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Inhibition of the lateral habenular CaMKⅡ abolishes naloxone-precipitated conditioned place aversion in morphine-dependent mice
Accepted Manuscript Title: Inhibition of the lateral habenular CaMKII abolishes naloxone-precipitated conditioned place aversion in morphine-dependent mice Authors: Jing Wang, Min Li, Ping Wang, Yunhong Zha, Zhi He, Zicheng Li PII: DOI: Reference:
S0304-3940(17)30415-9 http://dx.doi.org/doi:10.1016/j.neulet.2017.05.027 NSL 32834
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
15-2-2017 30-4-2017 15-5-2017
Please cite this article as: Jing Wang, Min Li, Ping Wang, Yunhong Zha, Zhi He, Zicheng Li, Inhibition of the lateral habenular CaMKII abolishes naloxoneprecipitated conditioned place aversion in morphine-dependent mice, Neuroscience Lettershttp://dx.doi.org/10.1016/j.neulet.2017.05.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
Intra-lateral habenula administration of TTX abolished naloxone-precipitated CPA in morphine-dependent mice.
Intra-lateral habenula administration of KN-62 abolished naloxone-precipitated CPA in morphine-dependent mice.
Chronic morphine treatment resulted in overexpression of CaMKⅡ in the lateral habenula.
Title: Inhibition of the lateral habenular CaMKⅡ abolishes naloxone-precipitated conditioned place aversion in morphine-dependent mice Author: Jing Wang1 *, Min Li1, 2 *, Ping Wang2, Yunhong Zha2, Zhi He1 and Zicheng Li1, 2 #, 1
Medical College of China Three Gorges University, Yichang 443002, China.
2
People's Hospital of China Three Gorges University, Yichang 443002, China.
* Jing Wang and Min Li contributed equally to this paper. #
Corresponding author:
Zicheng Li, Ph.D. Department of Biochemistry and Molecular Biology Medical College of China Three Gorges University 8 Daxue Road Yichang 443002 People’s Republic of China Phone: (86) (717) 6396818 e-mail: [email protected] Abstract Addictive substances mediate positive and negative states promoting compulsive drug use. However, substrates for aversive effects of drugs are not fully understood. We found that inactivation of the lateral habenula (LHb) by microinjection of tetrodotoxin (TTX) abolished naloxone-precipitated conditioned place aversion (CPA) in morphine-dependent mice. We also found that lateral habenular administration of KN-62, a specific inhibitor for calcium/calmodulin dependent protein kinase II (CaMKII), abolished naloxone-precipitated CPA in morphine-dependent mice. Furthermore, we found chronic morphine treatment induced overexpression of CaMKII in the LHb. In conclusion, our results suggest that the increased expression of CaMKII in the LHb is instrumental for morphine-driven aversive behaviors. Key words: morphine; calcium/calmodulin dependent protein kinase II (CaMKII); lateral habenula (LHb); conditioned place aversion (CPA);
1. Introduction Withdrawal from addictive substances, including morphine, produces negative symptoms that contribute to compulsive drug abuse [1]. The lateral habenula (LHb) inhibits monoaminergic systems via the GABAergic rostromedial tegmental nucleus (RMTg), encoding aversion-related stimuli [2]. Notably, functional modifications of the LHb are associated with neuropsychiatric disorders, including addiction such as cocaine [3, 4] and alcohol addiction [5]. However, it is unknown whether LHb is involved in the morphine withdrawal syndromes. Furthermore, the underlying molecular mechanisms mediating morphine-driven aversive behaviors are elusive. Calcium/calmodulin-dependent protein kinase II (CaMKII), a multiple functional enzyme, is one of the most abundant kinases in the brain, comprising about 2% of total proteins [6, 7]. At least four isoforms of CaMKII (α, β, γ and δ) are found to express in the brain and to function in the form of homomultimers or heteromultimers [6, 7]. Among them, the α and β isoforms are restricted in nervous tissues, whereas γ and δ are found in most tissues besides brain [6, 7]. CaMKII has been reported to play important roles in various neuronal adaptive processes such as drug addiction [8, 9]. For example, studies revealed that inhibition of CaMKII by microinjection of specific inhibitors KN-62 into the hippocampus decrease the morphine withdrawal syndromes induced by opiate antagonist naloxone [10] and suppress the development of formation and reactivation of morphine conditioned place preference (CPP) [10]. More studies imply that the expression of CaMKII in brain can be selectively regulated by acute or chronic morphine treatment [11-13].These data strongly suggest that CaMKII play important roles in the development of morphine addiction. However, there is no evidenc showing CaMKII in the LHb is involved in the development of morphine addiction. Considering the overexpression of CaMKII in the LHb is instrumental for processing of aversive information [14], so we hypothesized that the morphine withdrawal syndromes may attribute to upregulation of CaMKII in the LHb under chronic morphine treatment.
