Supplementary data
Figures 1-3. As presented in the main text for male mice, the first three figures in the
supplementary data show the same behavioral measurements in females. Similarly, the
low dose of CP-55,940 induced a decrease in anxiety, combined with an increased
exploration in all the groups except of the Glu-CB1-KO female mice (Figures S1a, S1b,
S2a, S2b, S3c and S3d). On the other hand, the high dose of the CB1 receptor agonist
promoted an anxiogenic-like response, accompanied by a reduction in exploration in all
the groups except of the GABA-CB1-KO female mice (Figures S1a, S1b, S2a, S2b, S3c
and S3d). Together with the absence of locomotor effects of none of the doses
administered, these experiments demonstrate not only the similarity of responses
between sexes, but also the dual role of CB1 receptors in the regulation of anxiety and
exploration.
Figures 4-7. As a complementary measurement in evaluating anxiety, exploration and
locomotion, the time the animals spend in the OA, the time they spend exploring the
holes in the HB test, and the frequency of risk assessment were scored. These
measures confirmed in most of the cases the previous observations both in males
(Figures S4a-d and S5a-b) and females (Figure S6a-d and S7a-b). The evaluation of the
horizontal activity revealed no effect of any of the doses of CP-55,940 on locomotion in
both sexes (Figures S5c-d and S7c-d).
Statistical evaluation of the sex factor
The three-way ANOVA (sex x genotype x treatment) for percentage of entries into the
open arms for GABA-CB1-KO mice revealed no interaction between the three factors
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1
(F2,83 = 0.555; p<0.576). The effect of the sex factor per se (F1,83 = 1.080; p<0.302) or
any of its interactions (sex x genotype: F2,83 = 2.401; p<0.097; sex x treatment: F2,83 =
0.206; p<0.814) was also negligible. The same result was obtained from GABA-CB1-KO
mice for the rest of parameters measured in the elevated plus-maze test and the
holeboard test (Table S1 and S2), indicating the absence of differences between sexes
in the anxiety response and the exploratory activity tested under our conditions.
Regarding the mouse line where the CB1 receptor was deleted from glutamatergic
forebrain neurons (Glu-CB1-KO), the absence of differences between sexes was
detected in all the parameters except for stretched-attend postures (females did it more
frequently; F1,83 = 5.818; p<0.018) and head dipping frequency and time (females were
less explorative; F1,83 = 4.317; p<0.041 and F1,83 = 5.317; p<0.024, respectively; Table
S2). However, in all these cases, the underlying biphasic pattern and its regulation by
CB1 receptor activation present in different subpopulations of neurons were still very
similar to males. As a matter of fact, the interaction between sex and any of the other
factors was not significant (Table S2). Thus, we decided to focus on the regulation of
this bimodal action induced by the treatment with exogenous cannabinoids, instead of
focusing on differences between sexes, which were much less prominent with our
protocol. Moreover, data extracted from vaginal smears revealed a synchronization
effect of group housing, leading to a percentage of estrus and pro-estrus states higher
than 80% in all the groups, suggesting a negligible effect of the estrous cycle, especially
under our conditions of acute treatment.
Figures 8-10. Secondary parameters were also measured during the dose curve which
we performed for the GABAB receptor positive allosteric modulator GS-39783. The
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highest doses (10 and 50 mg/kg) promoted a clear anxiolytic-like effect in most of the
parametters analyzed (Figures S8a-f and S10c-d) without locomotor effects (Figures
S8b and S9c-d). The combination of CP-55,940 (high dose) + GS-39783 (sub-thresold
dose) did not affect significantly locomotion (S9a-d), but counteracted the anxiogeniclike effect of the CP-55,940 alone (Figures S10a-b).
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Supplementary Figures
Figure S1 Behavioral responses of GABA-CB1-KO (left panels) and Glu-CB1-KO (right
panels) female mice and their corresponding GABA-CB1-WT and Glu-CB1-WT mice,
evaluated in the elevated plus-maze paradigm, treated with either vehicle or CP-55,940
(1 and 50 µg/kg). (a) and (b) Percentage of open arms (OA) entries [% open arms = 100
x open/(open+enclosed)] are presented, instead of absolute values in order to avoid any
underlying interaction of treatment or genotype on the locomotion activity. (c) and (d)
Total number of entries into open + enclosed arms. Data are presented as mean ± SEM
(6-10 animals per experimental group). Significant differences: *, p<0.05; **, p<0.01; ***,
p<0.001 when compared to vehicles (same genotype) (one-way ANOVA followed by
Bonferroni´s test); §, p<0.05; §§, p<0.01; §§§, p<0.001 when compared to wild-type
littermates (same treatment) (two-way ANOVA followed by Bonferroni´s test).
