Supplementary Material (doc 248K)
Distinct mu, delta and kappa opioid receptor mechanisms underlie low sociability and depressive-like behaviors during heroin abstinence
P.-E. Lutz 1,2 , G. Ayranci 1 , P. Chu-Sin-Chung 1 , A. Matifas 1 , P. Koebel 1 , D. Filliol 1 , K. Befort 1 , A.M.
Ouagazzal 1 , B.L. Kieffer 1,3
1 Translational Medicine and Neurogenetics, Institut de Génétique et de Biologie Moléculaire et
Cellulaire, INSERM U-964, CNRS UMR7104, Université de Strasbourg, Illkirch, F-67404 France
2 McGill Group for Suicide Studies, Douglas Institute Resear ch Centre, McGill University, Montréal,
Canada
3 Douglas Institute Research Centre, McGill University, Montréal, Canada
Corresponding authors
Brigitte L. Kieffer
Douglas Institute Research Centre, McGill University
6875, boulevard La Salle, Montréal (Québec) H4H 1R3 Canada
Phone: + 001 514-761-6131 ext. 3172
Fax: + 001 514 762-3033
E-mail addresses: brigitte.kieffer@douglas.mcgill.ca
P.-E. Lutz
McGill Group for Suicide Studies, Douglas Institute Research Centre, McGill University
6875, boulevard La Salle, Montréal (Québec) H4H 1R3 Canada
Phone: + 001 514-761-6131 ext. 6103
Fax: + 001 514 762-3023
E-mail addresses: pierreeric.lutz@gmail.com
Supplementary material and methods
Animals
All procedures followed ethical guidelines (EU 86/609/EEC, and IGBMC & ICS ethical comity,
ComEth saisine 2010-013). Mice were housed in groups of 3 –5 in a 12-h dark/light cycle under controlled conditions of temperature and humidity. Food and water were available ad libitum. In mu opioid receptor (MOR) conditional knock-out experiments, we used knock-in mice with a floxed
MOR gene (MOR fl/fl ) on a mixed 50% C57BL/6J-50% 129SvPas genetic background. KI mice were
generated as described previously (Weibel et al , 2013). Briefly, a 6.8 kb genomic clone (SalI/SpeI)
containing exons 2 and 3 of the MOR gene was isolated from 129Sv genomic DNA and cloned into pBluescript plasmid to generate the targeting vector. This clone was engineered to introduce a loxP site 600 bp upstream of exon 2, and sequenced to verify loxP sequence. In order to increase the homologous regions surrounding the targeted locus, the construct was extended in the 3’ direction with a 3.5 kb SpeI-SacI fragment isolated from another MOR genomic clone containing a region downstream of exon 3. A vector containing the floxed hygro cassette was obtained from D. Metzger
(IGBMC, Illkirch, France). The hygro cassette was removed from the cloning vector using NotI and
SalI, and the fragment filled in using the Klenow fragment to generate blunt ends. Similarly, the targeting construct was digested with SpeI, blunted and the floxed hygro cassette ligated into the open site. The final 12.5 kb construct were checked by restriction and sequence analysis before being linearized for electroporation into 129Sv derived embryonic stem (ES) cells, which were selected with hygromycin. Surviving cells were screened for homologous recombination by
Southern blotting with MfeI digests and using a 3' external probe (Suppl. Fig. 3A,B). Positive cells were transfected with a Cre recombinase-expressing plasmid for removal of the floxed-Hygro cassette. ES cells with the correct genotype were injected into C57BL/6J blastocysts, and resulting chimeric males were bred with C57BL/6J females to obtain germ line transmission. F1 heterozygous Oprm1fl/+ mice were intercrossed to generate the homozygous MOR fl/fl mouse line
(50% C57BL/6J-50% 129Sv genetic background). We verified that MOR fl/fl mice show intact MOR expression by using a [ 3 H]-DAMGO radioligand binding and a MOR agonist-stimulated [ 35 S]GTPγS
binding assays on brain membrane preparations (Gaveriaux-Ruff et al , 2011). The MOR ligand [
3 H]-
DAMGO bound similarly to brain membrane preparation from wild-type and MOR fl/fl mice, as shown by comparable Kd (1.53 ± 0.18 nM wild-type; 2.04 ± 0.31 nM MOR fl/fl , respectively) and Bmax (108 ±
11 pmol/mg protein wildtype; 109 ± 6 pmol/mg protein MOR fl/fl , respectively) values. DAMGO induced a similar dose-dependent increase of [ 35 S]GTPγS binding in preparations from the two genotypes (EC
50
315± 31 and 331 ± 44 nM; E max
178 ± 7% and 175 ± 5% of basal activity, respectively), indicating that insertion of LoxP sites did not disrupt gene transcription. Recently, our laboratory has been successfully using MOR fl/fl mice to induce the conditional knock-out (KO) of the
MOR gene in dorsal root ganglia, demonstrating that the floxed allele is accessible to Cre-mediated
recombination (Weibel et al , 2013).
