Variation in syndrome intensity
Neuropsychopharmacology, Tao et al, 2014
Supplemental S1: Effect of days of CLG pretreatment on extracellular 5-HT accumulation in the PFC
and POA following challenge injection of CLG+PRX
Materials and Methods: Rats received saline
Controls were also challenged with CLG+PRX. To
(control; 1 ml/kg, s.c.) or clorgyline (CLG; 2
assess the significant difference between days
mg/kg, s.c) once daily for 3-13 days and were
of CLG pretreatment, changes in 5-HT efflux are
examined on the next day after the last
expressed in two different ways. Typically, data
treatment. On experimental day, extracellular
were normalized into fold changes relative to
5-HT was extracted with a microdialysis probe
respective baseline, by which individual
pre-implanted in the prefrontal cortex (PFC) or
difference was minimized as shown in fig. 1B
preoptic/anterior hypothalamic area (POA).
(see details in Results). Additionally, data were
After two baselines, animals were challenged
expressed as absolute 5HT levels (pg/60 min
with paroxetine (PRX; 15 mg/kg, i.p) in
microdialysis sample; ±s.e.m) as follows.
combination with clorgyline (CLG; 2 mg/kg, s.c.).
Fig. S1. A, Determine 5-HT response in the PFC. Each group consisted of 4-8 animals. Basal 5-HT in
the control group was 1.2 ±0.4 pg/sample. After once daily CLG pretreatment for 3, 6, and 13 days, it
was gradually increased to 2.5, 4.0 and 4.2 pg/sample (±0.3-0.7 by s.e.m) on day 4, 7, and 14,
respectively. Injection of CLG+PRX to control rats caused an increase in 5-HT up to 77.1 ±19.9
pg/sample. Challenge injection to rats pretreated with CLG also increased 5-HT up to 123.6 ±21.5, 83.0
±15.5, and 79.3 ±6.3 pg/sample in groups of day 4, 7 and 14, respectively. There is no difference
between controls and days of CLG pretreatment (F(3,14) =0.431, P =0.7341). B, Determine 5-HT response
in the POA. Basal 5-HT in the control group was 0.7 ±0.1 pg/sample, gradually increasing to 2.7, 3.6 and
4.7 pg/sample (±0.4-0.5 by s.e.m) in the CLG groups of day 4, 7 and 14, respectively. In the control
group, injection of CLG+PRX caused an increase in 5-HT up to 122.0 ±17.3 pg/sample. The increase was
gradually reduced to 75.4 ±6.9, 68.2 ±25.3 and 48.9 ±7.9 pg/sample in groups of day 4, 7 and 14,
respectively. There is a significant difference between controls and days of CLG pretreatment (F(3,12)
=3.823, P =0.0392). However, there is no difference between days of CLG pretreatment (F(2,9) =1.175, P
=0.3522).
Data interpretation: Comparing with
individual variation. After normalizing the data,
controls, days of CLG pretreatment had no
both regions are shown to have a CLG
effect on PFC 5HT, but caused a reduction in
pretreatment-dependent reduction in 5HT (fig.
POA 5HT. However, there was no difference
1B). Regardless the ways of data analysis,
between days of CLG pretreatment. The results
changes in 5HT efflux were strikingly different
lead us to suggest that CLG pretreatment had
from changes in syndrome intensity (fig. 2B),
no effect on absolute 5HT levels in response to
arguing against the presynaptic hypothesis.
challenge injection. The conflicting results could
be explained after normalization to limit inter1
Variation in syndrome intensity
Neuropsychopharmacology, Tao et al, 2014
Supplemental S2. Test the hypothesis that CLG pretreatment functionally up-regulates activity of 5HT2A receptors
Materials and Methods: Animals were
thermoprobe inserted into the rectum.
divided into 4 groups in S2-A and 3 groups in
Experiments were not begun until completion
S2-B. Each group was assigned with 5 animals.
of 2 h habituation. Body-core temperature (Tcor)
Pretreatment was carried out with saline (SAL; 1
was recorded at 15 min intervals. Data
ml/kg, s.c.) or clorgyline (CLG; 2 mg/kg, s.c)
collection included 4 baselines and 6-9 postonce daily for 4 consecutive days. The last
drug samples. To minimize individual
injection took place on day 4, 2 h before the
difference, Tcor data were normalized into
tests. After last pretreatment, animals were
changes relative to its baseline, and then
placed on a test chamber at ambient
expressed as mean ∆Tcor ±s.e.m.
temperature of 23.0 ±1 °C and a flexible
Fig. S2. Basal Tcor was 37.8 ±0.2 °C in SAL pretreatment group and 37.5 ±0.1 °C in CLG pretreatment
groups. A, Determine the effect of CLG pretreatment. Comparing with respective SAL controls, challenge
injection of DOI at a dose of 0.5 mg/kg, s.c. caused a 0.5 °C increase in SAL pretreat/DOI challenge group
(±0.1 by s.e.m; F(1,8) =11.34, P =0.0098), and 1.4 °C increase in CLG pretreat/DOI challenge group (±0.2 by
s.e.m; F(1,8) =33.107, P =0.0004). The difference in Tcor response between SAL pretreat/DOI challenge and
CLG pretreat/DOI challenge was significant (F(1,8) =18.114, P =0.0028). ***p <0.001 vs. SAL pretreat/DOI
challenge. B, Test an involvement of 5-HT2A receptor upregulation in CLG pretreatment. M100907 was
administered 30 min before SAL or DOI. Comparing with SAL+SAL, M100907+SAL with the M100907
dose at 1 mg/kg, s.c., caused a significant reduction in Tcor (F(1,8) =125.866, P =0.0009). DOI injection
failed to induced the increase in Tcor (F(1,8) =0.012, P =0.9138). ***p <0.001 vs. CLG pretreat/SAL+SAL.
Data interpretation: Findings that the CLGpretreatment group had a great response to the
5HT2A/CR agonist DOI are consistent with the
view that increases in 5HT availability to
postsynaptic circuits functionally upregulate 5HT2A receptors. Such functional upregulation is
allostatic since the basal Tcor was kept at the
same level but inhibited by the 5HT2AR
antagonist M100907 (fig. S2 B; also fig. 6A). The
data are consistent with the CLG+PRX
experiments in support of the hypothesis that
functional up-regulation of 5-HT2A receptors
(postsynaptic mechanisms) is responsible for
the syndrome intensity.
2