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Figure S2 Behavioral responses of GABA-CB1-KO (left panels) and Glu-CB1-KO (right
panels) female mice and their corresponding GABA-CB1-WT and Glu-CB1-WT mice,
evaluated in the holeboard paradigm, treated either with vehicle or CP-55,940 (1 and 50
µg/kg). (a) and (b) Number of head dippings performed by the animals (only when the
mice were facing at least their snouts to the bottom of the holes). (c) and (d) Number of
square lines crossed by the animals. Data are presented as mean ± SEM (6-10 animals
per experimental group). Significant differences: *, p<0.05; **, p<0.01, ***, p<0.001 when
compared to vehicles (same genotype) (one-way ANOVA followed by Bonferroni´s test);
§, p<0.05; §§, p<0.01; §§§, p<0.001 when compared to wild-type littermates (same
treatment) (two-way ANOVA followed by Bonferroni´s test).
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Figure S3 Secondary behavioral parameters of GABA-CB1-KO (left panels) and GluCB1-KO (right panels) female mice and their corresponding GABA-CB1-WT and GluCB1-WT mice, evaluated in the elevated-plus maze and the holeboard paradigm,
treated either with vehicle or CP-55,940 (1 and 50 µg/kg). (a) and (b) Number of
stretched-attend postures performed by the mice. (c) and (d) Percentage of square lines
crossed in the internal area [% internal area = 100 x internal/(internal+external)]. Data
are presented as mean ± SEM (6-10 animals per experimental group). Significant
differences: *, p<0.05; **, p<0.01, ***, p<0.001 when compared to vehicles (same
genotype) (one-way ANOVA followed by Bonferroni´s test); §, p<0.05; §§, p<0.01; §§§,
p<0.001 when compared to wild-type littermates (same treatment) (two-way ANOVA
followed by Bonferroni´s test).
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Figure S4 Behavioral responses of GABA-CB1-KO (left panels) and Glu-CB1-KO (right
panels) male mice and their corresponding GABA-CB1-WT and Glu-CB1-WT mice,
evaluated in the elevated plus-maze paradigm, treated with either vehicle or CP-55,940
(1 and 50 µg/kg). (a) and (b) Percentage of time spent in the open arms. [% open arms
= 100 x open/(open+enclosed)]. (c) and (d) Frequency of risk assessment behavior
(exploratory posture in which the head is stuck out of the rim in an open arm, looking to
the floor) performed by the mice during the 5 minutes test. Data are presented as mean
± SEM (8-10 animals per experimental group). Significant differences: *, p<0.05; **,
p<0.01; ***, p<0.001, when compared to vehicles (same genotype)(one-way ANOVA
followed by Bonferroni´s test); §, p<0.05; §§, p<0.01; §§§, p<0.001, when compared to wildtype littermates (same treatment)(two-way ANOVA followed by Bonferroni´s test).
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Figure S5 Secondary behavioral parameters of GABA-CB1-KO (left panels) and GluCB1-KO (right panels) male mice and their corresponding GABA-CB1-WT and Glu-CB1WT mice, evaluated in the elevated plus-maze paradigm, treated with either vehicle or
CP-55,940 (1 and 50 µg/kg). (a) and (b) Total time spent by the subjects exploring the
holes (seconds). (c) and (d) Frequency of rearing behavior as an indicator of vertical
activity. Data are presented as mean ± SEM (8-10 animals per experimental group).
Significant differences: *, p<0.05; *, p<0.01; ***, p<0.001, when compared to vehicles
(same genotype)(one-way ANOVA followed by Bonferroni´s test); §, p<0.05; §§, p<0.01;
§§§, p<0.001, when compared to wild-type littermates (same treatment)(two-way ANOVA
followed by Bonferroni´s test).