In the present study, to confirm the role of CaMKII in the LHb in morphine withdrawal syndromes, we first investigated the effects of inactivation of the LHb by microinjection of tetrodotoxin (TTX), a Na+ channels blocker, on naloxone-precipitated conditioned place aversion (CPA) in morphine-dependent mice. We then observed the effects of microinjection of KN-62, a selective CaMK II inhibitor, into the LHb on CPA. Finally, the effects of chronic morphine treatment on the protein levels of CaMKII in the LHb were examined. 2. Materials and methods 2.1. Chronic morphine treatment Male C57BL/6 mice (7-8 weeks old for behavioral test and western blot analysis, 4-5 weeks old for patch-clamp recordings) were treated with morphine (Shenyang No.1 Pharmaceutical Factory) according to procedures described previously [15]. Briefly, morphine dependence was induced in mice by repeated intraperitoneal (i.p.) injections of morphine twice daily at 9:00 A.M. and 5:00 P.M. Morphine doses were progressively increased from 10 to 40 mg/kg: first day, 2 × 10 mg/kg; second day, 2 × 20 mg/kg; third day, 2 × 30 mg/kg; fourth and fifth days, 2 × 40 mg/kg. Control mice were treated with saline following the same procedure. 2.2. LHb slice preparation Male C57BL/6 mice (4–5 weeks old) were anesthetized with chloral hydrate (400 mg/kg, i.p.). All experimental procedures conformed to China Three Gorges University as well as international guidelines on the ethical use of animals. All efforts were made to minimize animal suffering and reduce the number of animals used. The brain was removed rapidly from the skull and placed in modified artificial cerebral spinal fluid (ACSF) containing （mM） 75 sucrose, 88 NaCl, 2.5 KCl, 1.25 NaH2PO4, 7 MgCl2, 0.5 CaCl2, 25 NaHCO3, and saturated with 95% O2 and 5% CO2 at 0 ˚C. Coronal 200 μm slices containing LHb were cut on a vibratome (VT-1200, Leica, Wetzlar, Germany) and transferred to normal ACSF containing (mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2.5 CaCl2, 25 NaHCO3, and 10 glucose at 32˚C.
Slices were incubated for 1 h before patch-clamp recording. 2.3.
Whole-cell patch-clamp recording LHb neurons were visualized under an upright microscope (BX50WI, Olympus,
Tokyo, Japan) using infrared differential interference contrast optics. Whole-cell current- and voltage-clamp recordings were made using an EPC10 amplifier and PatchMaster 2.54 software (HEKA, Lambrecht, Germany). Electrodes had a resistance of 3–4 MΩ when filled with the patch pipette solution. The internal pipette solution contained (mM) 130 K-gluconate, 8 NaCl, 0.1 CaCl2, 0.6 EGTA, 2 Mg-ATP, 0.1 Na3-GTP, and 10 HEPES (pH 7.4). Cells were held at 0 pA under a current-clamp mode to record spontaneous firing. The series resistance (Rs) was monitored by measuring the instantaneous current in response to a 5 mV voltage step command. Rs compensation was not used, but cells where Rs changed by >15% were discarded. 2.4. Surgery and intra-LHb microinjection Male C57BL mice (7-8 weeks) were used. According to whether the animals received i.p. injection of naloxone and intra-LHb microinjection of TTX (Research Institute of Aquatic Products of Hebei, China) or KN-62 (Sigma Aldrich), the animals were housed in groups of six (described in Fig. 1A and Fig. 1C) in a temperature- and humidity-controlled room and maintained on a standard 12 h light/dark cycle (lights on 7:00 AM to 7:00 PM) with food and water ad libitum. The mice were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) after pretreatment with atropine sulfate (0.5 mg/kg, i.p.) and placed in a stereotaxic instrument (Narishige). Two 26-gauge stainless-steel guide cannulae were implanted bilaterally 1 mm above the LHb. The coordinates were based on the atlas of Paxinos and Franklin (2001): AP, -1.58 mm from bregma; ML, 1.5 mm from midline; DV, 2.6 mm from skull surface with a 20˚ lateral angle. The cannulae were secured to the skull with two anchoring screws and dental cement. Wire plugs (28-gauge) were inserted into the cannulae to prevent occlusion. After surgery, animals were housed individually and were allowed to recover for more than a week. For microinjection, 32-gauge injection needles were
