Supplementary data
Materials and methods
HPLC determinations of 5-HT and 5-HIAA
Brain regions [amygdala, frontal cortex, hippocampus, ventral striatal region including
mainly the nucleus accumbens, ventral tegmental / substantia nigra (VTA/SN)] were
dissected on ice, weighed and homogenized in an extraction solution (0.1 M perchloric acid;
1.34 mM EDTA; sodium metabisulfite 0.05% w/v) by sonication for 15 s. Samples were then
centrifuged at 30,000 g for 20 min at 4°C and 10 µl of the supernatant was automatically
injected into the HPLC system. 5-HT and 5-HIAA were separated on an Ultrasphere IP C18
column (25 x 0.46 cm; Beckman) with a mobile phase (70 mM K2HPO4, 3.1 mM triethylamine, 0.1 mM EDTA, methanol 16 % v/v, 1.05 mM octane sulphonate, pH 3) at a flow
rate of 1 ml/min. Compounds were oxidized by a coulorimetric electrochemical detector
(ESA, Bedford, MA, USA) and the signal was analyzed by the acquisition software Empower 2
(Waters, Saint-Quentin en Yvelines, France). Results are expressed as ng/g of fresh tissue
(supplemental Table 1) and 5-HIAA/5-HT ratios (Table 1).
5-HT2CR immunohistochemistry coupled to c-fos mRNA in situ hybridization
ProtectRNA™
0.2%
and
DEPC-treated
components
were
used
throughout
the
immunohistochemistry procedure to prevent mRNA degradation before the in situ
hybridization assay, performed 3 days later.
Free floating brain sections were washed (3 x 5 min) in DEPC-PBS with slow orbital shaking,
incubated for 30 min in a solution of 1% H2O2, 50% ethanol and 49% DEPC-PBS (v/v), and
washed again (3 x 5 min) with DEPC-PBS. Brain sections were then incubated in blocking
solution A [3% normal goat serum (Jackson Immunoresearch, Newmarket, Suffolk, UK), 0.4%
Triton X-100 (Sigma-Aldrich), in DEPC-PBS] for 1 h at room temperature, before starting the
incubation with the 5-HT2CR primary antibody (1:200) diluted in a freshly prepared solution A
during 48 h at 4°C. To proceed, sections were washed in DEPC-PBS (3 x 5 min) and incubated
1 h in blocking solution B (1% normal goat serum, 0.3% Triton X-100, in DEPC-PBS), followed
by 2 h in the same (but fresh) solution supplemented with the secondary antibody (1:200
biotinylated goat anti-rabbit IgG from Vector, Philadelphia, USA). For the following steps,
sections were first exposed to the streptavidin-peroxidase complex prepared in DEPC-PBS +
0.3% Triton X-100 (1:200 of Vectastain ABC kit; Vector) for 2 h then to 3-3’-diaminobenzidine
(DAB; Sigma-Aldrich) diluted in DEPC-PBS with 1% H2O2 for 10 min, and 2% H2O2 for the
subsequent 20 min. The reaction was stopped by 4 washes in DEPC-PBS. Sections were
stored in 4% PFA at 4°C until the in situ hybridization.
The c-fos mRNA in situ hybridization assay was performed as previously described
(Mongeau et al., 2003). Immunolabeled sections were gently digested for 20 min using
proteinase K (10 µg/mL), fixed with 4% PFA and hybridized at 60°C with a cRNA digoxigeninlabeled probe specifically binding c-fos mRNA. The non-hybridized probe was washed off at
60°C and digested with RNAse A at 37°C for 30 min. Immunohistochemistry was performed
using the anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche, Meylan,
France). Development was made with the alkaline phosphatase substrate (0.18 mg/mL of 5bromo-4-chloro-3-indolyl phosphate, BCIP, + 0.5 mg/mL nitro blue tetrazolium chloride,
NBT; Roche) in recommended buffer (100 mM Tris-HCl, pH 9.5, 50 mM MgCl2, 100 mM NaCl,
2 mM levamisole) at 37°C until the blue color generated was sufficient to distinguish c-fos
positive cells (generally 5 hours). Sections were washed in the TBST (500 mM NaCl, 20 mM
Tris-HCl pH 9.5, 0.1% Tween 20) and PBS solutions. To stop the reaction, sections were
incubated 30 min in MABT (100 mM maleic acid, pH 7.5, 150 mM NaCl, 0.1% Tween 20) at
65°C, washed two times in MABT solution and incubated 30 min in an acidic glycine solution
(0.1 M glycine, pH 2.2). Finally, sections were washed in MABT and fixed in 4% PFA at 4°C,
before being mounted in 50% glycerol.
5-HT2CR mRNA quantification by real time RT-qPCR
RNA extraction was done using the RNeasy Kit (Qiagen, GmbH, Germany). Then, samples
were homogenized in lysis buffer containing 2-mercapto-ethanol and RNAse inhibitor.
Purified RNAs were eluted in “RNAse free” water, and RNA dosage was made by
spectrophotometry.