Drugs
Morphine, heroin (Francopia, France), and naloxone (Sigma-Aldrich) were prepared in 0.9% sodium chloride. Mice were anaesthetized using ketamine/xylazine (Virbac/Bayer, 100/10 mg/kg). Drugs were administered subcutaneously (naloxone) or intraperitoneally (i.p., heroin, morphine and anesthetics) in a volume of 10 ml/kg (except for fluoxetine, see below).
Fluoxetine treatment
The 10 mg/kg dose was chosen based on pilot studies and our previous work on morphine
abstinence (Goeldner et al , 2011). Briefly, the amount of fluoxetine (Sigma-Aldrich, Lyon, France)
supplemented to regular chow powder was based upon initial body weight of animals and daily average food intake. For example, a 30 g mouse consuming 4 g/day (dry weight) of chow supplemented with 0.3 mg fluoxetine received an approximate 10 mg/kg/24h dose.
Appropriate amount of fluoxetine was dissolved in drinking water (600ml) and then thoroughly mixed with regular chow powder (1Kg, Safe R04, Augy, France) until a homogenous “dough” was obtained. Pellets were formed and frozen at 20°C overnight. The chow pellets were then lyophilized for 48 h and stored at 4°C before use. Lyophilization allows monitoring of dry weight food intake during experiments, by weighing remaining pellets in feeder.
All groups of mice were habituated to control
lyophilized chow (0mg/kg fluoxetine) for 1 week before experiments, during which their daily food intake was measured. Fluoxetine treatment was stopped 48 hours before the first behavioral test, to avoid acute effects of the drug.
Behavioural testing
See http://www.ics-mci.fr/service_neurobiology_behaviour_tests.html
for additional information on our phenotyping facility and procedures. As an index of general opiate effects, mice are weighed daily during chronic injections and after 1, 4 or 7 weeks of abstinence ( Supplementary Fig.4
).
Open-field (OF). Mice were placed during 30 min in 44x44 cm open-field arenas (Panlab,
Barcelona, Spain), fitted with infrared beams allowing automated locomotor activity measures
(Actitrack, Barcelona, Spain). Arenas were indirectly lit at 150 lux. Distance and time spent in the center during the first 15 minutes of the test were recorded as locomotor activity and anxiety measures. This 30-mn exposition to the open-field also served as habituation to the environment of the social interaction test, performed on the following day.
Tail suspension (TS). Mice were suspended by the tail 50 cm above the floor. Activity was automatically monitored during the last 4 min of the 6-minute test (MED associates Inc, St Albans,
Latency to the first immobilization was also noted.