inserted into the cannulae. The injection needles were connected to a 1 μl microsyringe (Hamilton) by polyethylene tubing and controlled by a syringe pump (Harvard Apparatus). After the injection, the needles were left in place for another 1 min. 2.5. Conditioned place aversion The conditioned place aversion (CPA) test was conducted according to procedures described previously [16], with some modification. CPA was conducted with a three-compartment place conditioning apparatus (Med Associates). Preconditioning test (pre-test): On day 0, the mice were given a Pre-test. The animals were placed in the middle neutral area and were allowed to freely access both sides of the apparatus for 15 min. Mice with a strong preference (60%) for any compartment were discarded. Before conditioning, morphine dependence was induced by twice daily i.p. injections of morphine at 9:00 A.M. and 5:00 P.M. Chronic morphine treatment: The morphine dose was progressively increased by 10 mg/kg increments from 10 mg/kg on day 1 to 40 mg/kg on day 4, and this dose was maintained on day 5 and 6. Conditioning: On day 5, 2 h after 40 mg/kg morphine administration, mice were confined to either compartment for 20 min immediately after the i.p. injection of naloxone (1 mg/kg). On day 6, mice were confined to the opposite compartment for 20 min after the i.p. injection of saline. Postconditioning test (post-test): The post-test was conducted 24 h after conditioning on day 7. The mice were allowed to freely explore three compartments for 15 min and CPA score was calculated as difference between the time spent in the saline-paired compartment and the time spent in the naloxone-paired compartment (time in the naloxone-paired compartment minus time in the saline-paired compartment). After the testing sessions each mouse was deeply anaesthetized and 200 nl of a 4% methylene-blue solution was microinjected into the LHb to verify the injection sites under light microscope (MODEL BX53F, OLYMPUS, Tokyo, Japan), photographs were taken and analyzed using software OLYMPUS cellSens Entry (OLYMPUS, Tokyo, Japan). To observe the effect of inactivation of the LHb by TTX on naloxone-precipitated CPA, mice received
bilaterally microinjection of TTX (5 ng/200 nl/side) into the LHb 30 min before the administration of naloxone (1 mg/kg, i.p.). The control mice received bilaterally microinjection of saline solution (200 nl/side). In order to observe the effect of 1-[N,O-bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosy]-4-phenylpiperazine (KN-62) (Sigma Aldrich), a specific CaMK II inhibitor, on naloxone-precipitated CPA, mice received bilaterally microinjection of KN-62 (20 nmol/200 nl/side) into the LHb 30 min prior to each morphine treatment. The control mice received bilaterally microinjection of 0.1% Dimethyl Sulphoxide (DMSO) in saline solution (200 nl/side). 2.6.
Western blot Two hours after morphine administration on day 5, the mice were sacrificed.
Treated and control LHb were initially microdissected and stored at −80 °C until processing. To detect total protein level of α-CaMKⅡ and β-CaMKⅡ in the LHb, tissue was homogenized in ice-cold RIPA lysis buffer (Sigma-Aldrich). Proteins were quantified using Bradford assay (Bio-Rad Laboratories) and 20 μg per sample were loaded on 10% SDS-PAGE gel and transferred to nitrocellulose membrane using Trans-Blot Turbo Transfer System (Bio-Rad Laboratories) to perform western blot analysis. The blots were blocked by 5% BSA at room temperature and then incubated with a goat anti-α-CaMKⅡ (1:800, Santa Cruz) or a goat anti-β-CaMKⅡ (1:800, Santa Cruz) or a goat anti-β-actin (1:1500, Sigma-Aldrich). Following extensive washing, membranes were incubated with donkey anti-goat horseradish peroxydase-conjugated secondary antibody (1:3000, Santa Cruz) for 1 h at room temperature. The washings were repeated, and the blots were visualized with a chemiluminescence system (Bio-Rad Laboratories). The densities of the CaMKII bands were quantified by scanning densitometry with the Bio-Rad imaging densitometer. The immunoreactivity of α-CaMKⅡand β-CaMKⅡwas normalized to that of β-actin. To detect post-synaptic membrane proteins level of phospho-GluA1 (Ser845) and phospho-GluA1 (Ser831) in the LHb, The following method was used. Briefly, the LHb from mice were gathered, resuspended in the solution of 0.32 M sucrose, 4