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Figure S6 Behavioral responses of GABA-CB1-KO (left panels) and Glu-CB1-KO (right
panels) female mice and their corresponding GABA-CB1-WT and Glu-CB1-WT mice,
evaluated in the elevated plus-maze paradigm, treated with either vehicle or CP-55,940
(1 and 50 µg/kg). (a) and (b) Percentage of time spent into the open arms. [% open
arms = 100 x open/(open+enclosed)]. (c) and (d) Frequency of risk assessment behavior
(exploratory posture in which the head is stuck out of the rim in an open arm, looking to
the floor) performed by the mice during the 5 minutes test. Data are presented as mean
± SEM (6-10 animals per experimental group). Significant differences: *, p<0.05; **,
p<0.01; ***, p<0.001, when compared to vehicles (same genotype)(one-way ANOVA
followed by Bonferroni´s test); §, p<0.05; §§, p<0.01; §§§, p<0.001, when compared to wildtype littermates (same treatment)(two-way ANOVA followed by Bonferroni´s test).
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Figure S7 Secondary behavioral parameters of GABA-CB1-KO (left panels) and GluCB1-KO (right panels) female mice and their corresponding GABA-CB1-WT and GluCB1-WT mice, evaluated in the elevated plus-maze paradigm, treated with either vehicle
or CP-55,940 (1 and 50 µg/kg). (a) and (b) Total time spent by the subjects exploring the
holes (seconds). (c) and (d) Frequency of rearing behavior as an indicator of vertical
activity. Data are presented as mean ± SEM (6-10 animals per experimental group).
Significant differences: *, p<0.05; *, p<0.01; ***, p<0.001, when compared to vehicles
(same genotype)(one-way ANOVA followed by Bonferroni´s test); §, p<0.05; §§, p<0.01;
§§§, p<0.001, when compared to wild-type littermates (same treatment)(two-way ANOVA
followed by Bonferroni´s test).
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Figure S8 Behavioral responses of C57BL/6N male mice treated with 4 different doses
of the GABAB receptor positive allosteric modulator GS-39783 and its vehicle. Mice were
evaluated in the elevated-plus maze (left panels) and the holeboard paradigm (right
panels), 1 hour after drug administration (p.o.). During the EPM, (a) the percentage of
open arms (OA) entries, (c) risk assessment behavior, and (e) stretched-attend postures
were scored. In the HB test, (a) total ambulation was used as a measurement of
locomotion, and (d) percentage of internal ambulation and (f) head dipping frequency
indicated the level of exploration (8-9 mice per experimental group). Significant
differences: *, p<0.05; ***, p<0.001, when compared to vehicles (one-way ANOVA
followed by Bonferroni´s test).
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Figure S9 Locomotor responses of C57BL/6N male mice treated with GS-39783 alone
(lower panels) or in combination with CP-55,940 (upper panels). Mice were evaluated in
the elevated-plus maze (left panels) and the Holeboard paradigms (right panels), 1 hour
after oral administration of GS-39783 or its vehicle. (a) and (c) total entries into open and
enclosed arms entries were scored, as well as (b) and (d) frequency of rearing behavior
as indicators of general locomotion and vertical activity, respectively. Significant
differences: *, p<0.05; ***, p<0.001, when compared to vehicles (one-way ANOVA
followed by Bonferroni´s test).
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Figure S10 Behavioral responses of C57BL/6N male mice treated with GS-39783 alone
(lower panels) or in combination with CP-55,940 (upper panels). Mice were evaluated in
the elevated-plus maze (left panels) and the holeboard paradigm (right panels), 1 hour
after oral administration of GS-39783 or its vehicle. (a) and (c) Percentage of time
expend by the mice into the open arms (OA) was scored, as well as (b) and (d) head
dipping time as indicators of anxiety-like behavior and exploration, respectively.
Significant differences: *, p<0.05; ***, p<0.001, when compared to vehicles (one-way
ANOVA followed by Bonferroni´s test).