Precipitated withdrawal
Mice were injected with escalating doses of morphine or heroin (or saline as a control), twice daily for 5 days and received a single injection on day 6. To measure physical dependence, withdrawal was precipitated by naloxone (1 mg/kg), administered either 2 h after the last morphine
was chosen based on previous comparisons of morphine and heroin precipitated withdrawals
across multiple inbred mice strains (Kest et al
, 2002; Klein et al , 2008), and reflects the faster
pharmacokinetic properties of heroin (Andersen et al , 2009). Somatic signs of withdrawal were
evaluated immediately after antagonist injection during a period of 20 min. Each animal was scored individually. The number of head shakes and wet dog shakes, front paw tremors, scratches, jumps and sniffing episodes was counted. Body tremor, ptosis, mastication, and piloerection were scored 1 for appearance or 0 for nonappearance within 5 min bins. Locomotor activity over 5 min periods was rated from 0 for inactivity to 2 for increased activity. A global withdrawal score was calculated for each animal by giving each somatic sign a relative weight: 0.5 for each episode of head shake or wet dog shake, paw tremor, scratching, sniffing and jumping; and 1 for the presence of body tremor, ptosis, mastication and piloerection during each observation period of 5 min. A score for locomotor
Brain dissections
Mice were killed by decapitation. The brain was quickly excised, rinsed in ice-cold phosphatebuffered saline (PBS, Sigma) and placed upside down in a chilled metal matrix (ASI Instruments,
Warren, MI, USA). As described previously (Contet et al , 2008), brain slices were obtained using
chilled razor blades inserted into 1-mm-spaced coronal grooves. Then, based on the stereotaxic
mouse brain atlas (Paxinos and Franklin, 2001), the dorsal raphe nucleus (DRN), median raphe
nucleus and periaqueductal gray were dissected using tissue corers (Fine Science Tools, Foster
City, USA) of 1 mm diameter. Brain punches were stored at 80°c and used for MOR agoniststimulated [ 35 S]-GTP
γS binding (Sovago et al , 2001).
Agonist-stimulated [ 35 S]GTPγS binding assays
Brain punches were homogenized in 800 µl of ice-cold 0.32 M sucrose solution, aliquoted and freshly used. MOR coupling to G-proteins was measured using agonist-stimulated [ 35 S]-GTP γS
binding assay (Sovago et al , 2001) on brain homogenates. Protein content was determined using
the Bradford assay. In each well, 2 μg of proteins were incubated in assay buffer (50 mM Tris-HCl pH 7.4;3 mM MgCl2; 100 mM NaCl; 0.2 mM EGTA; 30 mM GDP; 0.05 nM [ 35 S]GTPγS) with
ascending concentrations (10 -7 , 10 -6 and 10 -5 M) of the specific MOR agonist DAMGO, in a total volume of 200 μl, at 25°C for 1 h. Nonspecific binding was defined as binding in the presence of 10
µM unlabeled GTPγS, and basal binding indicates binding in the absence of agonist (Pradhan et al ,
2009). Reaction was terminated by rapid filtration through Whatman GF/B filters with an ice-cold
buffer containing 50 mM Tris-HCl pH 7.4, 50 mM NaCl and 5 mM MgCl2, using a Filtermate
Harvester (Perkin Elmer). Bound radioactivity was determined with a liquid scintillation analyzer
(TopCount NXT, Perkin Elmer). Results are expressed as percentage of basal [ 35 S]GTPγS binding.
Production of viral vectors
Recombinant Adeno-Associated Viruses Serotype 2 (AAV) were produced following the instructions
2011; Del Boca et al , 2012). AAV-293 cells were transfected with 3 plasmids: 2 auxiliary plasmids
providing either Adenovirus genes (pHelper) or AAV serotype 2 genes involved in replication and encapsidation (pAAV-RC); and the expression plasmid encoding the reporter enhanced green fluorescent protein (eGFP) or the Cre recombinase-eGFP (Cre) fusion protein, driven by CMV promoter (pAAV-eGFP or pAAV-Cre-eGFP). Two days after transfection, cells were harvested and freeze/thawed three times. The crude lysate was then treated with Benzonase (100U/mL) for 30min at 37°C and clarified by centrifugation. AAV were purified by ultracentrifugation using a preformed
discontinuous Iodixanol gradient (Zolotukhin et al , 2002) and dialysed in DPBS 0.5mM MgCL2 by
ultrafiltration using a Amicon Ultra15-100K filter. AAV titers were determined by quantitative PCR using a linearized standard plasmid and adjusted to 3.10
12 viral genome per mL.