mM HEPES (pH 7.4) with protease/phosphatase inhibitors (Sigma-Aldrich) and homogenized. With utilization of centrifugations, lysis, and sucrose gradient, a postsynaptic density (PSD) pellet was made. LHb membrane proteins from the PSD pellet were extracted using the ProteoExtract Transmembrane Protein Extraction Kit (Millipore). Then 20 µg of each lysate was separated on SDS-PAGE gels and transferred to nitrocellulose membranes (Bio-Rad Laboratories) using a semi-dry electrotransfer system (Bio-Rad Laboratories). The blots were blocked by 5% BSA and then the blots were incubated overnight at 4 °C with primary antibodies, followed by washes and incubation with appropriate secondary antibodies, and visualized with a chemiluminescence system. We used the following primary antibodies: goat antiSer845 (1:800, Santa Cruz), goat anti-Ser831 (1:800, Santa Cruz) and rabbit anti-β-actin (1:1500, Sigma-Aldrich). Each experiment was repeated three times. Each sample is from at least 3 mice. 2.7. Data analysis Numerical data were expressed as mean ± s.e.m. Off-line data analysis was performed using the Mini Analysis Program (Synaptosoft), Clampfit (Axon Instruments), SigmaPlot (Systat Software), and Origin (Microcal Software). Spontaneous firing was analyzed using the Event detection function of Clampfit. Statistical significance was determined using Student’s t-test for comparisons between two groups. In the patch-clamp studies, n refers to the number of cells. Every cell was from a different slice, and a group of cells in each experiment was from at least five animals. Two-way ANOVAs were performed on the data from CPA with the between-subjects factors treatment (different drugs) and within-subjects factors test condition (pre-test and post-test). All post-hoc comparisons were made using Tukey’s test. Results with p < 0.05 were accepted as being statistically significant. 3. Results 3.1. The effect of inactivation of the LHb by TTX on naloxone-precipitated CPA in morphine-dependent mice
We observed the effect of TTX on naloxone-precipitated CPA in mice. TTX were locally injected into the LHb. A schematic of the experimental design for CPA and drug application is shown in Fig. 1A. As shown in Fig. 1B, two-way ANOVAs conducted on the CPA score revealed that there was a significant interaction of treatment (F(2,30) = 6.26; p = 0.005) and test condition (F(1,30) = 50.73; p < 0.001). Post-hoc analysis showed that after CPA training, the morphine + saline + naloxone group (n = 6 mice) exhibited significant CPA compared with the morphine + saline + saline group (n = 6 mice, p < 0.05), but in groups receiving TTX (morphine + TTX + naloxone group) (n = 6 mice), naloxone-precipitated CPA was absent (p < 0.01). These results suggest that intra-LHb injection of TTX abolishes naloxone-precipitated CPA. 3.2. The effect of the intra-lateral habenular administration of selective CaMKⅡ inhibitor KN-62 on naloxone-precipitated CPA in morphine-dependent mice We also observed the effect of KN-62 on naloxone-precipitated CPA in morphine-dependent mice. KN-62 was locally injected into the LHb. A schematic of the experimental design for CPA and drug application is shown in Fig. 1C. As shown in Fig. 1D, two-way ANOVAs conducted on the CPA score revealed that there was a significant interaction of treatment (F(2,30) = 8.32; p = 0.001) and test condition (F(1,30) = 71.92; p < 0.001). Post-hoc analysis showed that after CPA training, the morphine + DMSO + naloxone group (n = 6 mice) exhibited significant CPA compared with the morphine + DMSO + saline group (n = 6 mice, p < 0.05), but in groups receiving KN-62 (morphine + KN-62 + naloxone) (n = 6 mice), morphine-induced CPA was absent (p < 0.01). These results suggest that intra-LHb injection of KN-62 abolishes naloxone-precipitated CPA. For all the CPA tests, injection sites were verified under light microscope (Fig. 1E); the injection sites used for data analysis are shown in Fig. 1F. 3.3. The effect of chronic morphine treatment on the protein levels of α-CaMKⅡ and β-CaMKⅡ in the LHb
