SupplementaryInformation
αCaMKII controls the establishment of cocaine’s reinforcing effectsin mice and humans
Alanna C. Easton1, Anbarasu Lourdusamy1, Michael Havranek2, Keiko Mizuno3, Jalal Solati4,5, Yulia Golub4,Toni-Kim Clarke6, Homero Vallada7, Ronaldo Laranjeira8, Sylvane Desrivières1, Gunther H. Moll4, Rainald Mössner9, Johannes Kornhuber10, Gunter Schumann1,K. Peter Giese3, Cathy Fernandes1,Boris B. Quednow2, Christian P. Müller1,10
Materials and Methods
Animals
Male and female αCaMKIIT286Amice (Giese et al, 1998) were studied in sex balanced designs in all experiments. Mutants were generated using a gene-targeting strategy which utilizes a replacement vector containing the point mutation and a neo gene flanked by loxP sites. After homologous recombination the neo gene was removed by Cre recombination (Giese et al, 1998). R1 ES cells (F1 between 129/Sv and 129/Sv-CP) were used. A PCR was used to detect the loxP site which determining the genotype. A second PCR was used to identify the point mutations. The mice were subsequently backcrossed for at least eight generations into C57BL/6J and then crossed once with 129S2/SvHsd mice and kept in this mixed background by interbreeding for reasons of fecundity. Mice had gone through more than four rounds of interbreeding before testing. Mice were obtained from interbreeding of heterozygotes and genotypes were determined by PCR with tail biopsies as described (Irvine et al, 2005). In homozygous mutants, the autophosphorylation of αCaMKII is prevented by the insertion of a missense mutation (threonine-286 changed to alanine; T286A) within the autoinhibitory domain(Giese et al, 1998). This mutation blocks the autophosphorylation of CaMKII but does not affect the Ca2+ dependent activity(Giese et al, 1998).Animals were individually housed in Tecniplast cages (32cm x 16cm x 14cm), using Litaspen sawdust and nesting materials, (Sizzlenest, Datsand, Manchester UK). Mice were provided with food and water ad libitum, and kept on a 12:12 hour light: dark cycle (lights on at 7.00 am). Behavioral tests were performed during the light cycle between 09:00 and 16:00. Room temperature was maintained between 19°C and 22°C at a humidity of 55% (±10%).
Conditioned place preference (CPP)
Apparatus
The TSE Place Preference test boxes (Bad Homburg, Germany) were made of non transparent PVC with standard inside dimensions of 40cm (L) x 15cm (W) x 20cm (H). The apparatus was divided into 3 fully automated compartments; the outer chambers measured 17cm in length, and the centre chamber 6cm. The floor of the left chamber (compartment A) was covered with a smooth black rubber mat. The floor of the right chamber (compartment B) was covered with a patterned black rubber mat. The centre chamber was not covered and coloured white (compartment C). Activity was recorded in each compartment using high-resolution infrared sensors. The system automatically recorded the number of entries made and distance moved in each compartment for each trial. An unbiased design was used, i.e. half the mice were conditioned to their preferred compartment, and half to their non-preferred compartment (Easton et al, 2013a).
In-vivo Microdialysis
Surgery
Cocaine naïve animals were used for this test (Mt: n=8; WT: n=8; Ht: n=8). The mice were deeply anaesthetised using a mixture of 4.12ml saline (NaCl), 0.38ml Ketaset (containing 100mg/ml Ketamine) and 0.5ml Domitor (containing 1mg/ml Medetomidine hydrochloride) administered i.p. at 0.1ml per 10g body weight. In addition 0.01ml Rimadyl (5mg/kg Carprofen) analgesia was given subcutaneously (s.c.). The animal was placed in a Kopf stereotaxic frame. Two guide cannulas (Microbiotech/se AB, Stockholm, Sweden) were aimed at the PFC (A: +1.9; L: ±0.8; V: -1.3 angle ±10º from midline) and the NAcc (A: +1.2; L: ±1.6; V: -4.3 angle ±10º from midline) using coordinates relative to bregma (Franklin and Paxinos, 1997), and fixed in place using two anchor screws (stainless steel, d=1.4mm) and dental cement. Anaesthesia was reversed by administering a mixture of 3.9ml saline (NaCl) and 0.1ml Antisedan (containing 5mg/ml Atipamezole) at 0.08ml/ 10g body weight (s.c.) after approximately 45 minutes. Animals were kept warm and allowed to recover from the anaesthetic. Animals were then returned to their home cages and monitored daily, allowing at least 5 days for complete recovery (Stacey et al, 2012; Easton et al, 2013b).
