Supplementary Methods
Homecage CORT in the drinking water.
Stress exposure leads to the secretion of glucocorticoids (cortisol in the human; corticosterone (CORT) in the rodent). CORT was administered in the drinking water for 1 week as a noninvasive method to induce repeated and heightened elevations in glucocorticoids in intact (e.g., non-adrenalectamized) animals . An advantage of this method is that CORT dose can be determined from the daily fluid consumption, allowing for potential comparisons and increased replicability between laboratories in future work (e.g., the effects of restraint stress exposure can differ dramatically between labs).
Immunohistochemistry (IHC) procedure and quantification (mGluR5 and c-Fos).
Rats were anesthetized with pentobarbital and perfused with 0.1 M PBS, followed by 4% paraformaldehyde, 4°C; pH=7.4. The brains were removed from the skull and placed in the same fixative solution for approximately 24 h before being washed with PBS and sliced coronally on a vibratome into 40 µm sections. Tissue was stored (-20°C) in cryoprotectant until immunohistochemistry processing. Free-floating sections were blocked in 10% goat serum and
0.1% Triton X-100 in PBS, and then incubated in 3% goat serum, 0.1% Triton X-100 in PBS and rabbit anti-c-Fos antibody (1:20,000 dilution, Oncogene Research Products/Calbiochem) for 48 h or rabbit anti-mGluR5 (1:1000 dilution, Millipore) for 16 h at 4°C with agitation. For the c-Fos
IHC, sections were next incubated in biotinylated secondary anti-rabbit antibody for 1 h and then processed with avidin-biotin complex (Vector ABC kit, Vector Laboratories, USA) and placed in diaminobenzidine solution (Polysciences) containing 0.006% hydrogen peroxide, 0.005% cobalt, and 0.0075% nickel in order to visualize c-Fos immunoreactivity (IR). For the mGluR5 IHC, sections were next incubated in secondary antibody for 1 h using the Dako EnVision Kit (Dako,
Carpinteria, CA). Immunoreactivity (IR) was detected with nickel-enhanced diaminobenzidine (c-
Fos: Polysciences; mGluR5: Dako EnVision Kit) as a chromagen. IR was visualized using Olympus CX41 (Experiment 1) and BX51 (Experiment 2) light microscopes (Olympus America,
Center Valley, PA) and image analysis software (Bioquant Nova Advanced Image Analysis;
R&M Biometric, Nashville, TN). For consistency of staining across subjects, brain tissue for each antibody was processed simultaneously.
IR was visualized using Olympus CX41 (Experiment 1) and BX51 (Experiment 2) light microscopes (Olympus America, Center Valley, PA) and image analysis software (Bioquant
Nova Advanced Image Analysis; R&M Biometric, Nashville, TN). The microscope, camera, and software were background corrected and normalized to preset light levels to ensure fidelity of data acquisition. Analysis was conducted by a researcher blind to treatment conditions.
Western blot procedure and quantification (mGluR5).
Rats were anesthetized, decapitated, the brains immediately extracted and placed in isopentane. Brains were rapidly removed and flash frozen in isopentane (-40° C) and stored at
-80° C until analysis. Brain tissue was submerged in buffer (10mM Tris, 1% SDS, 1:100
Protease and Phosphatase Inhibitors) and homogenized (Branson Sonifier, Danbury, CT). After colorimetric detection and quantification (Pierce Biotechnology, Rockford, IL) of protein concentration, protein (10µg) was diluted with Sample Reducing Agent and LDS Sample Buffer
(Invitrogen, Carlsbad, CA), boiled, and loaded onto TGX (4-15%) gels (Biorad, Hercules CA) for separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Transfer of protein to a PVDF membrane (Invitrogen) occurred via dry blotting using iBlot
(Invitrogen, Carlsbad, CA). Membranes were blocked in 3% normal goat serum (Vector
Laboratories, Burlingame, CA) before overnight incubation at 4○C in polyclonal rabbit anti- mGluR5 (Millipore; 1:1000) and monoclonal mouse actin (Millipore; 1:5000) in blocking solution.