The effect of chronic morphine treatment on the protein levels of CaMKII in the LHb was detected by western blot analysis. The results indicated that the basal expression of β-CaMKⅡ (28.52% of β-actin) is relatively low to that of α-CaMKⅡ (76.85% of β-actin) (As shown Fig. 2A and 2B). After five consecutive days of morphine treatment, the α (167.73 ± 9.07% of control, n=3, p<0.05, Student’s t test) and β isoforms (333.73 ± 8.17% of control, n=3, p<0.01, Student’s t test) were significantly upregulated (As shown in Fig. 2A and 2B). Notably, β isoform showed more increase than α isoform (333.73 ± 8.17% of control for β isoform VS 167.73 ± 9.07% of control for α isoform). 3.4. The effect of chronic morphine treatment on neuronal spontaneous firing in the LHb To investigate the effect of chronic morphine treatment on the activity of the LHb, the spontaneous action potential (AP) of the lateral habenular neurons were recorded by using in vitro whole-cell patch-clamp recording. There were only three cells which had spontaneous firing in twenty neurons recorded in five saline-treated mice. However, there were ten cells which had spontaneous firing in twenty neurons recorded in six chronic morphine-treated mice. Furthermore, the average of firing frequency in chronic morphine-treated mice (n=10, 2.60 ± 0.29 Hz) was much higher than that in saline-treated mice (n=3, 0.92 ± 0.32 Hz) (p<0.05, student’s t test) (As shown in Fig. 3). The above results suggest that chronic morphine treatment induces lateral habenular hyperactivity. 3.5.
The effect of chronic morphine treatment on the protein levels of
phospho-GluA1 (Ser831) and phospho-GluA1 (Ser845) in the LHb The effect of chronic morphine treatment on the protein levels of Ser831 and Ser845 in the LHb was detected by western blot analysis. After five consecutive days of morphine treatment, no any significant change of the protein levels of Ser831 and Ser845 in the LHb (As shown in Fig. 2C and 2D) was observed, which is different from cocaine-driven change reported previously [17].
4. Discussion Considering aversive emotional state driven by drugs abuse is one of the most important reasons contribute to addiction [1] and the LHb is the negative emotional center [18], so we addressed that LHb may be implicated in chronic morphine treatment-induced withdrawal syndromes. Despite the fact that the LHb play important roles in cocaine [3, 4] and alcohol addiction [5], there are no evidences showing the LHb is crucial for morphine withdrawal syndromes. We found that the inactivation of LHb by microinjection of TTX completely eliminated the naloxone-challenge induced withdrawal manifestations in morphine-dependent mice as evidenced by no aversion for naloxone-paired compartment in the morphine+TTX +naloxone group compared with the morphine+saline +naloxone group. This result indicates that LHb is a crucial brain region implicated in naloxone-precipitated withdrawal syndrome in morphine-dependent mice, which is in agree with the finding that LHb in mice is crucial for cocaine-evoked negative symptoms [17]. These results strongly suggest that LHb is a vital brain region mediating drug-driven aversive behaviors. Then, what could be the molecular mechanisms underlie LHb-mediating naloxone-precipitated CPA? Accumulating evidences show that acute and chronic morphine can regulate activity and expression of CaMKII in different brain regions such as hippocampus [11, 12], frontal cortex [11], piriform cortex [11] and amygdala [11]. Furthermore, microinjection of KN-62 into hippocampus decreased withdrawal signs induced by naloxone [10]. These results suggest that CaMK II is an important candidate for morphine addiction. However, there is no evidence showing CaMKII in the LHb play important roles in naloxone-precipitated withdrawal syndrome. In present study, we found that dependence-inducing morphine treatment induced strong overexpression of both α and β isoform of CaMKII in the LHb. More important, microinjection of KN-62 into the LHb completely abolished morphine withdrawal syndrome. These results suggest that upregulated CaMKII in the LHb is one of the most important substrates underlying aversive effects of chronic morphine treatment.
