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Kachroo et al.
Supplemental Figure Legends
Supplemental Fig 1. MPEP induces fine motor activity in WT but not mGlu5 KO mice.
WT mice (n=3) and their KO littermates (n=5) received sequential injections of saline
(x2), MPEP at doses of 5 and then 20 mg/kg, and finally saline administered 1 h apart.
Comparisons between WT vs KO at the peak drug effect for fine movement with MPEP
5mg/kg and MPEP 20 mg/kg showed significant attenuation in KO mice (* p<0.05,
unpaired t-test).
Supplemental Fig 2. Locomotion (first 10 min block) in reserpinized (2 mg/kg) mice
plotted against their striatal dopamine content (as a % of that in untreated control mice).
Pearson coefficient of 0.81 indicates a strong correlation between degree of lesion and
hypolocomotive response.
Supplemental Fig 3. (A) Characterizing a cage warming system for minimizing
hypothermia in mice. Time course (left graph) to achieve steady state temperature of
cages placed on heating pads (Homedics, model # HP-150 Thera-P Moist/Dry Heating
Pad, standard size, Las Vegas, Nevada) set on high (n=4) and medium (n=4) settings
shows rapid warming and equilibration of the ambient temperature in test cages.
Subsequent time course (right graph) of mouse core body temperature taken with a rectal
temperature probe (AD instruments, model # MLT 1404, Colorado Springs, CO).
C57BL/6NCrl mice reserpinized (1.5mg/kg) 24 h prior, were placed in activity (test)
cages heated on the high setting. Mice were hypothermic 24 h after reserpine treatment
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(31.9+2oC; * p<0.001 post- versus pre-reserpine temperature; one-way ANOVA) and
within 50 min in the high heated cages became hyperthermic. (B) Locomotor responses
to vehicle/antagonists of mice in warmed test cages. To assess the potential confound of
reserpine-induced hypothermia, C57BL/6NCrl mice (n=5/group) reserpinized (1mg/kg)
24 h prior to the test day were habituated in activity cages heated on pads on the medium
setting for 2 h (based on findings in (A) above). The mGlu5 antagonist MPEP (5mg/kg)
or saline was administered i.p. in combination with either vehicle or the A2A antagonist
KW-6002 at 0.3 mg/kg and locomotion monitored for a further 30min. At the last
timepoint (at which time core body temperatures were also assessed) the motor response
to KW+MPEP (versus MPEP, KW-6002, or vehicle) was significantly increased (*
p<0.05, p<0.05, and p<0.01 respectively; one-way ANOVA). (C) Core body temperature
during peak locomotor response to vehicle/antagonists. Rectal temperatures of the mice
in (B) were taken pre- and 24 h post reserpine in home cages, then 2 h after placement in
warmed test cages (immediately preceding antagonist/vehicle treatment) and then 30min
later. Mouse core body temperature was slightly reduced 24 h after reserpine (37.7+0.1
o
C versus 38.7+0.1 oC, p<0.01, one-way ANOVA repeated measures test; n=20 total). At
the time of antagonist/vehicle treatment 2 h after placement into warmed test cages, the
isolated mice displayed marked hypolocomotion (as shown in B) and a further reduction
in core body temperature (36.7+0.1 oC; p<0.001 versus pre-reserpine, one-way ANOVA
repeated measures test). For all measurements prior to drug administration there was no
difference in temperature between groups. At the 30 min post-drug timepoint, rectal
temperature after KW treatment was significantly increased compared to the vehicle
control and MPEP treatments (*p<0.01, one-way ANOVA) demonstrating a clear
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dissociation between a synergistic motor response and thermal changes after combined
antagonist treatment.
Supplemental Fig 4. Western immunoblotting using specific antibodies for the A2A
receptor was performed on striata for the conditional A2A KO (n=8; lanes 1-4) and their
littermate controls (n=7; lanes 5-8) used in Fig 4. A mouse monoclonal anti-A2AR
antibody (Upstate, Lake Placid, NY; cat. # 05-717; raised against the 3rd intracellular
loop, which is encoded by exon 3 and expected to be present in the protein product of the
recombined A2A gene) was used at a 1:500 dilution. Striata from a fully WT mouse and a
global A2A KO mouse (Chen et al, 1999) were run in parallel (in the indicated lanes) as a
positive and negative control respectively. The presence of a band at approximately
45kDa (A2A receptor protein – arrow) in the control and WT mice and lack thereof in the
conditional and global KO mice confirms the depletion of striatal A2A receptor protein in
the KO lines.
Supplemental Fig 5. D1 agonist-induced fine movement does not require A2A or D2
receptors. Mice were treated with the D1 agonist SKF 38393 (15 mg/kg) on the test day 2
h post habituation, and fine movement was recorded for one more hour. Post vs pre-drug
treatment, * p<0.01; paired t-test. @ p<0.01 vs double WT (pre-drug).
Supplemental Fig 6. Alternative models hypothesizing the role of adenosine A2A
receptors on mGlu5 regulation of striatal neurons and motor function. (A) Two schematic
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diagrams depict glutamatergic activation of mGlu5 receptors on striatopallidal neurons in
WT and A2A KO mice. A ‘mandatory’ model of the A2A receptor’s role in striatopallidal
neuron function hypothesizes the dependence of mGlu5 receptor effects on the A2A
receptor due to the mGlu5 receptor acting immediately upstream of the A2AR (potentially
in a heteromeric receptor complex). A ‘facilitative’ model hypothesizes the A2AR (in an
A2A-mGlu5 heteromer) acting on the mGlu5 receptor to increase the tonic action of
endogenous glutamate on the mGlu5 receptor. (B) Observed and predicted effects on
motor activity according to models hypothesized in (A) explain current observations and
predict differential responses to an mGlu5 agonist in the absence of the A2A receptor.
Note however that an alternative ‘facilitative’ model in which a heteromeric A2A receptor
directly facilitates the output of the mGlu5 (i.e., its 2nd messenger generation) would also
predict that the loss of mGlu5 agonist efficacy in A2A KO mice.
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