Supplementary Figure Legends
Figure S1: R-baclofen rescued social approach deficits in the BTBR mouse model of autism in a
second independent cohort of B6 and BTBR mice, replicating findings in Cohort 1, Figure 1.
Replication in Cohort 2: (A) B6 mice displayed normal sociability on the chamber time parameter,
spending more time in the side chamber with the novel mouse as compared to the side chamber with the
novel object, after treatment with saline and at each dose of R-baclofen. (B) BTBR mice exhibited
characteristic lack of sociability on the chamber time parameter after vehicle treatment, spending
approximately equal time in the side chamber with the novel mouse and the side chamber with the novel
object. R-baclofen at the 3 mg/kg dose reversed the sociability deficits in BTBR. (C) B6 mice treated
with saline or R-baclofen exhibited characteristic sociability on the directed sniffing parameter.
Automated sniffing was measured by Noldus Ethovision 9.0XT software. (D) BTBR exhibited its
characteristic lack of sociability, i.e. did not spend more time sniffing the novel mouse versus the novel
object, after vehicle treatment. R-baclofen at doses of 1 and 3 mg/kg reversed the social sniffing deficits
in BTBR. Number of entries into the side chambers was unaffected by R-baclofen treatment in (E) B6
and (F) BTBR, indicating the absence of confounding increased hyper or hypo exploratory locomotion
during the social approach task. * p < 0.05, novel mouse versus novel object. See Supplementary Figure
S2 for validation of the automated directed sniff times by manual scoring of social sniffing.
Figure S2: R-baclofen rescued social approach sniffing in BTBR in two independent cohorts of
BTBR mice when digital videotapes were scored by a trained human observer.
Validation of R-baclofen rescue of the automated social sniffing results shown in Figures 1 and S1 were
compared to scoring of digital videos by a trained human observer. (A) B6 mice treated with saline or Rbaclofen exhibited characteristic sociability on the sniffing parameter.
(B) BTBR exhibited its
characteristic lack of sociability and did not spend more time sniffing the novel mouse versus the novel
object, after treatment with saline. R-baclofen at doses of 1 and 3 mg/kg, reversed the social sniffing
deficits in BTBR. (C) In the replication Cohort 2, B6 mice treated with saline or R-baclofen exhibited
characteristic sociability on the sniffing parameter. (D) In the replication Cohort 2, BTBR exhibited its
characteristic lack of sociability and did not spend more time sniffing the novel mouse versus the novel
object, after treatment with saline. R-baclofen at the 3 mg/kg dose, reversed the social sniffing deficits in
BTBR. See Figures 1 and S1 in the main and supplementary text, showing qualitatively similar treatment
effects when the same sessions were scored by Noldus Ethovision 9.0XT automated videotracking
software.
Figure S3: R-baclofen treatment caused no deleterious effects in C58 social approach.
(A) C58 displayed normal sociability on the chamber time parameter, spending more time in the chamber
with the novel mouse as compared to the novel object, after treatment with vehicle and at each dose of Rbaclofen tested. (B)
C58 mice treated with R-baclofen exhibited characteristic sociability on the
automated directed sniffing parameter at each dose tested. (C) No significant difference in the number of
entries into the side chambers was observed in C58 following treatment with R-baclofen, indicating the
absence of confounding increases or decreases in exploratory locomotion during the social approach task.
*p < 0.05, novel mouse versus novel object.
Figure S4:
R-baclofen had no significant effect on male-female social interactions and
vocalizations.
Social interactions between a subject male and an estrous B6 female were assayed in the Noldus
PhenoTyper arena filled with a small amount of bedding. The 5 minute test session began 60 minutes
after vehicle or R-baclofen. The test session was video recorded and subsequently scored for parameters
of social interaction by an observer, blind to treatment condition, using Noldus Observer 9.0XT software.
B6 displayed normal sociability, including high levels of nose-to-nose sniffing (A), nose-to-anogenital
sniffing (C), following (E), and high numbers of ultrasonic calls (G). BTBR exhibited its characteristic
low sociability, including lower levels of nose-to-nose sniffing (B), nose-to-anogenital sniffing (D),
following (F) and low ultrasonic call emissions (H). R-baclofen at the 1 mg/kg dose did not change time
spent engaged in male-female social interaction, on several standard behavioral parameters, or number of
ultrasonic vocalization emissions in B6 or BTBR.
Figure S5: R-baclofen had no effect on olfactory ability in B6 and BTBR.
Olfactory habituation/dishabituation for B6 and BTBR was evaluated in a standard empty mouse cage.