Then, what could be the functional results of this upregulated expression for CaMKII? The previous evidences showed that overexpression of CaMKII in the RMTg-projecting neurons in the LHb significantly increase spontaneous firing rate of lateral habenular neurons [14] and it has been suggested that neuronal excitability is closely related to CaMKII [19]. In consistent with these researches, our results showed that chronic morphine treatment strongly upregulate neuronal firing frequency in the lateral habenular neurons. Therefore, the observed upregulated expression of CaMKII in the LHb could at least partially contribute to increased neuronal firing in the LHb. Of course, distinct LHb circuits encode different motivational signatures [2], so more researches should be done to clarify the specific projection neurons in the LHb underlie the plastic change. Then, what could be the cellular mechanism by which α- and β-CaMKII overexpression alters LHb neuron activity and function? We further studied the downstream molecular targets for upregulated CaMKII in the LHb using western blot method. It is well known that CaMKII is important for transporting of AMPA glutamate receptors to synapses [20-22] and trafficking of AMPA receptors is important for controlling neuronal excitability [23, 24]. In fact, the previous evidences showed that overexpression of CaMKII in RMTg-projecting neurons resulted in upregulated GluA1-type AMPAR in the LHb [14]. It also has been demonstrated that phosphorylation of serine 845 (Ser845) on the GluA1 C terminus in the LHb are instrumental for cocaine-driven aversive behaviors [17]. So, it is logical to hypothesize that morphine-induced hyperexcitability in the LHb could be at least partially attributed to modification of GluA1 in the LHb. However, we observed no any changes for phosphorylation of both Ser845 and Ser831 on the GluA1 C terminus in the LHb, which may suggest different mechanisms underlie morphine addiction and cocaine addiction. Our results are consistent with the idea that there are some different mechanisms between morphine and cocaine addiction [25]. Our results therefore suggest that there are some alternative mechanisms mediating morphine-induced hyperexcitability in the LHb. Considering HCNs play important roles in regulating neuronal excitability in the LHb [26] and the expression of HCNs
can be regulated by morphine [27] and CaMKII [28], more studies can be done in the future to clarify whether HCNs are the dowstream targets of CaMKII in the LHb under morphine addiction. Whenever, these results suggest that the LHb is crucial for chronic morphine treatment-mediated aversive component in the development of morphine dependence. Lateral habenular CaMKII is one of the most important candidates involved in morphine withdrawal syndromes. Notably, our results indicate that the change of β isoform was more prominent than α isoform. In addition, in consistent with our findings, other investigators reported that β-CaMKII but not α-CaMKII is a powerful regulator of lateral habenular neuronal function and a key molecular determinant regulating negative emotional states [14]. These findings could imply more important roles of β isoform in the LHb in the development of morphine addiction. Therefore, the future researches should be done to identify the isoform of CaMKII in the LHb involved in morphine-driven aversive behaviors. Inhibition of this kinase or finding out the dowstream targets of CaMKII may have some therapeutic benefit in the treatment of opiate dependence.
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Figure legends Fig. 1. The effects of inactivation of the LHb by TTX and inhibition of the lateral habenular CaMKⅡby locally administration of KN-62 on naloxone-precipitated CPA in morphine-dependent mice. (A) and (C) Schematics of the experimental design for CPA and administration of drugs. (B) Influence of intra-LHb injection of TTX on naloxone-precipitated CPA in morphine-dependent
mice.
Averaged
CPA
score
of
preconditioning
and
postconditioning in different groups (n = 6 mice in each group; *p < 0.05, i.p. injection of naloxone group compared with i.p. injection of saline group, ##p < 0.01, i.p. injection of naloxone plus intra-LHb injection of TTX group compared with i.p. injection of naloxone plus intra-LHb injection of saline group). (D) Influence of intra-LHb injection of KN-62 on naloxone-precipitated CPA in morphine-dependent mice. Averaged CPA score of preconditioning and postconditioning in different groups (n = 6 mice in each group; **p < 0.01, i.p. injection of naloxone group compared with i.p. injection of saline group, ##p < 0.01, i.p. injection of naloxone plus intra-LHb injection of KN-62 group compared with i.p. injection of naloxone plus intra-LHb injection of 0.1% DMSO in saline group). (E) Representative photograph of cannula tips terminating in the LHb under light microscope (MODEL BX53F, OLYMPUS, Tokyo, Japan), photographs were captured and analyzed using software OLYMPUS cellSens Entry (OLYMPUS, Tokyo, Japan). (F) Schematic representation of injection sites of LHb. The injection sites were outlined with thick black line.
Fig. 2. The effect of chronic morphine treatment on the protein levels of CaMKⅡand phospho-GluA1 in the LHb. (A) and (C) Representative of western blotting showing CaMK Ⅱ
and
phospho-GluA1 expression in the LHb. (B) and (D) Averaged density of the corresponding protein bands after normalization by β-actin protein in the chronic-saline-treated and chronic-morphine-treated mice. n = 3 samples in each
group; *P<0.05, **p < 0.01, chronic-morphine-treated mice compared with chronic-saline-treated mice).
