Supplementary Methods:
Mice:
Inbred strains C57BL/6J (B6) and BTBR T+ tf/J (BTBR) were purchased from The
Jackson Laboratory (Bar, Harbor, ME) and bred in a conventional mouse vivarium at the
National Institute of Mental Health (NIMH), Bethesda, MD, using harem breeding trios. Male
and female mice were used in all studies in approximately equal proportions. Experiments were
conducted in dedicated behavioral testing rooms during the standard light phase, usually
between 1000 and 1500 hr. All procedures were conducted in strict compliance with the NIH
Guidelines for the Care and Use of Laboratory Animals and approved by the National Institute of
Mental Health Animal Care and Use Committee.
Drug Administration:
MPEP (10.0 and 30.0 mg/kg, Sigma Aldrich, St. Louis, MO) was dissolved in saline
(0.9% NaCl). Risperidone (0.125, 0.250, and 0.500 mg/kg, Sigma Aldrich, St. Louis, MO) was
dissolved in saline (0.9% NaCl) containing 1% acetic acid. Adult male and female B6 and
BTBR mice weighing 25-40 grams received an intraperitoneal (i.p.) injection of MPEP, its saline
vehicle, risperidone, or its saline and acetic acid vehicle, at an injection volume of 10 ml/kg, 30
minutes before the start of behavioral test sessions for the open field behavioral task. Previous
behavioral data and in vivo receptor occupancy studies (Anderson et al, 2003; Carvalho et al,
2003; Yan et al, 2005) were used to design the 30 minute post treatment interval for both MPEP
and risperidone Testing was conducted at ages 8-10 weeks. Drug doses were coded to
ensure that the raters were blind to the treatment condition.
Open Field Locomotion:
Individual mice were placed in a VersaMax Animal Activity Monitoring System
(AccuScan Instruments, Columbus, OH, USA) for a 30-minute test session. The testing room
was illuminated with overhead lighting at ~ 200 lux. The chambers consisted of clear Plexiglas
sides and floor, approximately 40 x 40 x 30.5 cm. Mice were placed in the center of the open
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field at the initiation of the testing session. Photocells at standard heights for recording
horizontal activity were aligned 8 to a side, dividing the chamber into 64 equal squares. Vertical
activity was assessed by an additional 8 photocells located above the horizontal photocells.
Horizontal activity, total distance, vertical activity, and center time were automatically collected
using the Versamax activity monitor and analyzer software system.
Supplementary Results:
Figure S1 illustrates the four parameters assessed in the open field exploratory
locomotion task in B6 following an injection of the two higher doses of MPEP or vehicle.
Horizontal activity over the 30 minute test period was reduced, as expected, representing
habituation to the novel open field (Figure S1 Panel A; F (5, 21) = 115.47, p < 0.001). Horizontal
activity did not differ across B6 groups treated with vehicle, MPEP 10 mg/kg, and MPEP 30
mg/kg (F (2, 21) = 0.86, p > 0.05). The time course for total distance traversed in the novel open
field over a 30 minute time period was highly significant, as expected, representing habituation
to the novel open field (Figure S1 Panel B; F (5, 20) = 66.92, p < 0.0001). Total distance scores
were increased in the B6 group treated MPEP at the 30 mg/kg dose (F (2, 21) = 4.318, p < 0.05)
using a stringent Tukey’s post-hoc comparison. Vertical activity did not differ across the time
course of testing in B6 (Figure S1 Panel C; F (5, 21) = 1.58, p > 0.05). Vertical activity did not
differ across B6 groups treated with vehicle, MPEP 10 mg/kg, and MPEP 30 mg/kg (F (2, 21) =
0.01, p > 0.05). Time spent in the center of the test arena did not differ over the time course in
B6 (Figure S1 Panel D; F (5, 21) = 1.16, p > 0.05). Time in the center of the arena did not differ
across B6 groups treated with vehicle, MPEP 10 mg/kg, and MPEP 30 mg/kg (F
(2, 21)
= 0.10, p >
0.05).
Figure S2 illustrates the four parameters assessed in the open field exploratory
locomotion task in BTBR following a single injection of the two higher doses of MPEP or vehicle.