The test session began 60 minutes after saline vehicle or R-baclofen. Habituation is indicated by
decreased time spent in sniffing a sequence of three identical odors. Dishabituation is indicated by
increased time sniffing a novel odor, different from the previous three presentations. (A) B6 mice showed
normal habituation and dishabituation to nonsocial and social odor cues in saline or R-baclofen treated
groups. (B) BTBR mice showed normal habituation and dishabituation to nonsocial and social odor cues
in groups treated with saline or the 1 mg/kg dose of R-baclofen. In BTBR treated with the highest dose of
3 mg/kg of R-baclofen, neither nonsocial nor social odors elicited habituation or dishabituation. This
deficit is likely due to a combination of the mildly sedating effects of this higher dose, and competing
self-grooming behavior. S. (1) odor 1 = first presentation of social odor 1, S. (1) odor 2 = second
presentation of social odor 1, and S. (1) odor 3 = third presentation of social odor 1. S. (2) odor 1 = first
presentation of social odor 2, S. (2) odor 2 = second presentation of social odor 2, and S. (2) odor 3 =
third presentation of social odor 2. Social odor stimuli were prepped by swabbing across a soiled cage of
unfamiliar mice of the same sex of a different strain. Note: y-axis varies by strain.
Figure S6: R-baclofen reduced marble burying repetitive behavior in BTBR and jumping in C58 in
a second independent cohort of each strain, replicating findings in Cohort 1, Figure 2.
Replication in Cohort 2: (A) B6 mice displayed their normally low levels of self-grooming after
administration of saline or R-baclofen. (B) A trend was seen but R-baclofen did not significantly reduce
the BTBR high levels of repetitive self-grooming. (C) B6 mice treated with saline vehicle or R-baclofen
displayed their normally low levels of the repetitive behavior marble burying. (D) In BTBR Cohort 2, Rbaclofen again reduced the typical high number of marbles buried after treatment with the 3 mg/kg dose.
* p < 0.05 versus saline. (E) Stereotyped vertical jumping in a second cohort of C58 was significantly
reduced after 3 mg/kg of R-baclofen. * p < 0.05 versus saline.
Figure S7: R-baclofen treatment showed sedative effects at high doses and when tested 30 minutes
before behavioral testing in B6 and BTBR.
Exploratory locomotion measured by total distance traversed and horizontal activity in B6 and BTBR
following administration of saline vehicle or R-baclofen, 30-minutes before the start of the test session.
(A) R-baclofen treatment, 5 mg/kg, decreased total distance traversed when administered 30-minutes
prior to the test session in B6. (B) R-baclofen treatment, 3 and 5 mg/kg, similarly decreased total distance
traversed when administered 30-minutes prior to the test session in BTBR. (C) Horizontal activity in B6
mice was reduced by R-baclofen treatment, 5 mg/kg, when assayed 30-minutes post administration. (D)
Horizontal activity in BTBR was reduced by R-baclofen treatment, 3 and 5 mg/kg, when tested 30minutes post administration. Data are shown in 5 minute time bins. *p < 0.05 as compared to saline.
Lower Ns were used for the 5 mg/kg dose due to the clear presence of sedation at this dose.
These
results showing sedation at the 30 minute time point but not at the 60 minute time point provided the
rationale for using the 60 minute post-injection interval for the primary behavioral experiments.
Figure S8: No anxiolytic effects of R-baclofen using the standard elevated plus-maze.
Elevated plus-maze anxiety-like behavior in B6 and BTBR was evaluated using a standard automated
Med Associates maze that detects photo beam breaks. The 5-minute test session began 60 minutes after
saline vehicle or R-baclofen. (A) In B6, R-baclofen did not reduce time spent in the open arm. (C) Rbaclofen at 3 mg/kg reduced number of entries onto the open arms and (E) the number of total entries on
both the open and closed arms of the maze in B6, indicating sedating effects. (B) In BTBR mice, Rbaclofen reduced time spent in the open arms, (D) number of entries onto the open arms, and (F) the total
entries on both the open and closed arms of the maze, similarly indicating sedating effects in BTBR on
this task. *p < 0.05 as compared to saline.
Figure S9: No anxiolytic effects of R-baclofen using the standard light ↔ dark conflict assay.
Light ↔ dark anxiety-like behavior in B6 and BTBR was evaluated using a standard light ↔ dark box
with automated National Instruments software that detects photo beam breaks. The 10-minute test session
began 60 minutes after vehicle or R-baclofen. (A) In B6, R-baclofen increased time spent in dark
compartment of the two-chambered box and (C) reduced transitions between chambers of the assay. (D)
In BTBR, R-baclofen at 3 mg/kg reduced transitions between chambers of the assay but did not increase
the time spent in the dark chamber (B). *p < 0.05 as compared to saline.
Figure S10: S-baclofen at comparable doses did not reduce repetitive behaviors in BTBR and C58.
(A) S-baclofen had no effect on the normal levels of self-grooming in B6. (B) S-baclofen had no effect
on the high levels of repetitive self-grooming in BTBR. (C) S-baclofen did not alter the normal levels of
marbles buried by B6. (D) S-baclofen did not reduce the high number of marbles buried by BTBR. (E)
Stereotyped vertical jumping in C58 mice was not significantly reduced following S-baclofen treatment.
Figure S11: S-baclofen did not induce hyperactivity or sedation in the open field.
Exploratory locomotion measured by total distance traversed and horizontal activity across a 30 minute
test session assayed 60-minutes following administration of saline vehicle or doses of S-baclofen. (A)
total distance and (B) horizontal activity were unaffected by S-baclofen treatment in B6. Data are shown
in 5-minute time bins.