Following incubation, membranes were washed and incubated 1h at room temperature in hrp- conjugated goat anti-rabbit and anti-mouse secondary antibodies, respectively (Jackson
Immunoresearch Labs, Inc.; 1:10,000). Alcohol Discrimination Training and Testing Procedures.
Training. Alcohol (1 g/kg) or water was administered IG before daily training sessions
(M-F). Rats had a 10-min delay period prior to the start of the 15-min session at which time the house light was illuminated and both levers were introduced. On an alcohol training day, completion of an FR10 on the alcohol-appropriate lever (e.g., left lever) resulted in the presentation of the sucrose solution. On a water training day, completion of 10 responses on the water-appropriate lever (e.g., right lever) resulted in sucrose delivery. Responses on the inappropriate lever were recorded, but produced no programmed consequences.
Testing. Test sessions were similar to training sessions except that they were 2-min in duration (after 10-min delay), and 10 responses on either lever resulted in sucrose delivery.
Cumulative dosing procedures were used as indicated in the Experiments. To determine a cumulative alcohol dose response curve (0.1, 0.3, 1.0, 1.7 g/kg) the rat initially received 0.1 g/kg alcohol and was placed in the chamber for a 10-min delay period followed by a 2-min test session. At the end of that session, the rat immediately received a subsequent alcohol administration of 0.2 g/kg (0.3 g/kg cumulative dose) and another delay/test session. This procedure was repeated with two subsequent administrations of 0.7 g/kg alcohol, which are additive to produce the stated dose range. This procedure allows for the entire alcohol dose curve to be determined in approximately 48 min. The 1.0 g/kg alcohol dose (i.e., training dose) which generally produces greater than 90% alcohol-appropriate responding during regular training sessions, tends to produce about 60-70% alcohol-appropriate responding under the cumulative dosing procedure . For the studies in Experiment 4 and 5, a baseline cumulative alcohol session was conducted prior to the initiation of the 7-day CORT or Water procedure
(see Tables 2 and 3 and Figures 4 and 6).
Cannulae Implantation Surgery and Microinjection Procedures. Rats were anesthetized with an isoflurane/oxygen combination and received bilateral implantation of 26-gauge guide cannulae (Plastics One Inc., Roanoke, VA) aimed to terminate 2 mm above the nucleus accumbens core. Rats were given at least 1 week of recovery, after which discrimination training resumed. Bilateral injections were made with Hamilton syringes
(Hamilton, Reno, NV) connected to 33-gauge injectors (Plastics One) that extended 2 mm below the guide cannulae. A pump (Harvard Apparatus, model 22, Natick, MA) was used to deliver a volume of 0.5 µl into each side over 1 min; the injectors were left in place for another
1.5 min after the injections to allow for diffusion.
Cannulae verification. At the end of the experiment, rats were deeply anesthetized with pentobarbital and transcardially perfused with 0.1 M buffered saline (PBS) followed by 4% formaldehyde (pH=7.4). The brains were sliced into 40-μm coronal sections and stained with cresyl violet. Cannulae placement was verified using an Olympus CX41 light microscope
(Olympus America, Center Valley, PA). Only data from rats with cannulae determined to be in the target brain regions were used in the analyses and the sample sizes reported in the experiments are in accordance with the analyses.
Supplementary Results
Experiment 3: Effect of mGluR5 positive allosteric modulator on alcohol sensitivity.
Two different groups of rats were used for this assessment (one to test the low CDPPB dose range (Low – Vehicle, 1, 10 mg/kg) and the other to test the high CDPPB dose range (High –
Vehicle, 20, 40 mg/kg). The data from the two groups was analyzed by separate two-way repeated measures ANOVAs. There were no significant differences between the vehicle pretreatment conditions of the two groups in relation to alcohol-appropriate responses and response rates (compared by two-way ANOVA with alcohol dose as a repeated factor), and therefore, for ease of presentation, the data are graphically presented together (Figure 3). Experiment 5: Effect of intra-accumbens mGluR5 agonist on alcohol sensitivity following CORT exposure.