Surgery and Virus Delivery
With a stereotaxic apparatus (Unimecanique, France), AAV-eGFP and AAV-Cre viruses were infused into the dorsal raphe nucleus (Paxinos and Franklin, 2001) under ketamine/xylazine
anesthesia. Coordinates for the injections, adapted from(Land et al , 2009; Thevenot et al , 2003),
were: anteroposterior, -0.3 mm from lambda; mediolateral, 0.0 mm from midline; dorsoventral, +3.55 mm from dura skull surface. A 5-ml microsyringe (SGE Analytical Science, Australia) was used for
viral infusions (1.5 ml/15 min), and was held in place for an additional 10 min before being removed.
Following surgery, mice were single housed for 48 h and then placed back in their home cage for 4 weeks prior to the quantification of MOR coupling efficiency, precipitated withdrawal or behavioral testing (see experimental timeline in Fig.4
). This 4-week duration was chosen based on previous experiments run in our lab to induce a shRNA-mediated knock-down of the MOR. Results indicated that the maximal decrease in MOR function is already obtained after 4 weeks, in agreement with
published studies (Lasek et al
, 2007; Zhang et al , 2009), and likely reflecting the MOR half-life.
Tissue preparation and stereotaxy validation
To determine the distribution of eGFP after DRN stereotaxy, mice were anaesthetized
(k etamine/xylazine) and intracardially perfused with 10 ml Dulbecco’s Phosphate Buffered Saline 1x
(DPBS PAA cat No:G1001,30130) at the speed of 5 mL/min followed by 60 ml 4% paraformaldehyde (Sigmaaldrich No:158127) in Dulbecco’s PBS 1x at the same speed. Brains were then postfixed for 24 hours at 4°C in the fixative solution. The tissue was cryoprotected at 4°C in a 30% sucrose in Dulbecco’s PBS 1x until the tissue sank. Tissue was then frozen in
Cryomatrix™ Frozen Embedding Medium (Thermo scientific No:6769006) and stored at -80°C until cut. Coronal sections were cut at 30 µM in a cryostat, mounted on Superfrost® Plus glass slides
(Thermo scientific No:J1800AMNZ) with Mowiol®/DAPI(1µg/mL) medium (Mowiol Calbiochem cat#475904, DAPI Roche ref:10 236 276 001) and cover slides, and observed under fluorescent microscopy. Finally, expression patterns of eGFP were observed independently by 2 observers.
Stereotaxy was considered successful when eGFP was largely restricted to the DRN, and extended rostro-caudally over at least 400 µm.
Supplementary Figures
Supplementary Figure 1. Physical dependence to morphine and heroin chronic treatments.
We measured physical dependence induced by a chronic heroin regimen in comparison with our earlier morphine protocol (a) . Morphine and heroin treated groups (n=8/group) both showed strong naloxone-precipitated withdrawal (One-way ANOVA, F(2,29)=199.1; p<0.001), with high global withdrawal scores compared to saline controls (n=16, panel b ), confirming that the heroin treatment induces severe physical dependence. Notably, withdrawals from morphine and heroin differed in 4
(paw tremors, body tremor, jumps and teeth chattering) out of 9 individual withdrawal signs (see panel b and also Supplementary Table1 ), suggesting qualitative differences in physical dependence achieved by these 2 drugs. Although morphine and heroin both exert their addictive-
like effects through the MOR (Le Merrer et al , 2009), combined results from this report and our
previous study (Goeldner et al , 2011) reveal differences in both acute and long-term effects of the
two drugs. While the global severity of physical dependence was similar, strong differences were detected for individual withdrawal signs. Also, despair-like behavior could be observed in the tail
suspension test after four weeks for morphine abstinence (Goeldner et al , 2011), but for heroin was
only detected after 7 weeks in the forced swim test (see main text, Fig.1
). Heroin, the synthetic diacetylated form of morphine, has poor agonist activity at MOR, in contrast with its metabolites 6-
MAM and morphine (Selley et al , 2001). Upon systemic administration, heroin acts as a lipophilic
pro-drug that easily crosses the blood brain barrier, triggering faster and stronger MOR activation
than morphine in the brain (Andersen et al , 2009; Gottas et al , 2012). A recent comprehensive
pharmacokinetic report showed that following heroin systemic injection, brain levels of 6-MAM, but
not of morphine, remarkably correlate with heroin-induced hyperlocomotion (Andersen et al , 2009).