Fig. 3. The effect of chronic morphine treatment on the neuronal spontaneous firing in the LHb. (A) Representative spontaneous firing traces in the LHb from chronic-saline-treated mice and chronic-morphine-treated mice. (B) Average frequency of spontaneous firing in the lateral habenular nerons from chronic-saline-treated mice and chronic-morphine-treated mice. (n = 3 cells in chronic-saline-treated mice and n=10 cells in chronic-morphine-treated mice, *p < 0.05, chronic-morphine-treated mice compared to chronic-saline-treated mice.).
Acknowledgments: This work was supported by Project of Foundation of National Natural Science of China (31200814); Project of Outstanding Young Talent, Educational Commission of Hubei Province, China (Q20121310) and Natural Science Foundation for the Youth Scholars of Hubei Provincial Department of Education, China (T201403). We gratefully thank Dr Ping Zheng at the Fudan University for his critical help in sharing the research platform.
S0304-3940(17)30415-9 http://dx.doi.org/doi:10.1016/j.neulet.2017.05.027 NSL 32834
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
15-2-2017 30-4-2017 15-5-2017
Please cite this article as: Jing Wang, Min Li, Ping Wang, Yunhong Zha, Zhi He, Zicheng Li, Inhibition of the lateral habenular CaMKII abolishes naloxoneprecipitated conditioned place aversion in morphine-dependent mice, Neuroscience Lettershttp://dx.doi.org/10.1016/j.neulet.2017.05.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
Intra-lateral habenula administration of TTX abolished naloxone-precipitated CPA in morphine-dependent mice.
Intra-lateral habenula administration of KN-62 abolished naloxone-precipitated CPA in morphine-dependent mice.
Chronic morphine treatment resulted in overexpression of CaMKⅡ in the lateral habenula.
Title: Inhibition of the lateral habenular CaMKⅡ abolishes naloxone-precipitated conditioned place aversion in morphine-dependent mice Author: Jing Wang1 *, Min Li1, 2 *, Ping Wang2, Yunhong Zha2, Zhi He1 and Zicheng Li1, 2 #, 1
Medical College of China Three Gorges University, Yichang 443002, China.
2
People's Hospital of China Three Gorges University, Yichang 443002, China.
* Jing Wang and Min Li contributed equally to this paper. #
Corresponding author:
Zicheng Li, Ph.D. Department of Biochemistry and Molecular Biology Medical College of China Three Gorges University 8 Daxue Road Yichang 443002 People’s Republic of China Phone: (86) (717) 6396818 e-mail: [email protected] Abstract Addictive substances mediate positive and negative states promoting compulsive drug use. However, substrates for aversive effects of drugs are not fully understood. We found that inactivation of the lateral habenula (LHb) by microinjection of tetrodotoxin (TTX) abolished naloxone-precipitated conditioned place aversion (CPA) in morphine-dependent mice. We also found that lateral habenular administration of KN-62, a specific inhibitor for calcium/calmodulin dependent protein kinase II (CaMKII), abolished naloxone-precipitated CPA in morphine-dependent mice. Furthermore, we found chronic morphine treatment induced overexpression of CaMKII in the LHb. In conclusion, our results suggest that the increased expression of CaMKII in the LHb is instrumental for morphine-driven aversive behaviors. Key words: morphine; calcium/calmodulin dependent protein kinase II (CaMKII); lateral habenula (LHb); conditioned place aversion (CPA);
1. Introduction Withdrawal from addictive substances, including morphine, produces negative symptoms that contribute to compulsive drug abuse [1]. The lateral habenula (LHb) inhibits monoaminergic systems via the GABAergic rostromedial tegmental nucleus (RMTg), encoding aversion-related stimuli [2]. Notably, functional modifications of the LHb are associated with neuropsychiatric disorders, including addiction such as cocaine [3, 4] and alcohol addiction [5]. However, it is unknown whether LHb is involved in the morphine withdrawal syndromes. Furthermore, the underlying molecular mechanisms mediating morphine-driven aversive behaviors are elusive. Calcium/calmodulin-dependent protein kinase II (CaMKII), a multiple functional enzyme, is one of the most abundant kinases in the brain, comprising about 2% of total proteins [6, 7]. At least four isoforms of CaMKII (α, β, γ and δ) are found to express in the brain and to function in the form of homomultimers or heteromultimers [6, 7]. Among them, the α and β isoforms are restricted in nervous tissues, whereas γ and δ are found in most tissues besides brain [6, 7]. CaMKII has been reported to play important roles in various neuronal adaptive processes such as drug addiction [8, 9]. For example, studies revealed that inhibition of CaMKII by microinjection of specific inhibitors KN-62 into the hippocampus decrease the morphine withdrawal syndromes induced by opiate antagonist naloxone [10] and suppress the development of formation and reactivation of morphine conditioned place preference (CPP) [10]. More studies imply that the expression of CaMKII in brain can be selectively regulated by acute or chronic morphine treatment [11-13].These data strongly suggest that CaMKII play important roles in the development of morphine addiction. However, there is no evidenc showing CaMKII in the LHb is involved in the development of morphine addiction. Considering the overexpression of CaMKII in the LHb is instrumental for processing of aversive information [14], so we hypothesized that the morphine withdrawal syndromes may attribute to upregulation of CaMKII in the LHb under chronic morphine treatment.