Intra-accumbens administration of CHPG was tested to determine whether mGluR5 activation at the doses found to restore sensitivity to alcohol would potentiate the discriminative stimulus effects of a low alcohol dose (0.3 g/kg, IG) under “normal” conditions (i.e., absence of CORT).
The following are the percent alcohol-appropriate responses reported as mean±SEM: aCSF –
33.88±16.83; 5 µg CHPG – 37.43±16.82; 10 µg CHPG – 40.75±16.61. The percentage of alcohol-appropriate responding at the dose selected (0.3 g/kg, IG) after aCSF injection was similar to that observed following CORT exposure in the CORT assessment of this experiment
(34% and 40%, respectively). CHPG pretreatment did not alter sensitivity to alcohol (0.3 g/kg,
IG) as measured by alcohol-appropriate responding and did not alter response rate (aCSF –
47.06±2.25; 5 µg CHPG – 49.36±5.11; 10 µg CHPG – 41.50±6.05. These findings are consistent with previous work in which intra-accumbens administration of 10 µg CHPG did not alter the discriminative stimulus effects of the same alcohol dose (0.3 g/kg, IG) . However, in that work CHPG treatment potentiated the discriminative stimulus effects of a higher (0.5 g/kg) alcohol dose .
Supplementary Discussion
In general CDPPB (positive allosteric modulator) and CHPG (orthosteric agonist) had similar functional behavioral effects – both restored sensitivity to alcohol following CORT exposure.
Unlike agonists that compete with the endogenous ligand (glutamate in this case), positive allosteric modulators act through allosteric sites on the receptor (distinct from the orthosteric site). Consequently, positive allosteric modulators can functionally enhance the effects of the endogenous ligand and tend to be devoid of activity in the absence of the endogenous ligand
(see . Therefore, it is interesting and important that both compounds restored sensitivity to alcohol as such a finding further supports the role for mGluR5.
Supplementary Table 1. Experiments 1 and 2: Mean (±S.E.M.) daily fluid consumption and CORT dose consumed for each group during CORT/Water exposure. Daily fluid Daily CORT consumption dose consumed (ml) (mg/kg) Experiment 1 – IHC groups
Water 19.1±0.8 n/a
CORT 20.0±0.9 21.5±0.9
Experiment 1- WB groups
Water 23.9±1.0 n/a
CORT 27.5±2.0 29.3±2.6
Experiment 2
WATER
Supplementary Table 2.Vehicle Experiment29.9 4±:1.2 Mean (±S.E.M.)n/a baseline alcohol discrimination performance for each group prior to CORT/Water exposure and daily consumption measures during CORT/Water exposure. Alcohol-appropriate Responses (%) Response Rate (resp/min) Daily CumulativeAlcohol Alcohol26.7± 1.5Dose (g/kg, IG)n/a Cumulative Alcohol Dose (g/kg, IG) Daily fluid CORT consumption dose CORT (ml) (mg/kg) WATER 0.1 0.3 1.0 1.7 0.1 0.3 1.0 1.7 Vehicle 6.1±2.3 Vehicle27.8±10.630.9±80.1±7.01.9 29.887.3±5.0±1.6 75.1±7.0 68.1±5.2 60.5±2.8 50.1±5.0 21.5±1.4 n/a 10 CDPPB 7.2±3.3 18.7±9.1 55.8±12.6 97.5±1.3 65.0±2.4 58.8±2.8 