Procedure
On the day of the experiment, microdialysis probes of a concentric design were inserted into the guide cannulae under a short (3-5min) Isoflurane anaesthesia (O2 at 1L/min, Isoflurane at 3% to induce and 2% to sustain). Membrane lengths were 2mm for the PFC (MAB 6.14.2.) and 1mm (MAB 6.14.1.) for the NAcc. After probe insertion, the animal was placed into an open field (21x21x30cm) of a Truscan system (Coulbourn Instruments, Allentown, USA). Food and water were given ad libitum and room temperature maintained between 19-22 ºC. The microdialysis probes were connected to a microinfusion pump (CMA 400, Carnegie, Sweden) via a swivel mounted on a balanced arm above the chamber, and were perfused with artificial cerebrospinal fluid (aCSF) (containing Na+ 147 mmol, K+ 4 mmol, Ca2+ 2.2 mmol, Cl- 156 mmol, pH = 7.4) at room temperature (Müller et al, 2007). The flow rate was set to 1.5μl/min and allowed to stabilise for at least two hours until a stable baseline was obtained. Samples were collected every 20 minutes into vials containing 2.73μl of antioxidant (0.1 M perchloric acid and 500 pg dihydroxybenzylamine (DHBA) as internal standard). Three samples were taken during the first testing hour of the experiment to measure baseline quantities of the neurotransmitters DA, 5-HT and NA (Pum et al, 2007, 2008).An injection of cocaine was then administered i.p. (20 mg/kg, vinj=10 ml/kg). A further nine samples were collected simultaneously to behavioural data collection. Once microdialysis experiments were complete, animals were sacrificed by cervical dislocation. Brains were fixed in 4% formaldehyde solution and stored at 4°C. Brains were sliced on a microtome and stained with cresyl violet for analysis of probe placement.
HPLC-ED analysis
All samples were analysed using HPLC-ED to measure DA, 5-HT and NA levels in response to cocaine administration. The column was an ET 125/2, Nucleosil 120-5, C-18 reversed phase column (Macherey–Nagel, Germany) perfused with a mobile phase composed of 75 mM NaH2PO4, 4 mM KCl, 20μM ethylenediamine tetraacetic acid (EDTA), 1.5 mM sodium dodecyl sulfate, 100 μl/l diethylamine, 12% methanol, and 12% acetonitrile adjusted to pH 6.0 using phosphoric acid. The electrochemical detector (Intro, Antec, The Netherlands) was set at 500 mV vs. an in situ Ag/AgCl (ISAAC) reference electrode (Antec, Leyden, Netherlands) at 30°C. This setup allows the simultaneous measurement of DA, 5-HT and NA. The detection limit of the assay was 0.1pg for all neurotransmitters with a signal–noise ratio of 2:1. Neurochemical data were not corrected for recovery (Pum et al, 2007, 2008).
c-Fos activation after cocaine treatment
Naïve animals were used for this test (Mt: n=8; Ht: n=7, WT: n=8). Animals were transferred from the homecage to a temporary cage and injected with either saline or cocaine (15 mg/kg, i.p.). Mice were left undisturbed for 70 minutes after injection. Thereafter, mice were culled under isoflurane narcosis and transcardially perfused with 0.1M PBS for 10 minutes and then fixed with 4% paraformaldehyde (PFA) solution for a further 10 min (flow rate 4ml/min). The brain was removed and left in 4% PFA solution overnight at 4°C. Brains were then transferred to a 30% saccharose solution and stored at 4°C until brains were fully submerged. Brains were then snap frozen in isopentane at -60°C and stored at -80°C until the whole brain was cut into 40 µm coronal sections by cryosectioning. All sections were collected and then stored at -20°C in an anti-freezing solution until processed for immunohistochemical staining. The floating coronal sections were incubated with an anti-c-Fos rabbit polyclonal antibody (1:30.000, Calbiochem, Germany) for 20 hours. c-Fos immunoreactive cells were visualized using a biotinylated donkey anti-rabbit secondary antibody (1:500, Santa Cruz, Germany) and the avidin-biotin complex (ABC-Elite kit rabbit, Vector Laboratories, Germany) (Easton et al, 2013b).
