic pathway from the PL to the nucleus
accumbens core, rats were given an intra-NAc infusion of CTb prior to
extinction
• Once extinction criteria was met (< 15 lever presses), rats received one of
the following reinstatement tests was performed:
• Vehicle (10% EtOH)/Cocaine (2.5 mg/kg, i.p.)
• CORT/Saline (2 mg/kg, ip)
• Coc (2.5 mg/kg, i.p.)/ Cort(2 mg/kg, ip)
• Vehicle/Saline
• Rats were perfused immediately after the 2 hr reinstatement test.
• Brains were removed, frozen and sectioned to 25 μm
• CTb and c-Fos were visualized using a double-stained
immunohistochemistry using a DAB stain followed by a cell count for CTb
and c-Fos alone or present in the same cell.
Rats undergo self administration for 14 days followed by Cholera Toxin subunit B (CTb) injections into the
nucleus accumbens core and then are given five days to recover. These rats go through extinction and are
subjected to one of four reinstatement conditions. Rats are perfused 90 min after the start of reinstatement
testing and their brains are collected and processed for double immunohistochemistry.
CONCLUSIONS
0
20
40
60
80
Extinction
Reinstatement
SHAM
CocaineLeverResponses
0
20
40
60
80
Extinction
Reinstatement
ADX
CocaineLeverResponses0
20
40
60
80
Extinction
Reinstatement
CocaineLeverResponses
EFS
Reinstatement
Testing
15 min 2 hrs
i.p. cocaine (2.5 mg/kg or
saline)
EFS - - + +
COC - + - +
EFS - - + +
COC - + - +
CORT - - + +
COC - + - +
A B
C
*
*
0
10
20
30
40 Extinction
Reinstatement
CocaineLeverResponses
COC + -
+Intra-PL CORT - +
+
*
Double immunohistochemistry for CTb and c-Fos using a DAB stain from the prelimbic cortex at a 20X magnification. A. Brain tissue of a rat under
corticosterone/ saline reinstatement condition. B. Brain tissue of a rat under vehicle/ cocaine reinstatement condition.
Prelimbic Cortex
Nucleus Accumbens Core
Prelimbic Cortex
Nucleus Accumbens Core
CTb
c-fos
Reinstatement
A B
Research funded by NIDA grant DA038663 & DA15758
References
• Graf EN, Wheeler RA, Baker DA, Ebben AL, Hill JE, McReynolds JR, Robble MA, Vranjkovic O, Wheeler DS, Mantsch JR, Gasser PJ. 2013. Corticosterone acts in the nucleus accumbens to
enhance dopamine signaling and potentiate reinstatement of cocaine seeking. J Neurosci 33(29): 11800-11810.
• Mahler SV & Aston-Jones GS. 2012. Fos activation of selective afferents to ventral tegmental area during cue-induced reinstatement of cocaine seeking in rats. J Neurosci 32(38):
13309-13325.
Self-Ad
14 DAYS
CTb injection
into nucleus
accumbens core
Recovery
5 DAYS
Extinction Reinstatement
40 min 90 min
i.p. cocaine (2.5
mg/kg or saline)
CORT Reinstatement Testing
perfusion
Neurons projecting from the prelimbic cortex (PL) to the nucleus accumbens core
are identified by visualization of the retrograde tracer cholera toxin B (CTb) that
was injected into the nucleus accumbens core. The image is a 20X magnification
from the prelimbic cortex.
REINSTATEMENT
CONDITIONS:
• SALINE ONLY
• COCAINE (COC) ONLY
• CORTICOSTERONE
(CORT) ONLY
• CORT + COC
Fig. 3 Cortico- Accumbens Pathway
Example of outcome from performing a double immunohistochemistry for CTb
and c-Fos using a DAB stain. CTb positive neurons are indicated with the red
arrow. C- Fos positive neurons are indicated by the blue arrow. Neurons that
are positive for CTb and Fos are indicated by the black arrow (40X
magnification).
PL
Representative of the injection of the retrograde tracer, cholera toxin subunit B, into the
nucleus accumbens core.
Nucleus
Accumbens
Core
Nucleus
Accumbens
Shell
Projections to the nucleus accumbens (NAc) core are examined using a double-label immunohistochemical
approach. Cholera Toxin subunit B (CTb) is a retrograde tracer that is injected into the NAc core and will be
identified with a DAB stain. In addition, neuronal activation in the prelimbic cortex will be identified by the
presence of c-Fos, an immediate early gene, in neurons that project to the NAc core and will be identified by
a Ni-DAB stain.
CTb+ Fos+ CTb/Fos+
Corticosterone/ Saline Vehicle/ Cocaine