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Variable
Factor / Interaction
SEX
% OA
entries
SEX * GENOTYPE
SEX * TREATMENT
SEX * GENOTYPE * TREATMENT
Error
SEX
%OA time
SEX * GENOTYPE
SEX * TREATMENT
SEX * GENOTYPE * TREATMENT
Error
SEX
Total entries
SEX * GENOTYPE
SEX * TREATMENT
SEX * GENOTYPE * TREATMENT
Error
SEX
Risk
assessment
SEX * GENOTYPE
SEX * TREATMENT
SEX * GENOTYPE * TREATMENT
Error
Stretchedattend
postures
SEX
SEX * GENOTYPE
SEX * TREATMENT
SEX * GENOTYPE * TREATMENT
Error
GABA-CB1-KO
df
F
p
1 1.080 .302
2 2.401 .097
2
.205 .814
2
.555 .576
83
1 1.897 .172
2 2.535 .085
2 1.374 .259
2 1.693 .133
83
1
.164 .687
2 1.285 .260
2
.705 .497
2
.805 .451
83
1
.161 .690
2
.868 .354
2 1.426 .246
2
.704 .498
83
1
.407 .525
2
.023 .880
2
.755 .473
2 2.321 .105
83
df
1
2
2
6
83
1
2
2
6
83
1
2
2
6
83
1
2
2
6
83
1
2
2
6
83
Glu-CB1-KO
F
p
.509
.478
.005
.942
1.079 .345
.463
.631
.117
.070
.794
.551
.734
.792
.456
.578
3.515
.018
.624
.138
.064
.893
.538
.871
.002
2.221
.056
.327
.961
.140
.946
.722
5.818
.200
.464
.845
.018 *
.656
.631
.433
Table S1 Statistical analysis of sex influence on biphasic effects induced by CB1
receptor agonist treatment on anxiety. Panel shows the statistical values for F
distribution and the correspondence p value for significance of sex factor and all of its
interactions with the rest of the factors (genotype and treatment) from the three-ways
ANOVA performed on every parameter studied on the elevated plus-maze paradigm.
Only in one case, a significant difference (*, p<0.05) was found, and in all of these
situations there was no interaction between the rest of the factors.
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Variable
Factor / Interaction
SEX
Head
dipping
frequency
SEX * GENOTYPE
SEX * TREATMENT
SEX * GENOTYPE * TREATMENT
Error
SEX
Total
ambulation
SEX * GENOTYPE
SEX * TREATMENT
SEX * GENOTYPE * TREATMENT
Error
SEX
Head
dipping
time
SEX * GENOTYPE
SEX * TREATMENT
SEX * GENOTYPE * TREATMENT
Error
SEX
% Internal
ambulation
SEX * GENOTYPE
SEX * TREATMENT
SEX * GENOTYPE * TREATMENT
Error
SEX
Rearing
SEX * GENOTYPE
SEX * TREATMENT
SEX * GENOTYPE * TREATMENT
Error
GABA-CB1-KO
df
F
p
1
3.197 .077
2
.169
.682
2
.385
.681
2
.085
.918
83
1
3.723 .057
2
.672
.415
2
1.981 .144
2
.163
.850
83
1
2.268 .136
2
.302
.584
2
.024
.976
2
.335
.716
83
1
.748
.389
2
1.441 .233
2
1.271 .286
2
.164
.849
83
1
1.115 .294
2
.092
.763
2
1.251 .292
2
.358
.700
83
Glu-CB1-KO
df
F
p
1
4.317 .041 *
2
.191
.664
2
1.787 .174
6
.312
.733
83
1
1.774 .186
2
1.000 .320
2
1.191 .309
6
1.319 .273
83
1
5.317 .024 *
2
.011
.917
2
1.834 .166
6
.711
.494
83
1
.415
.521
2
.670
.415
2
.007
.993
6
.305
.738
83
1
.131
.718
2
1.788 .185
2
.223
.801
6
1.451 .240
83
Table S2 Statistical analysis of sex influence on biphasic effects induced by CB1
receptor agonist treatment on exploratory activity evaluated. Right panel shows the
statistical values for F distribution and the correspondence p value for significance of sex
factor and all of its interactions with the rest of the factors (genotype and treatment) from
the three-ways ANOVA performed on every parameter studied on the holeboard
paradigm. Only in two cases, a significant difference (*, p<0.05) was found, and in all of
these situations there was no interaction between the rest of the factors.
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