Statistical Analysis
Microdialysis: Baseline neurochemical data were analysed using one-way ANOVA and planned Fisher’s LSD tests. Cocaine induced neurochemical effects were expressed as a percentage of the mean of the three baseline samples which were taken as 100%. Sex differences have been well established in clinical and rodent studies of cocaine-related behaviors. However, we did not see significant sex differences in this study. Therefore, data were collapsed for analysis. Data were compared using one-way and two-way ANOVAs for repeated measures followed by planned pairwise comparisons using Fisher’s LSD tests.
Results
Sensitisation effects of cocaine are not altered by genotype
CaMKII was shown to be involved in the acute and sub-acute locomotor activation after cocaine (Licata et al, 2003). The acute locomotor responses to cocaine and saline were identical in all genotype groups (p>0.05). Over seven cocaine treatments, a two-way ANOVA revealed a significant time effect (F1,42=28.25, p<0.0001), indicating a sensitization of cocaine-induced hyperlocomotion, but no genotype effect or interaction (p>0.05). Planned pairwise comparisons confirmed a significant increase in activity after seven cocaine pairings compared to activity levels after only one pairing for all genotypes (WT: p=0.01; Ht: p=0.003; Mt: p=0.01; Fig. 1B). This effect was not seen in the saline-paired compartment (Fig 1C; p>0.05). These data suggest that acute cocaine-induced hyperlocomotion as well as its sensitization over seven treatment trials do not depend on αCaMKII autophosphorylation.
Conditioned cocaine-induced hyperactivity is not altered by genotype
Repeated pairing of an environment with the hyperlocomotor effects of cocaine may lead to conditioned hyperlocomotion (Carey et al, 2005). We found conditioned hyperlocomotion in the conditioning compartment of the CPP box, but not in the saline-paired compartment (Fig. 1D and 1E). However, this effect was not genotype dependent. A two-way ANOVA revealed a significant time effect (F4,152=10.60, p<0.0001) of cocaine-induced hyperactivity in the cocaine-paired compartment, but no effect of genotype or interaction (p>0.05). Planned comparisons vs. baseline revealed an increase in locomotion in test trial one (p=0.019), trial 3 (p<0.0001), and trial seven (p=0.001). These data suggest that the αCaMKII autophosphorylation does not influence the establishment of conditioned hyperactivity.
Altered catecholamine levels in αCaMKII autophosphorylation deficient mice
We found that DA levels in the NAcc were comparable between WT and αCaMKIIT286A mice but were significantly elevated in Ht mice (F2,55=16.25, p<0.001;LSD: Ht vs. WT, p<0.001; Ht vs. Mt, p<0.001; Fig. 2A). DA levels in the PFC (Fig. 2B) were significantly greater in Ht and Mt mice compared to WT levels (F2,60=4.13, p=0.02; LSD: WT vs. Ht, p=0.03; WT vs. Mt, p=0.01).
Baseline 5-HT levels were elevated in the NAcc (Fig. 2C) in Ht and αCaMKIIT286A mice compared to WT mice (F2,59=5.43, p=0.007; LSD: WT vs. Ht, p=0.006; WT vs. Mt, p=0.004).Levels of 5-HT in the PFC (Fig. 2D) were higher in the αCaMKIIT286A mice compared to both WT and Ht mice (F2,64=10.45, p<0.001; LSD: Mt vs. WT, p=0.003; Mt vs. Ht, p=0.001).
NA levels in the NAcc (Suppl. Fig. 1A) were comparable between αCaMKIIT286A and WT mice, although levels of NA in Ht mice were elevated compared to other groups (F2,58=4.25, p=0.02; LSD: Ht vs. WT, p=0.006 ; Ht vs. Mt, p=0.05). Basal NA levels in the PFC (Suppl. Fig.1B) were significantly lower in αCaMKIIT286A mice (F2,61=5.31, p=0.007; LSD: Mt vs. WT, p=0.006; Mt vs. Ht, p=0.006). These findings suggest that a complete lack of αCaMKII autophosphorylation may enhance basal DA and 5-HT levels, and reduce basal NA levels in the PFC.