Similar to B6, BTBR exhibit a significant reduction in horizontal activity across the 30 minute
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task, indicating the expected habituation to the novelty of the open field (Figure S2 Panel A; F (5,
21)
= 59.13, p < 0.001). Horizontal activity did not differ across BTBR groups treated with
vehicle, MPEP 10 mg/kg, and MPEP 30 mg/kg (F (2, 21) = 2.78, p > 0.05). Total distance
traversed in the novel open field over a 30 minute time period was highly significant in BTBR
(Figure S2 Panel B; F (5, 21) = 65.99, p < 0.0001). Total distance scores were increased in the
BTBR group treated with MPEP at the 30 mg/kg dose (F (2, 21) = 6.02, p < 0.01) using a Tukey’s
posthoc comparison. Vertical activity was significantly reduced over time in BTBR (Figure S2
Panel C; F (5, 21) = 5.54, p < 0.001). Vertical activity did not differ across B6 groups treated with
vehicle, MPEP 10 mg/kg, and MPEP 30 mg/kg (F (2, 21) = 1.19, p > 0.05). Time spent in the
center of the arena across the testing session was reduced in the BTBR mice (Figure S2 Panel
D; F (5, 21) = 15.93, p < 0.001). Time in the center of the arena did not differ across B6 groups
treated with vehicle, MPEP 10 mg/kg, and MPEP 30 mg/kg (F (2, 21) = 0.17, p > 0.05).
Figure S3 illustrates the four parameters assessed in the open field exploratory
locomotion task in B6 following a single injection of risperidone or vehicle. Horizontal activity
over the 30 minute test session was reduced in B6 (F (5, 40) 11.69, p < 0.001). Unlike MPEP, a
single injection of risperidone significantly reduces horizontal activity in B6 (Figure S3 Panel A,
F (3, 40) = 28.7, p < 0.001). A significant drug x horizontal activity interaction was detected (F (3, 15)
= 4.68, p < 0.01). Tukey’s posthoc comparisons reveal significant reductions in horizontal
activity after risperidone treatment in B6, at each dose compared to vehicle, at each of the 5minute time bins. (0.125 mg/kg, q = 7.47, p < 0.001; 0.25 mg/kg, q = 7.86, p < 0.001; 0.5 mg/kg,
q = 12.70, p < 0.001). Total distance traversed in the novel open field over a 30 minute time
period was highly significant, as expected, representing habituation to the novel open field in B6
(Figure S3 Panel B, F (5, 40) = 9.04, p < 0.0001). A significant effect of risperidone was detected
in B6 (F (3, 40) = 27.20, p < 0.05). The interaction of risperidone x distance traversed was
significant in B6 (F (3, 15) = 4.49, p < 0.01). Tukey’s posthoc comparisons reveal significant
reductions in total distance scores after risperidone treatment in B6, at each dose compared to
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vehicle during the 30 minute test session, at each of the 5-minute time bins (0.125 mg/kg, q =
8.01, p < 0.05; 0.25 mg/kg, q = 7.90, p < 0.05; 0.5 mg/kg, q = 12.15, p < 0.05). Vertical activity
did not differ over the 30 minute test in B6 (Figure S3 Panel C). Risperidone reduced vertical
activity in B6 (F (3, 40) = 33.78, p < 0.001). Tukey’s posthoc comparisons revealed significant
reductions in vertical activity after risperidone treatment in B6, at each dose compared to
vehicle, at each of the 5-minute time bin intervals (0.125 mg/kg, q = 9.34, p < 0.001; 0.25 mg/kg,
q = 10.63, p < 0.001; 0.5 mg/kg, q = 12.63, p < 0.001). Time spent in the center across the 30
minute test session was significant in B6 (Figure S3 Panel D; F (5, 40) = 3.25, p<0.01). Time
spent in the center of the arena in B6 was increased by risperidone administration (F (3, 40) =
14.09, p < 0.001). A significant drug x center time interaction was also significant (F
(3, 15)
=
2.49, p < 0.01). Tukey’s posthoc comparisons revealed that administration of 0.5 mg/kg
risperidone significantly increased time spent in the center of the arena in B6 compared to
vehicle (q = 7.00, p < 0.001), 0.125mg/kg (q = 8.03, p < 0.001) and 0.25 mg/kg (q = 7.59, p <
0.001) at each of the 5-minute time bin intervals. Suggesting that the 0.5 mg/kg treatment
resulted in gross reductions in locomotion. The mice moved very little from the area in which
they were placed by the experimenter.