Similarly in our model 6-MAM may recruit anatomically distinct receptor pools or engage distinct
MOR-associated signaling effectors (Pradhan et al , 2012) compared to morphine. The combined
actions of 6-MAM and morphine at MORs after heroin injections would thus lead to slightly distinct withdrawal and emotional syndromes, compared to morphine alone.
Supplementary Figure 2. Locomotor activity and anxiety-like behaviours are not modified following 1, 4, or 7 weeks of heroin abstinence. (a) In the open-field test (OF), locomotor activity was modified by heroin treatment (2-way ANOVA; (F(1;135)=7,8; p=0,006)), with a significant effect of abstinence duration (F(2;135)=34,8; p<0,001), but no interaction between the 2 factors
(F(2;135)=0,75; p=0,47). (b) In this test also, the percentage of time spent in the centre, a classical measure of anxiety-like behaviours in rodents, was not affected by heroin treatment [F(1;135)=1,9; p=0,17], while there was an effect of abstinence duration [F(2;135)=9,0; p<0,001] but no interaction
[F(2;135)=1,3; p=0,27]. Every pair-wise comparisons (1 vs 4 week, p=0.04; 1 vs 7 week, p=0.04; 4 vs 7 week, p<0.001) were significant, in particular for the 7-week time point. Therefore, reminiscent of our previous morphine study, heightened anxiety-like level was observed in both saline- and heroin-treated animals during early abstinence. This effect may result from the stress associated with twice-daily handling and intraperitoneal injections, still detectable 1 and 4 weeks later, but is nonetheless unrelated to heroin treatment per se. + p<0.01, ANOVA main effect of heroin; # p<0.001, ANOVA main effects of abstinence duration.
Supplementary Figure 3. Effects of 1, 4, or 7 weeks of heroin abstinence on despair-like behaviours, spatial working memory and sucrose preference. Immobility in the tail suspension test (TS), a classical measure of depressive-like behaviours in rodents (a) , was not modified by heroin treatment (F(1;89)=0,46; p=0,50) or the duration of abstinence (F(1;89)=0,009; p=0,92), and there was no interaction between the 2 factors (F(1;89)=0,45; p=0,50).
Latency to first immobilization, a secondary index of behavioural despair (b) , was similarly unaffected by heroin treatment (F(1;89)=0,044; p=0,83) or abstinence duration (F(1;89)=2,5; p=0,12), without an interaction (F(1;89)=3,7; p=0,058).
In the sucrose preference test, heroin treatment modified sucrose intake [F(1;130)=6,8; p=0,010] (c) , with an effect of abstinence duration [F(2;130)=16,7; p<0,001] and a significant interaction [F(2;130)=3,9; p=0,024]. Post-hoc comparisons showed that sucrose consumption is significantly increased in heroin abstinent mice compared to saline controls after 1 week of abstinence (p<0.001), but not later on during abstinence (4 weeks, p=0.76; 7 weeks, p=0.64). While the hedonic tone is not disrupted in heroin-treated mice (see Fig.1f
), this increased
sucrose intake after 1 week of abstinence likely reflects the recovery from heroin-induced body weight loss, which is not fully achieved at this time point (see Supplementary Fig.4
). In the Y-maze task (YM), exploratory locomotor activity (d) was affected by heroin treatment (F(1;135)=4.7; p=0.032), with no effect of abstinence duration (F(1;135)=1,6; p=0,20) and no interaction
(F(2;135)=2,4; p=0,098). Post-hoc comparisons showed that this heroin effect is significant at the 4week (p=0,027), but not 1- (p=0,75) or 7-week (p=0,67) time points, suggesting that impaired working memory during heroin abstinence ( Fig.1g) isn’t merely the result of increased exploratory drive. n.d., not determined. * p<0.05, post-hoc effects of heroin for specific abstinence durations.