αCaMKII autophosphorylation is not required for cocaine-induced NA increases
NA levels in the NAcc increased in all genotype groups after cocaine treatment, suggesting that this effect was not dependent on αCaMKII autophosphorylation (Suppl.Fig. 1C). WT animals showed a significant NA increase vs. baseline in all intervals after injection (20-100 min and 140 min: p<0.01, 120 min and 160-180 min: p<0.05). In Ht mice there was also a significant increase in all intervals (20 min: p<0.05, 60 min and 100-180 min: p<0.01, 40 min and 80-100 min: p<0.001). In αCaMKIIT286A mice, the NA increase was statistically significant vs. baseline 20 min after injection (p=0.04). A two-way ANOVA showed a significant effect of time (F11,176=14.5, p<0.001), but no effect of genotype or interaction (p>0.05). A similar effect emerged in the PFC (Suppl.Fig. 1D). WT animals showed a significant NA increase vs. baseline in all intervals after injection (20 min, 80 min, 120 min, p<0.01, all other intervals: p<0.05). In Ht mice there was also a significant NA increase vs. baseline 60 min and 80 min (p<0.05) after injection. In αCaMKIIT286A mice, the NA increase was statistically significant vs. baseline in all intervals after injection (20-60 min, 120 min and 180 min: p<0.05; all other intervals: p<0.01). A two-way ANOVA revealed a significant effect of time (F11,187=10.62, p<0.001), but no effect of genotype or interaction (p>0.05). However, planned comparisons showed a difference between WT vs. Ht animals 20 min after cocaine injection (p=0.012). These findings suggest no major role for αCaMKII autophosphorylation in the acute NA response to cocaine.
Supplementary Figure 1.Baseline extracellular noradrenaline levels in A.the nucleus accumbens and B.prefrontal cortex (mean+SEM; Mt - αCaMKIIT286A, Ht – heterozygous, WT – wildtype; **p0.01, vs. WT; ##p0.01, vs. Ht).Acute cocaine effects on noradrenergic activity in the nucleus accumbens and prefrontal cortex represented as percent of baseline (mean+SEM). Extracellular levels in the C.nucleus accumbens, and in D. prefrontal cortex after acute cocaine (20 mg/kg, i.p.) treatment.Arrows indicate time of cocaine injection(p<0.05 WT vs. Ht; for more statistical details: see text).
Supplementary Figure 2. Showing the LD between 12 SNPs genotyped in CAMK2A. Shading represents r2 and the numbers inside the squares represent D’, where no number is present, D’=1. LD information taken from the HapMap CEU population (HapMap Data Release 28, Phase II & III). Haplotype blocks are defined according to the methods described by Gabriel and colleges.13
Supplementary Table 1. Association of CAMK2A SNPs with Kt, an index for the fast transition to dependent cocaine use, in a Brazilian sample of cocaine dependent individuals. Significant association results are shown in bold (Bonferroni-corrected).
SNP / Position (bp) / Minor Allele / MAF / Stat / Beta / P valuers2163766 / 149579741 / A / 0.09 / -0.451 / -0.066 / 0.650
rs4958469 / 149585309 / T / 0.23 / -1.048 / -0.102 / 0.295
rs17656349 / 149586187 / T / 0.47 / 0.675 / 0.054 / 0.500
rs2053053 / 149589586 / T / 0.32 / -0.042 / -0.004 / 0.966
rs2288799 / 149611606 / A / 0.46 / -0.683 / -0.057 / 0.495
rs3776823 / 149617798 / T / 0.27 / 2.934 / 0.275 / 0.003
rs874083 / 149631118 / A / 0.20 / -0.870 / -0.089 / 0.385
rs6881743 / 149640783 / C / 0.46 / -1.087 / -0.088 / 0.278
rs1432832 / 149653734 / C / 0.45 / 0.070 / 0.006 / 0.944
Supplementary Table 2. Association of threeCAMK2A SNPs with cocaine hair concentration-based Kthair in a Swiss sample of recreational and dependent cocaine users. Significant association results are shown in bold (Bonferroni-corrected).
SNP / Position (bp) / Minor Allele / MAF / F / df/dferr / P valuers4958469 / 149585309 / T / 0.25 / 1.540 / 2/138 / 0.218
rs3776823 / 149617798 / T / 0.26 / 0.669 / 2/138 / 0.514
rs6881743 / 149640783 / C / 0.29 / 5.495 / 2/138 / 0.005
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