Figure S4 illustrates the four parameters assessed in the open field exploratory
locomotion task in BTBR following a single injection of risperidone. Horizontal activity over the
30 minute test session was reduced in BTBR (F (5, 33) = 7.33, p < 0.001). Unlike MPEP, a single
injection of risperidone significantly reduces horizontal activity in BTBR (Figure S4 Panel A, F (3,
33)
= 16.03, p < 0.001). A significant drug x horizontal activity interaction was found in BTBR (F
(3, 15)
= 4.42, p < 0.01). Tukey’s posthoc comparisons revealed significant reductions in
horizontal activity in the open field in BTBR at the two higher doses of risperidone (0.25 mg/kg,
q = 4.53, p < 0.05; 0.5 mg/kg, q = 9.70, p < 0.001). Subsequent Tukey’s posthoc comparisons
in BTBR revealed that the 0.5 mg/kg dose differed from vehicle at each of the 5-minute time bin
intervals. The 0.25 mg/kg dose differed from vehicle only during the first (q = 8.43) and second
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(q = 5.74) 5 minute intervals. Total distance traversed in the novel open field over a 30 minute
time period was highly significant, as expected, representing habituation to the novel open field
in BTBR (Figure S4 Panel B, F (5, 33) = 9.55, p < 0.0001). A significant effect of risperidone was
detected in BTBR (F (3, 33) = 11.31, p < 0.001). Tukey’s posthoc comparisons reveal significant
reductions total distance scores in BTBR at each dose of risperidone (0.125 mg/kg, q = 3.84, p
< 0.05; 0.25 mg/kg, q = 4.69, p < 0.05; 0.5 mg/kg, q = 8.19, p < 0.05). Additional Tukey’s
posthoc comparisons in BTBR revealed that the 0.5 mg/kg dose differed from vehicle at the first
(q = 10.70), second (q = 7.40), third (q = 5.602), fourth (q= 5.327) and sixth (q = 3.739) 5-minute
intervals. In the BTBR, the two lower doses of risperidone reduced locomotion during the first
(0.125 mg/kg, q = 4.18; 0.25 mg/kg, q = 9.16) and second 5-minute intervals (0.125 mg/kg, q =
4.35; 0.25 mg/kg, q = 4.88). Vertical activity did not differ over the 30 minute test in BTBR
(Figure S4 Panel C). Risperidone reduced vertical activity in BTBR (F (3, 33) = 9.24, p < 0.001).
BTBR exhibited a significant drug x vertical activity interaction (F
(3, 15) =
2.69 p < 0.01). Tukey’s
posthoc comparisons revealed significant reductions in vertical activity after risperidone
treatment in BTBR, at each dose compared to vehicle (0.125 mg/kg, q = 4.57, p < 0.05; 0.25
mg/kg, q = 4.84, p < 0.01; 0.5 mg/kg, q = 7.31, p < 0.001) during the 30 minute test session.
Additional Tukey’s posthoc comparisons revealed that the 0.5 mg/kg dose differed from vehicle
at the each of the 5-minute time bins. The 0.25 mg/kg dose differed from vehicle only during the
first (q = 7.58), second (q = 5.72) and third (q = 4.33) 5-minute time session intervals. The
0.125 mg/kg dose differed from vehicle only during the first (q = 6.25) and second (q = 4.96) 5minute time session intervals. Time spent in the center across the 30 minute test session was
significant in BTBR (Figure S4 Panel D; F (5, 33) = 3.29, p < 0.01). Time in the center of the
arena did not differ across BTBR groups treated with vehicle, risperidone 0.125 mg/kg, 0.25
mg/kg and 0.5mg/kg (F (3, 33) = 0.39, p > 0.05).
5
Supplementary References:
Anderson JJ, Bradbury MJ, Giracello DR, Chapman DF, Holtz G, Roppe J, et al (2003). In
vivo receptor occupancy of mGlu5 receptor antagonists using the novel radioligand
[3H]3-methoxy-5-(pyridin-2-ylethynyl)pyridine). Eur J Pharmacol 473(1): 35-40.