56.5±5.3 52.6±5.0 22.9±1.3 n/a 20 CDPPB 6.6±4.4 Alcohol5.4±4.131.3±54.3±17.51.3 29.798.7±1.3±1.0 62.1±3.3 61.8±4.5 58.9±7.1 53.1±5.5 23.5±1.5 n/a CORT Vehicle 10.2±2.3 26.9±9.5 61.6±11.5 84.4±9.0 74.7±3.9 65.6±1.4 63.5±3.1 57.3±3.3 25.8±1.6 23.5±1.6 10 CDPPB 7.2±3.3 18.7±9.1 55.8±12.6 97.5±1.3 65.0±2.4 58.8±2.8 56.5±5.3 52.6±5.0 28.3±2.1 26.0±2.1 20 CDPPB 4.2±3.3 11.2±8.3 71.1±9.8 82.6±10.6 62.2±2.8 60.0±1.5 57.4±3.4 47.3±4.2 22.2±1.2 20.1±1.0 Supplementary Table 3. Experiment 5: Mean (±S.E.M.) baseline alcohol discrimination performance for each group prior to CORT/Water exposure and daily consumption measures during CORT/Water exposure. Daily Daily fluid CORT Alcohol-appropriate Responses (%) Response Rate (resp/min) consumption dose Cumulative Alcohol Dose (g/kg, IG) Cumulative Alcohol Dose (g/kg, IG) (ml) (mg/kg) WATER 0.1 0.3 1.0 1.7 0.1 0.3 1.0 1.7 aCSF 16.8±4.4 37.0±13.5 66.9±17.5 95.1±3.1 55.4±2.8 56.4±3.0 61.2±4.7 54.6±5.5 21.1±1.0 n/a 5 CHPG 7.9±5.8 19.6±12.4 62.6±15.7 87.9±11.0 52.9±4.0 44.6±6.7 51.5±3.8 47.1±5.6 22.0±2.0 n/a 10 CHPG 11.0±5.2 30.6±13.3 83.3±12.2 94.9±2.9 68.6±4.1 60.2±6.8 65.2±3.9 58.9±4.7 21.3±1.1 n/a CORT aCSF 16.9±7.4 22.5±14.1 63.4±15.0 86.9±5.9 57.0±5.1 53.6±3.2 53.3±2.8 52.2±1.6 21.8±1.6 20.2±1.5 5 CHPG 12.4±4.3 8.1±5.7 84.3±12.2 87.5±11.3 47.1±6.2 48.8±4.6 49.4±3.5 38.8±7.1 22.9±1.4 20.9±1.3 10 CHPG 10.2±4.7 21.3±10.6 66.6±13.7 89.6±5.9 61.7±3.6 58.4±4.2 56.1±5.2 51.4±5.9 22.3±1.2 20.2±1.2
a b 100 Pre-Water 80
Pre-CORT ) n i
m e
80 / t ) p 60 a i s r % e ( p r
( o s
r 60 e e p t s p a 40 n A R o -
l p 40 e o s s h e n o R o c
l 20 p A
20 s e R 0 0 0.1 0.3 1.0 1.7 0.1 0.3 1.0 1.7 Cumulative Alcohol Dose (g/kg, IG) Cumulative Alcohol Dose (g/kg, IG)
Supplementary Figure 1. Similar baseline discrimination performance prior to
CORT/Water exposure for the rats in Experiment 4. (a) Baseline discrimination performance collapsed across Water or CORT exposure and (b) response rate were similar prior to the initiation of the 7-day CORT/Water exposure. Horizontal dashed line (>80%) represents full expression of the discriminative stimulus effects of alcohol. Values on graphs represent mean ± s.e.m. a b 100 Pre-Water 80
Pre-CORT ) n i
m e 80 / t ) p
a 60 i s r % e ( p r
( o s
r 60 e e p t s p a
n 40 A R o -
l p
40 e o s s h e n o R o c
l 20 p A
20 s e R 0 0 0.1 0.3 1.0 1.7 0.1 0.3 1.0 1.7 Cumulative Alcohol Dose (g/kg, IG) Cumulative Alcohol Dose (g/kg, IG)
Supplementary Figure 2. Similar baseline discrimination performance prior to
CORT/Water exposure for the rats in Experiment 5. (a) Baseline discrimination performance collapsed across Water or CORT exposure and (b) response rate were similar prior to the initiation of the 7-day CORT/Water exposure. Horizontal dashed line (>80%) represents full expression of the discriminative stimulus effects of alcohol. Values on graphs represent mean ± s.e.m. Supplementary References
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