SUPPLEMENTARY MATERIALS
Antidepressant-like and anxiolytic-like effects following activation
of the - opioid receptor heteromer in the nucleus accumbens
Kabli, Nguyen, Balboni, O’Dowd and George
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LIST OF SUPPLEMENTARY MATERIALS
Figure S1. Effect of the OR carboxyl tail peptide minigene on -agonist SNC80 modulation of
µ-agonist binding to, and internalization of, the -OR heteromer.
Table S1. Effect of the OR carboxyl tail peptide minigene on -agonist binding to the receptor ligand binding pocket within the -OR heteromer.
Table S2. Effect of the OR carboxyl tail peptide minigene on - and -agonist binding to the
-receptor ligand binding pocket in the OR homomer or on -agonist binding to the -receptor
ligand pocket within the OR homomer.
Figure S2. Cannula placement in the rat nucleus accumbens core
Figure S3. Dose response curves of UFP-512’s antidepressant-like effects in the forced swim
test following intra-accumbens administration.
Figure S4. Dose response curves of UFP-512’s anxiolytic-like and antidepressant-like effects in
the novelty-induced hypophagia test following intracerebroventricular administration.
Figure S5. Effect of the -agonist UFP-512 on analgesia in the tail immersion nociceptive assay.
Figure S6. Effect of the -agonist UFP-512 or TAT-conjugated peptides on locomotor activity.
Supplementary Methods
References
2
Supplemental Figure 1. Effect of the OR carboxyl tail peptide minigene on -agonist
SNC80 modulation of µ-agonist binding to, and internalization of, the -OR heteromer.
Binding (A-C): SNC80 -agonist detection of the  ligand binding pocket using the µ-agonist
[3H]-DAMGO in cells co-expressing - and -OR in the absence or presence of the interfering
(A) or control peptide minigene (B), or in cells expressing OR alone (C). Grey and black
arrowheads point to the high and low ligand binding affinities, respectively. An F-test was used
to compare the coefficients of the goodness-of-fit and to determine whether a two-site (biphasic
competition curve with high and low affinity sites) or a one-site (monophasic competition curve
with one high or low affinity site) analysis was a statistically significant better fit for the
radioligand competition binding curves. Results shown are mean + S.E.M. and curves are
representative of n = 3-6 experiments performed in duplicate. Internalization (D): SNC80 agonist-induced internalization of cell surface OR in cells co-expressing - and -ORs in the
absence and presence of the interfering peptide minigene or pre-treatment with either the antagonist naltrindole (NALT) or the µ-antagonist CTOP, or in cells expressing OR alone.
Agonist-induced reduction in [3H]-DAMGO labeling in intact HEK 293T served as an index of
OR internalization. Data shown represent drug-induced loss of cell surface receptors as a
percentage of cell surface receptors in vehicle-treated control cells, and are expressed as mean +
S.E.M. for n = 3-4 experiments performed in triplicate.
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Supplemental Table 1. Effect of the OR carboxyl tail peptide minigene on -agonist modulation of µ-agonist binding to the
-OR heteromer
-OR
KH (nM)
KL (nM)
% HA
85 + 21
24 + 2.0
UFP-512 0.5 + 0.1
SNC80 12 + 2.4 27,374 + 6775 45 + 2.9
-OR + PEP
OR
KI (nM)
KI (nM)
79 + 7.2
16,809 + 4125
-OR + CTRL PEP
KH (nM)
KL (nM)
% HA
83 + 3.1
0.7 + 0.2
77 + 17
22 + 3.7
13,111 + 1835 11 + 3.1 15,056 + 4981 44 + 7.6
-agonist detection of the  ligand binding pocket in cells co-expressing - and -OR in the absence or presence of the inhibitory or
control peptide minigene, or in cells expressing OR alone. Competition radioligand binding was performed using [3H]-DAMGO.
Values shown represent mean + S.E.M. of n = 3 – 6 experiments performed in duplicate. KI = agonist-detected affinity site binding
constant, KH = agonist-detected high affinity site binding constant, KL = agonist-detected low affinity site binding constant, % HA =
percentage of receptors in the agonist-detected high affinity state. KI values are provided when single affinity binding sites are
detected. An F-test was used to compare the coefficients of the goodness-of-fit and to determine whether a two-site (high and low
affinity sites) or a one-site (one high or low affinity site) analysis was a statistically significant better fit for the radioligand
competition binding curves.
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Supplemental Table 2. Effect of the OR carboxyl tail peptide minigene on - and -agonist modulation of µ-agonist binding
to the µOR homomer and -agonist binding to the OR homomer.
OR
OR + PEP
KI (nM)
KI (nM)
UFP-512 83 + 3.1
DAMGO 7.4 + 0.2
82 + 10
7.7 + 0.2
OR
KH (nM)
KL (nM)
OR + PEP
% HA
KH (nM)
KL (nM)
% HA
0.7 + 0.1 781 + 18 58 + 2.0 0.6 + 0.1 495 + 186 50 + 4.3
n/a
n/a
n/a
n/a
n/a
n/a
Agonist detection of the  or  ligand binding pocket in OR- or OR-expressing cells, respectively, in the absence or presence of the
inhibitory peptide minigene. Competition radioligand binding was performed using [3H]-DAMGO in cells expressing µOR or [3H]diprenorphine in cells expressing OR. Values shown represent mean + S.E.M. of n = 3 experiments performed in duplicate. K I =
agonist-detected affinity site binding constant, KH = agonist-detected high affinity site binding constant, KL = agonist-detected low
affinity site binding constant, % HA = percentage of receptors in the agonist-detected high affinity state. K I values are provided when
single affinity binding sites are detected. An F-test was used to compare the coefficients of the goodness-of-fit and to determine
whether a two-site (high and low affinity sites) or a one-site (one high or low affinity site) analysis was a statistically significant better
fit for the radioligand competition binding curves.