Supplementary Figure 4. Weight evolution during chronic intermittent heroin exposure, and following either 1 (a), 4 (b) or 7 (c) weeks of abstinence. Repeated ANOVA indicated that heroin treatment reduced body weight across all 3 cohorts of mice (1-week abstinence (F(1;230)=151,2; p<0,001); 4-week abstinence, (F(1;230)=36,6; p<0,001) and 7-week abstinence, (F(1;230)=98,31; p<0,001)). In addition, across these 3 cohorts we found significant (i) effects of time (1-week abstinence (F(1;230)=54,5; p<0,001); 4-week abstinence, (F(1;230)=112,6; p<0,001), and 7-week abstinence, (F(1;230)=319,2; p<0,001) and (ii) significant interactions between time and treatment
(1-week abstinence (F(1;230)=23,2; p<0,001); 4-week abstinence, (F(1;230)=11,1; p<0,001), and 7week abstinence, (F(1;230)=17,7; p<0,001)). Importantly, post-hoc comparisons show that herointreated mice have decreased body weights compared to saline controls, as soon as on Day 2 of chronic injections up to the end of the first week of abstinence (p<0.05). Later on during abstinence, heroin-treated mice recovered body weight no longer different from saline controls (p>0.05). * p<0.05, post-hoc effects of heroin at specific time points.
Supplementary Figure 5. Anxiety- and depressive-like behaviours are not modified following
4 weeks of heroin abstinence in delta (DOR) and kappa (KOR) opioid receptor knock-out
(KO) mice. In DOR KO mice (a-c) , the percentage of time spent in the centre (a) of the open-field
(OF) was significantly decreased compared to wild-type (WT) controls (F(1,51)=4.07; p=0.049), while there was no main effect of previous heroin exposure (F(1,51)=0.28; p=0.60) and no
interaction between factors (F(1,51)=0.90; p=0.35). This anxiogenic-like effect of the mutation is
effect of genotype or heroin treatment on 2 despair-like measures in the tail suspension test (TS):
(b) duration of immobility (heroin, (F(1,51)=3.5; p=0.067); genotype, (F(1,51)=0.029; p=0.87), interaction (F(1,51)=0.69; p=0.41)), and (c) latency to first immobilization (heroin, (F(1,51)=0.74; p=0.39); genotype, (F(1,51)=0.12; p=0.73); interaction, (F(1,51)=0.33; p=0.57)). These results contrast with our previous report of increased behavioural despair in adult naïve DOR KO mice,
using the forced swim test (see (Filliol et al , 2000)). In the present study, the chronic injection
regimen, and associated handling, may have limited our ability to detect this phenotype.
Alternatively, forced swim and tail suspension tests use different stressors and explore non-identical emotional dimensions, which may show divergent DOR-dependent regulation. In KOR KO mice (df) , there was no effect of the mutation or the heroin treatment in any of these behavioural measures.
The percentage of time spent (d) was not affected by heroin (F(1,62)=0.005; p=0.95) or genotype
(F(1,62)=0.070; p=0.79), with no interaction (F(1,62)=0.49; p=0.48). Similarly, we found no effect of heroin (F(1,62)=0.070; p=0.79) or genotype (F(1,62)=0.070; p=0.79), and no interaction
(F(1,62)=0.070; p=0.79), on the duration of immobility (e) in the TS. Finally, no effect was detected on the latency to the first immobilization (f) for heroin (F(1,62)=0.016; p=0.90), genotype
(F(1,62)=1.39; p=0.24), or their interaction (F(1,62)=1.63; p=0.21) . ǂ p<0.05, ANOVA main effect of genotype. DOR KO and WT littermates were bred independently from KOR KO mice and WT littermates.
Supplementary Figure 6. Four weeks of heroin abstinence and chronic fluoxetine treatment do not modify measures of locomotor activity, anxiety-like behaviours and despair-like behaviours. Due to a technical error, data were available and analysed for 44 mice only in the
Open-field (OF) test. In this test, statistical analysis revealed no effect on locomotor activity (a) of heroin (F(1;44)=0.002; p=0.96) or fluoxetine (F(1;44)=3.7; p=0.067) treatments, and no interaction
(F(1;44)=0.70; p=0.41). Similarly, the percentage of time spent in the centre of the OF (b) was not modified upon heroin (F(1,44)=1.1; p=0.30) or fluoxetine (F(1,44)=0.013; p=0.91) treatment, with no
interaction (F(1,44)=0.23; p=0.63). In the tail suspension test (TS), in agreement with our kinetic experiments ( Supplementary Fig.3a-b ), despair-like behaviours were not affected after 4 weeks of heroin abstinence. In the analysis of duration of immobility (c) , we detected no effect of heroin
(F(1;63)=0.68; p=0.41) or fluoxetine (F(1;63)=0.27; p=0.61), while there was no interaction
(F(1;63)=0.006; p=0.94),. Also, latency of immobilization (d) was unchanged following either heroin
(F(1;63)=1.53; p=0.22) or fluoxetine (F(1;63)=1.04; p=0.31) exposures, with no interaction
(F(1;63)=0.55; p=0.46).