Carvalho RC, Silva RH, Abilio VC, Barbosa PN, Frussa-Filho R (2003). Antidyskinetic
effects of risperidone on animal models of tardive dyskinesia in mice. Brain Res Bull
60(1-2): 115-124.
Yan QJ, Rammal M, Tranfaglia M, Bauchwitz RP (2005). Suppression of two major
Fragile X Syndrome mouse model phenotypes by the mGluR5 antagonist MPEP.
Neuropharmacology 49(7): 1053-1066.
6
Supplementary Figure Legends:
Figure S1: MPEP did not reduce exploratory parameters in a novel open field in B6.
Horizontal activity, total distance, vertical activity and center time were assayed in 5minute time bins across a 30 minute session in a novel open field arena, following MPEP
administration in B6. A) MPEP had no effect on horizontal activity at doses of 10 mg/kg
and 30 mg/kg. B) MPEP administration produced an overall significant increase in total
distance traversed for the 30 mg/kg treatment group as compared to the vehicle
treatment group. MPEP had no effect on C) vertical activity or D) time spent in the
center of the arena at doses of 10 mg/kg and 30 mg/kg. *p < 0.05 as compared to
vehicle. N= 8 per dose.
Figure S2: MPEP did not reduce exploratory parameters in a novel open field in BTBR.
Horizontal activity, total distance, vertical activity and center time were assayed in 5minute time bins across a 30 minute session in a novel open field arena, following MPEP
administration in BTBR. A) MPEP had no effect on horizontal activity at doses of 10
mg/kg and 30 mg/kg. B) MPEP increased locomotion at the 30 mg/kg dose, during the
first, second, fifth and sixth 5-minute time bins. MPEP had no effect on C) vertical
activity and D) time spent in the center of the arena at doses of 10 mg/kg and 30 mg/kg.
*p < 0.05 as compared to vehicle. N= 7-9 per dose.
Figure S3: Risperidone reduced exploratory parameters in a novel open field in B6.
Horizontal activity, total distance, vertical activity and center time were assayed in 5minute time bins across a 30 minute session in a novel open field arena, following
risperidone administration in B6. Risperidone administration resulted in significant
reductions in A) horizontal activity, B) total distance, and C) vertical activity at doses of
0.125 mg/kg, 0.25 mg/kg and 0.5 mg/kg at each of the 5-minute time bins as compared
to vehicle. D) Time spent in the center of the arena was significantly increased by
7
risperidone. Risperidone administration at the 0.5 mg/kg dose significantly increased
time spent in the center of the arena in B6 compared to vehicle, 0.125mg/kg and 0.25
mg/kg at each of the 5-minute time bin intervals. +p<0.01 as compared to 0.5mg/kg. N=
10-14 per dose.
Figure S4: Risperidone reduced exploratory parameters in a novel open field in BTBR.
Horizontal activity, total distance, vertical activity and center time were assayed in 5minute time bins across a 30 minute session in a novel open field arena, following
risperidone administration in BTBR. A) Risperidone administration produced significant
reductions in horizontal activity at the two higher doses of risperidone, 0.25 mg/kg and
0.5 mg/kg. The 0.5 mg/kg dose differed from vehicle at each of the 5-minute time bin
intervals. The 0.25 mg/kg dose differed from vehicle only during the first and second 5minute intervals. B) Risperidone administration produced significant reductions in total
distance at doses of 0.125 mg/kg, 0.25 mg/kg and 0.5 mg/kg at each of the 5-minute
time bins, compared to vehicle. C) Risperidone administration produced significant
reductions in vertical activity at each dose compared to vehicle during the 30 minute test
session. The 0.5 mg/kg dose differed from vehicle at the each of the 5-minute time bin
intervals. The 0.25 mg/kg dose differed from vehicle only during the first, second and
third 5-minute time session intervals. The 0.125 mg/kg dose differed from vehicle only
during the first and second 5-minute time session intervals. D) Risperidone had no
effect on time spent in the center of the arena at doses of 0.125 mg/kg, 0.25 mg/kg and
0.5 mg/kg. *p<0.05 as compared to vehicle. N= 9-10 per dose.
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