CREB modulates excitability of nucleus accumbens neurons
Dong et al.
Supplementary On-line Material
ACKNOWLEDGEMENTS
We thank E. Sura and W. Ju for technical assistance and J. Trimmer, R.C. Yu and R. Axel for
providing constructs. This work was supported by grants from NIDA and NIMH.
Methods
Viral vectors.
Recombinant Sindbis pseudovirions expressing caCREB-IRES-GFP (Y134F mutation in
CREB), dnCREB-IRES-GFP (S133A mutation in CREB) or GFP alone were generated as
previously reported1. Viral supernatant was harvested 36 hours after transfection of the BHK
cells and concentrated as described1. A bicistronic HSV amplicon2 was constructed to express
Kir2.1 from an IE4/5 promoter and eGFP from a CMV promoter. The amplicon was packaged
according to published methods3.
NAc slice culture and in vitro viral-mediated gene transfer.
Sprague-Dawley rats (p13-14) were deeply anaesthetized with isoflurane and decapitated.
Coronal NAc slices (200 m thick) were obtained in ice-cold sterile low Ca2+ solution containing
(in mM): 126 NaCl, 1.6 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 0.625 CaCl2, 18 NaHCO3, and 11
glucose, and then placed on Millicell Millipore culture plate inserts in wells containing
Neurobasal-A Media with 4% B-27 and 1% Glutamax-I Supplements (Invitrogen). Concentrated
virus solution (1 μl) was carefully dropped onto the area of the slices containing the NAc 1-3
hours after their preparation. Electrophysiology experiments were performed 15-20 hours after
virus infection.
Cocaine administration.
A modified “binge” cocaine administration protocol was used4. Briefly, male rats (p2127) were allowed to acclimate to their home cages for 2 days and then received 3 daily injections
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(separated by 1 hour) of either cocaine (15 mg/kg i.p.) or the same volume of saline for three
consecutive days. The in vivo injection of virus was performed one day later and the
electrophysiology was performed 15-20 hours after virus injection. At this time point, cocainetreated animals exhibit sensitized locomotor responses to cocaine4,5.
In vivo viral injections
Sprague-Dawley rats (p24-27) were anesthetized and prepared for sterotaxic injection
according to standard procedures (approved by IACUCs of Stanford University and U.T.
Southwestern). Briefly, animals were anesthetized with a mixture of ketamine (50 mg/kg i.p.)
and xylazine (4.5 mg/kg i.p.). After immobilization on a stereotaxic instrument, a hole was
drilled (1 mm diameter) at 3 mm anterior and 1.5 mm lateral to bregma for injection into the
NAc shell. Using a stainless steel cannula (Plastic One) at a depth of 6.4 mm, viral solution (0.5
μl) was injected using a Harvard Apparatus pump at a flow rate of 0.1 μl/min.
Electrophysiological or behavioral experiments were performed 15-40 hours later. Acute NAc
slices were prepared as previously described6.
Electrophysiology.
Slices were transferred to a holding chamber containing artificial cerebrospinal fluid
(ACSF, in mM: 126 NaCl, 1.6 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 18 NaHCO3, and 11
glucose) and equilibriated at 31–34°C for at least 1 hour. All solutions were bubbled with 95%
O2/5% CO2 and perfused over the slice at a rate of 2.5 ml/min. Cells were visualized with an
upright microscope using infrared or epifluorescent illumination, and whole-cell voltage-clamp
recordings were made with an Axopatch 1D amplifier (Axon Instruments). Electrodes (2–6 MΩ)
contained in mM: 130 K-methansulfate, 10 KCl, 10 HEPES, 0.4 EGTA, 2.0 MgCl2, 2.5 MgATP,
0.25 Na3GTP (pH 7.2–7.4; 275–285 mOsm). Some cells were filled with Alexa 568 (0.2 mg/ml,
Molecular Probes, OR).
Whole-cell current clamp recording, as previously described7, was used for all electrophysiology
experiments. Neurons located in the NAc shell (ventral-medial area) were preferentially selected
for recording. For experiments illustrated in Figures 2a and b, Ca2+ channels were blocked by
adding Cd2+ (0.4 mM) to the ACSF; Na+ channels were blocked by adding TTX (1 μM) to the
ACSF; K+ channels were blocked by adding TEA (10 mM) and 4-AP (3 mM) to the ACSF and
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using a Cs+ based pipette solution (in mm: 117 Cs-methanesulfate, 20 HEPES, 0.4 EGTA, 2.8
NaCl, 5 TEA-Cl, 2.5 MgATP, 0.25 Na3GTP [pH 7.2–7.4; 275–285 mOsm]). The resting
membrane potential was maintained at -70 mV for all experiments except those illustrated in
Figure 2a during which membrane potential was maintained at –90 mV. Some recordings
(~20%) were made in the presence of NBQX (5 μM), D-AP5 (50 μM) and picrotoxin (100 μM)
to block fast excitatory and inhibitory synaptic transmission. There was no difference in action
potential threshold and firing pattern in the absence and presence of these drugs and therefore
data using the two different ACSF solutions were combined.
Locomotor activity.
Male Sprague-Dawley rats (Harlan, Houston), 250-300 g were used for behavioral
experiments. Rats were housed on a 12/12 hr light/dark schedule and testing was conducted
during the latter half of the light phase. All animals were housed in an AAALAC accredited
facility and procedures were in accordance with the NIH Guide for the care and use of laboratory
animals.
Locomotor activity was measured in a circular corridor (10 cm wide, 60 cm in diameter
and 30 cm high; Med-Associates, St. Albans VT) as described8. Horizontal ambulatory activity
registered via 4 photoelectric cells located every 90 along the circle. Rats were allowed to
habituate to the chambers for 120 min on post-surgical days 2 and 3. To acclimate rats to the
injection procedure, saline was injected (1 ml/kg) 60 min into the session on each day and
locomotor activity measured for the ensuing 60 min. On post-surgical day 4, animals received
either saline or cocaine (15 mg/kg).
Statistical analysis.
Over 70% of the data were collected and analyzed without knowledge of the molecular
manipulation the slices/animals had received. There were no differences in the results from
blinded and nonblinded experiments and therefore results were combined. Recordings from
control, GFP-expressing, and uninfected neurons were interleaved throughout the course of all
experiments. The controls presented in each bar graph represent the cells obtained during the
time period when the experimental manipulations shown in that graph were performed. All
values are expressed as mean + SEM. Statistical significance was assessed using ANOVA for
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evoked action potential firing, I-V relationships, and K+-dependent voltage traces (Fig. 2h), and
2-tailed t-test for all other experiments.
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