Supplementary Figure 7. Anxiety-like and depressive-like behaviours are not modified during a 4-week abstinence period or conditional knock-out of the mu opioid receptor (MOR) in dorsal raphe nucleus (MOR cKO). In the open-field test (OF), we found no effect of heroin treatment (F(1,25)=0.17; p=0.69) or genotype (F(1,25)=0.16; p=0.69), and no interaction
(F(1,25)=0.04; p=0.84), on the total distance travelled (a) . Similarly, the percentage time spent in the centre of the arena (b) was not modified by either heroin treatment (F(1,25)=0.78; p=0.39) of the genotype (F(1,25)=0.048; p=0.83), and there was no interaction (F(1,25)=0.18; p=0.67). In the forced swim test (FS), duration of immobility (c) was not affected by previous heroin exposure
(F(1,39)=0.08; p=0.78), or by the genetic manipulation (F(1,39)=0.61; p=0.44), and we found no significant interaction (F(1,39)=0.23; p=0.63). In addition, heroin treatment (F(1,39)=0.30; p=0.59) and genotype (F(1,39)=0.20; p=0.66) had no effect on latency to first immobilization (d) , with no interaction (F(1,39)=0.81; p=0.38). M, molar concentration.
References
Andersen JM, Ripel A, Boix F, Normann PT, Morland J (2009). Increased locomotor activity induced by heroin in mice: pharmacokinetic demonstration of heroin acting as a prodrug for the mediator 6monoacetylmorphine in vivo. The Journal of pharmacology and experimental therapeutics 331 (1):
153-161.
Contet C, Filliol D, Matifas A, Kieffer BL (2008). Morphine-induced analgesic tolerance, locomotor sensitization and physical dependence do not require modification of mu opioid receptor, cdk5 and adenylate cyclase activity. Neuropharmacology 54 (3): 475-486.
Darcq E, Befort K, Koebel P, Pannetier S, Mahoney MK, Gaveriaux-Ruff C , et al (2012). RSK2 signaling in medial habenula contributes to acute morphine analgesia. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 37 (5): 1288-1296.
Darcq E, Koebel P, Del Boca C, Pannetier S, Kirstetter AS, Garnier JM , et al (2011). RSK2 signaling in brain habenula contributes to place aversion learning. Learn Mem 18 (9): 574-578.
Del Boca C, Lutz PE, Le Merrer J, Koebel P, Kieffer BL (2012). Cholecystokinin knock-down in the basolateral amygdala has anxiolytic and antidepressant-like effects in mice. Neuroscience .
Filliol D, Ghozland S, Chluba J, Martin M, Matthes HW, Simonin F , et al (2000). Mice deficient for delta- and mu-opioid receptors exhibit opposing alterations of emotional responses. Nature genetics
25 (2): 195-200.
Gaveriaux-Ruff C, Nozaki C, Nadal X, Hever XC, Weibel R, Matifas A , et al (2011). Genetic ablation of delta opioid receptors in nociceptive sensory neurons increases chronic pain and abolishes opioid analgesia. Pain 152 (6): 1238-1248.
Goeldner C, Lutz PE, Darcq E, Halter T, Clesse D, Ouagazzal AM , et al (2011). Impaired emotionallike behavior and serotonergic function during protracted abstinence from chronic morphine.
Biological psychiatry 69 (3): 236-244.