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Supplemental Figure 2. Cannula placement in the rat nucleus accumbens core. Schematic
representation of micro-injection sites into the nucleus accumbens core (filled circles). Each dot
represents a cannula placement in the nucleus accumbens; one dot is used despite overlap in the
location of the cannulae. Co-ordinates are relative to Bregma according to the Atlas of Paxinos
and Watson (1).
Supplemental Figure 3. Dose response curves of UFP-512’s antidepressant-like effects in
the forced swim test following intra-accumbens administration. Rats were micro-injected
bilaterally into the NAc with either saline or various doses of UFP-512. Rat behavior was
scored, whereby the predominant behavior for each interval was recorded according the Lucki
method (2). Immobility represented no activity except that which is required to keep the rat’s
head above water. Statistical significance was determined using one-way ANOVA followed by
Dunnett post-hoc analysis [*** p<0.001 relative to saline-treated rats; n = 3 – 11 animals per
group].
Supplemental Figure 4.
antidepressant-like
effects
Dose response curves of UFP-512’s anxiolytic-like and
in
the
novelty-induced
hypophagia
test
following
intracerebroventricular administration. Rats were micro-injected into the lateral ventricle
with either saline or various doses of UFP-512. The latency to drink from palatable milk
solution in a novel and stressful environment (a measure of anhedonia) was used as an index of
anhedonia. Statistical significance was determined using one-way ANOVA followed by Dunnett
post-hoc analysis [* p<0.05 *** p<0.001 relative to saline-treated rats; n = 3 – 4 animals per
group].
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Supplemental Figure 5. Effect of the -agonist UFP-512 on analgesia in the tail immersion
nociceptive assay. Rats received bilateral intra-accumbens micro-injections of saline or UFP512 and the latency to tail withdrawal from warm water (52oC) was measured at baseline (BL)
and following saline or drug injection. A cut-off of 4 x BL latency was imposed to minimize
tissue damage. % Maximum Possible Effect was calculated according to the equation: %
Maximum Possible Effect = (Latency - BL latency) / (Cut-off Latency – BL latency) x 100.
Statistical significance was determined using one-way ANOVA for n = 5 – 6 animals per group.
Supplemental Figure 6. Effect of the -agonist UFP-512 or TAT-conjugated peptides on
locomotor activity. Rats received bilateral intra-accumbens micro-injections of saline, UFP512, TAT-conjugated interfering peptide (PEP) or scrambled peptide (PEP SCR). Locomotor
behaviour was assessed using automated activity monitors equipped with motion sensors. The
total horizontal distance traveled was used as an index of locomotor activity. Statistical
significance was determined using one-way ANOVA followed by Dunnett post-hoc analysis for
n = 5 – 9 animals per group.
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SUPPLEMENTARY METHODS
Verification of Cannula Placement: Rats were perfused transcardially with saline followed by
10% formalin fixative under brief isoflurane anesthesia. The brain was harvested and post-fixed
in fixative solution overnight. Brain samples were then transferred to 30% sucrose in 0.1 M
phosphate buffer (pH 7.4) for 48 hours and stored at 4oC. Brain samples were subsequently
frozen at -70oC and sectioned into 20 µm slices using a cryostat. Sections were collected onto
gelatin-coated slides and allowed to dry. Slides were viewed on the LSM 510 Zeiss confocal
microscope (Carl Zeiss, Toronto, ON, Canada).
Tail Immersion Antinociception Assay: Rats were restrained gently and the distal 5 cm of
their tail dipped in a bath of water maintained at 52oC. The latency to tail withdrawal from water
was measured. Three pre-drug baseline (BL) measures were obtained and averaged. A cut-off
of 4 x BL latency was imposed to minimize tissue damage. Rats were habituated to the tail
immersion assay before testing. % Maximum Possible Effect was calculated according to the
equation: % Maximum Possible Effect = (Latency - BL latency) / (Cut-off Latency – BL
latency) x 100.
Locomotor activity: Locomotor activity was assessed using automated activity monitors
equipped with motion sensors (Accuscan Instruments, Columbus, OH, USA). Rats were placed
in activity monitor chambers (45 cm x 45 cm) and their movement monitored for 60 min. Total
horizontal distance travelled was used as an index of locomotor activity.
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REFERENCES
1.
2.
Paxinos G, Watson CR. The Rat Brain in Stereotaxic Coordinates. Fourth ed. New York:
Academic Press; 1998.
Lucki I, Singh A, Kreiss DS. Antidepressant-like behavioral effects of serotonin receptor
agonists. Neurosci Biobehav Rev 1994;18(1):85-95.
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