Single-stranded DNAs were synthetized from pure RNA by SuperScript™ III reverse
Transcriptase (Invitrogen, Cergy-Pontoise, France). Real-time PCR amplification was done in
triplicate in a device type ABI Prism 7300 (Applied Biosystems, Courtaboeuf, France;
Reaction mixture ABgene® Absolute QPCR ROX Mix ABgene, Courtaboeuf, France), with
primers specific of the HTR2C gene (Mm00434127_m1) and Taqman probes (Applied
Biosystems, France). PCR amplification of a housekeeping gene (GAPDH) also proceeded in
parallel. The program of assay amplification consisted of a polymerase activation step (95°C,
15 min), followed by 40 cycles of denaturation (95°C, 15 s), annealing and elongation (60°C,
60 s). 5-HT2CR mRNA quantification was made using the RQ Study Software (version 1.2,
Applied Biosystems).
Measurements of pre-mRNA editing with nested PCR coupled to capillary electrophoresis
Reverse transcription
Mice were decapitated, their brain was immediately dissected on ice and the frontal cortex
was isolated. Purified mRNA samples were extracted and treated with DNAse (DNAse kit,
Invitrogen). Total RNA was linearized before reverse transcription using reverse
transcriptase kit (Thermoscript RT-PCR system, Invitrogen).
Nested-PCR
Nested-PCR was performed as previously described (Poyau et al., 2007). cDNA were
amplified (with the platinium Taq DNA Polymerase kit) in two steps. First, cDNA of 5-HT2C
receptors was amplified using two primers at 5’ and 3’ ends of exons IV-V of the HTR2C gene
sequence. Because the space between them was too short to be amplified, after the first
PCR reaction, a second PCR was performed to optimize the size of cDNA fragment and to
label 5’ ends of the forward and reverse strand with FAM and VIC fluorophores, respectively.
SSCP analyses and capillary electrophoresis
Samples were then denatured and immediately placed on ice. Separation was performed by
non-denaturing electrophoresis with the ABI PRISM® 3100-Avant Genetic Analyzer (Applied
Biosystems) on a 80 cm long capillary, filled with 7% of “POP™ Conformational Analysis
Polymer” (Applied Biosystems), borate-EDTA 1X. This technique relies on the fact that
hydrodynamic volume differs among cDNA sequences. After a pre-run performed at 15 kV
during 3 min, samples were injected at 2 kV during 15 s, and electrophoresis proceeded at
15 kV during 105 min, at a fixed temperature of 24°C. In these conditions, each isoform is
identified as a single peak with a specific retention time (Poyau et al., 2007).
Identification and relative quantification of each isoform
For each electrophoresis profile of fluorophores-labeled DNA fragments, the software
Peakfit® (v4.11,s Ritme Informatique, Paris, France) allowed determination of normalized
values to establish the percentage of each isoform relatively to the total (100%).
References :
Mongeau R, Miller GA, Chiang E, Anderson DJ (2003) Neural correlates of competing fear
behaviors evoked by an innately aversive stimulus. J Neurosci 23: 3855–3868.
Poyau A, Vincent L, Berthommé H, Paul C, Nicolas B, Pujol J-F, Madjar J-J (2007)
Identification and relative quantification of adenosine to inosine editing in serotonin 2C
receptor mRNA by CE. Electrophoresis 28: 2843–2852.
Supplementary results
5-HT
5-HIAA
N. accumbens
Genotype/group
5-HTT+/+
5-HTT-/-
Genotype/group
5-HTT+/+
5-HTT-/-
Naïve
476 ± 23
171 ± 12
Naïve
231 ± 16
116 ± 10
Stress
451 ± 17
165 ± 10
Stress
299 ± 19†
133 ± 13
+/+
-/-
Genotype/group
5-HTT+/+
5-HTT-/-
Amygdala
Genotype/group
5-HTT
5-HTT
Naïve
706 ± 29
269 ± 23
Naïve
290 ± 37
149 ± 19
Stress
743 ± 38
258 ± 45
Stress
528 ± 36†
235 ± 33†
+/+
-/-
Genotype/group
5-HTT+/+
5-HTT-/-
Frontal cortex
Genotype/group
5-HTT
5-HTT
Naïve
277 ± 27
106 ± 4
Naïve
136 ± 9
62 ± 9
Stress
285 ± 22
97 ± 13
Stress
169 ± 11†
71 ± 7
Genotype/group
5-HTT+/+
5-HTT-/-
Genotype/group
5-HTT+/+
5-HTT-/-
Naïve
501 ± 18
118 ± 4
Naïve
316 ± 21
124 ± 4
Stress
542 ± 22
107 ± 6
Stress
383 ± 23†
143 ± 8 †
Genotype/group
5-HTT+/+
5-HTT-/-
Genotype/group
5-HTT+/+
5-HTT-/-
Naïve
980 ± 40
259 ± 17
Naïve
768 ± 33
407 ± 24
Stress
935 ± 63
287 ± 22
Stress
818 ± 68
482 ± 33
Hippocampus
VTA/SN
Supplemental Table 1: 5-HT and 5-HIAA levels in various brain areas of 5-HTT-/- versus 5HTT+/+ mice – Effects of stress
5-HT and 5-HIAA levels were measured by HPLC-ED in nucleus accumbens (n=6-8), amygdala
(n=6-7), frontal cortex (n=7), hippocampus (n=7-8) and ventral tegmental area/subtantia
nigra (VTA/SN, n=6-8) of 5-HTT+/+ and 5-HTT-/- mice under basal conditions or after a
restraint stress of 45 minutes. Values, expressed as ng per g of fresh tissue, are mean ±
S.E.M. † p≤0.05 Student’s t-test comparing stressed versus naïve mice of the same genotype.