Goeldner C, Reiss D, Kieffer BL, Ouagazzal AM (2010). Endogenous nociceptin/orphanin-FQ in the dorsal hippocampus facilitates despair-related behavior. Hippocampus 20 (8): 911-916.
Gottas A, Oiestad EL, Boix F, Ripel A, Thaulow CH, Pettersen BS , et al (2012). Simultaneous measurement of heroin and its metabolites in brain extracellular fluid by microdialysis and ultra performance liquid chromatography tandem mass spectrometry. Journal of pharmacological and toxicological methods 66 (1): 14-21.
Kest B, Palmese CA, Hopkins E, Adler M, Juni A, Mogil JS (2002). Naloxone-precipitated withdrawal jumping in 11 inbred mouse strains: evidence for common genetic mechanisms in acute and chronic morphine physical dependence. Neuroscience 115 (2): 463-469.
Klein G, Juni A, Waxman AR, Arout CA, Inturrisi CE, Kest B (2008). A survey of acute and chronic heroin dependence in ten inbred mouse strains: evidence of genetic correlation with morphine dependence. Pharmacology, biochemistry, and behavior 90 (3): 447-452.
Land BB, Bruchas MR, Schattauer S, Giardino WJ, Aita M, Messinger D , et al (2009). Activation of the kappa opioid receptor in the dorsal raphe nucleus mediates the aversive effects of stress and reinstates drug seeking. Proceedings of the National Academy of Sciences of the United States of
America 106 (45): 19168-19173.
Lasek AW, Janak PH, He L, Whistler JL, Heberlein U (2007). Downregulation of mu opioid receptor by RNA interference in the ventral tegmental area reduces ethanol consumption in mice. Genes
Brain Behav 6 (8): 728-735.
Le Merrer J, Becker JA, Befort K, Kieffer BL (2009). Reward processing by the opioid system in the brain. Physiol Rev 89 (4): 1379-1412.
Le Merrer J, Befort K, Gardon O, Filliol D, Darcq E, Dembele D , et al (2011). Protracted abstinence from distinct drugs of abuse shows regulation of a common gene network. Addict Biol .
Paxinos G, Franklin KBJ (2001). The Mouse Brain in Stereotaxic Coordinates. 2nd ed Academic
Press .
Pradhan AA, Becker JA, Scherrer G, Tryoen-Toth P, Filliol D, Matifas A , et al (2009). In vivo delta opioid receptor internalization controls behavioral effects of agonists. PLoS One 4 (5): e5425.
Pradhan AA, Smith ML, Kieffer BL, Evans CJ (2012). Ligand-directed signalling within the opioid receptor family. British journal of pharmacology 167 (5): 960-969.
Selley DE, Cao CC, Sexton T, Schwegel JA, Martin TJ, Childers SR (2001). mu Opioid receptormediated G-protein activation by heroin metabolites: evidence for greater efficacy of 6monoacetylmorphine compared with morphine. Biochem Pharmacol 62 (4): 447-455.
Sovago J, Dupuis DS, Gulyas B, Hall H (2001). An overview on functional receptor autoradiography using [35S]GTPgammaS. Brain Res Brain Res Rev 38 (1-2): 149-164.
Thevenot E, Cote F, Colin P, He Y, Leblois H, Perricaudet M , et al (2003). Targeting conditional gene modification into the serotonin neurons of the dorsal raphe nucleus by viral delivery of the Cre recombinase. Mol Cell Neurosci 24 (1): 139-147.
Weibel R, Reiss D, Karchewski L, Gardon O, Matifas A, Filliol D , et al (2013). Mu opioid receptors on primary afferent nav1.8 neurons contribute to opiate-induced analgesia: insight from conditional knockout mice. PLoS One 8 (9): e74706.
Zhang Y, Landthaler M, Schlussman SD, Yuferov V, Ho A, Tuschl T , et al (2009). Mu opioid receptor knockdown in the substantia nigra/ventral tegmental area by synthetic small interfering
RNA blocks the rewarding and locomotor effects of heroin. Neuroscience 158 (2): 474-483.
Zolotukhin S, Potter M, Zolotukhin I, Sakai Y, Loiler S, Fraites TJ, Jr.
, et al (2002). Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2): 158-
167.
