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Experiment 1: Contribution of NMDAR subunits in formation of Fix-C and Esc-C memory

To determine whether NMDAR subunits were differentially regulated in mice
conditioned by Fix-C and Esc-C (Itzhak & Anderson, 2012) we performed quantitative
real-time polymerase chain reaction (qPCR) and immunoblot analyses (n=3-4
mice/group). Bilateral hippocampus was dissected 24h after conditioning and
subsequently analyzed. In the hippocampus NR2A and NR2B are the predominant NR2
subunits that comprise the NMDAR (Monyer et al., 1994). The NMDAR subunits NR1,
NR2A and NR2B are encoded by the genes Grin1, Grin2a and Grin2b, respectively. We
focused on the hippocampus because of its role in spatial/contextual memory.
Experimental groups for Fix-C and Esc-C included the following: Coc-Paired: saline was
given in one compartment at mornings and cocaine in the other compartment 3h later. Coc-
Unpaired: saline was given in one compartment at mornings and 3h later mice were reexposed
to the conditioning apparatus in the absence of drug; cocaine was administered
(30min later) in the home cage. Saline-Paired: saline given in both compartments. For
qPCR the saline, paired and unpaired (Fix-C and Esc-C) groups were analyzed whereas for
western blot analyses, only the saline controls and paired groups of Fix-C and Esc-C were
analyzed.
qPCR
Twenty-four hours following conditioning sessions, mice were tested for CPP and
were sacrificed 20 min later. Bi-lateral hippocampus was dissected and stored in RNAlater
(Qiagen). Equal amounts of total RNA were reverse-transcribed and subjected to qPCR
analysis (n=4 mice/group). Custom designed qPCR arrays (Qiagen) were used to evaluate
changes in gene expression profiles in response to different experimental conditions.
Cycle threshold (Ct) values were used to compare differences in expression levels among
the groups. Since saline groups from both Fix-C and Esc-C experiments received the same
treatment and there was no significant difference between them, they were combined to
serve as control.

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