Supplemental Information
Glutamatergic Mechanisms of Comorbidity Between Acute Stress and Cocaine
Self-administration
Constanza Garcia-Keller, Yonatan Kupchik, Cassandra D Gipson, Robyn M Brown, Sade
Spencer, Flavia Bollati, Maria A Esparza, Doug Roberts-Wolfe, Jasper Heinsbroek, Ana-Clara
Bobadilla, Liliana M Cancela and Peter W Kalivas.
Supplemental Figure 1
Acute stress-induce increase in dendritic spine density in the NAcore.
a-d)
Representative examples of dendritic spine measurements from medium spiny
neurons in the NAcore. Panels a and b show neurons from a sham rat and stress rat,
respectively, and panels c and d show the corresponding magnification of the
indicated dendritic segment. Bar in panels a, b= 50 µm, and in panels c, d= 10 µm.
e) Stress induced an increase in spine density on MSNs in NAcore compared with
sham. Student’s t-test t(65)= 2.13, * p< 0.05. f) Stress did not affect spine head
diameter. N is shown in the graph legend.
Supplemental Experimental Procedures
Animal Housing and Stress. Male Sprague-Dawley rats (250 g; Charles River
Laboratories) were double housed with a 12:12 hr dark/light cycle. The animals
were approximately 2 month old (± 1 week). All experimentation occurred in the
light cycle. Rats received food and water ad libitum and were allowed at least 1
week to acclimate to the vivarium before any treatment. The acute stress group was
restrained for 2 hours (anytime between 10:00 and 14:00 h) in restraining devices,
while sham animals were left undisturbed in their home cages. The Plexiglas
cylinders were designed so that the rats’ tails emerged from the rear. The animals
appeared healthy as shown by their coat texture and no difference in body weight
was detected between sham and stress exposed rats at the time animals were used
for behavior or sacrificed for the different measurements. All procedures were in
accordance with the NIH Guide for the Care and Use of Laboratory Animals and the
Assessment and Accreditation of Laboratory Animal Care.
Measurement of Dendritic Spine Morphology. As previously described1, NAcore was
labeled with DiI in coronal slices that were imaged on an LSM 510 Zeiss confocal
microscope, and analyzed using the IMARIS software package (Bitplane, St Paul,
MN). Dendritic segments analyzed were 45-55 microns in length, chosen to be
between 75 and 200 microns from the soma and distal to the first branch point. 810 segments (1 segment per neuron) were analyzed from 4 animals in each group
(sham and stress).
Statistics. All statistical analyses were performed using Graphpad Prism 6.0. Twotailed unpaired t-tests and 1- or 2-way ANOVAs were used as all data were normally
distributed according to a D’Agostino-Pearson omnibus normality test. The spine
morphology experiments were performed with experimenter blinded to condition.
No randomization was used. Statistical tests is indicated in the text or figure legend.
All experiments were replicated at least twice. Total number of animals per group
was determined by an a priori power analysis conducted to engender 95% power
assuming a moderate effect size (0.5).
Supplemental References
1.
Shen HW, Toda S, Moussawi K, Bouknight A, Zahm DS, Kalivas PW. Altered
dendritic spine plasticity in cocaine-withdrawn rats. J Neurosci 2009; 29(9):
2876-2884.