In the present study, to confirm the role of CaMKII in the LHb in morphine withdrawal syndromes, we first investigated the effects of inactivation of the LHb by microinjection of tetrodotoxin (TTX), a Na+ channels blocker, on naloxone-precipitated conditioned place aversion (CPA) in morphine-dependent mice. We then observed the effects of microinjection of KN-62, a selective CaMK II inhibitor, into the LHb on CPA. Finally, the effects of chronic morphine treatment on the protein levels of CaMKII in the LHb were examined. 2. Materials and methods 2.1. Chronic morphine treatment Male C57BL/6 mice (7-8 weeks old for behavioral test and western blot analysis, 4-5 weeks old for patch-clamp recordings) were treated with morphine (Shenyang No.1 Pharmaceutical Factory) according to procedures described previously [15]. Briefly, morphine dependence was induced in mice by repeated intraperitoneal (i.p.) injections of morphine twice daily at 9:00 A.M. and 5:00 P.M. Morphine doses were progressively increased from 10 to 40 mg/kg: first day, 2 × 10 mg/kg; second day, 2 × 20 mg/kg; third day, 2 × 30 mg/kg; fourth and fifth days, 2 × 40 mg/kg. Control mice were treated with saline following the same procedure. 2.2. LHb slice preparation Male C57BL/6 mice (4–5 weeks old) were anesthetized with chloral hydrate (400 mg/kg, i.p.). All experimental procedures conformed to China Three Gorges University as well as international guidelines on the ethical use of animals. All efforts were made to minimize animal suffering and reduce the number of animals used. The brain was removed rapidly from the skull and placed in modified artificial cerebral spinal fluid (ACSF) containing （mM） 75 sucrose, 88 NaCl, 2.5 KCl, 1.25 NaH2PO4, 7 MgCl2, 0.5 CaCl2, 25 NaHCO3, and saturated with 95% O2 and 5% CO2 at 0 ˚C. Coronal 200 μm slices containing LHb were cut on a vibratome (VT-1200, Leica, Wetzlar, Germany) and transferred to normal ACSF containing (mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2.5 CaCl2, 25 NaHCO3, and 10 glucose at 32˚C.
Slices were incubated for 1 h before patch-clamp recording. 2.3.
Whole-cell patch-clamp recording LHb neurons were visualized under an upright microscope (BX50WI, Olympus,
Tokyo, Japan) using infrared differential interference contrast optics. Whole-cell current- and voltage-clamp recordings were made using an EPC10 amplifier and PatchMaster 2.54 software (HEKA, Lambrecht, Germany). Electrodes had a resistance of 3–4 MΩ when filled with the patch pipette solution. The internal pipette solution contained (mM) 130 K-gluconate, 8 NaCl, 0.1 CaCl2, 0.6 EGTA, 2 Mg-ATP, 0.1 Na3-GTP, and 10 HEPES (pH 7.4). Cells were held at 0 pA under a current-clamp mode to record spontaneous firing. The series resistance (Rs) was monitored by measuring the instantaneous current in response to a 5 mV voltage step command. Rs compensation was not used, but cells where Rs changed by >15% were discarded. 2.4. Surgery and intra-LHb microinjection Male C57BL mice (7-8 weeks) were used. According to whether the animals received i.p. injection of naloxone and intra-LHb microinjection of TTX (Research Institute of Aquatic Products of Hebei, China) or KN-62 (Sigma Aldrich), the animals were housed in groups of six (described in Fig. 1A and Fig. 1C) in a temperature- and humidity-controlled room and maintained on a standard 12 h light/dark cycle (lights on 7:00 AM to 7:00 PM) with food and water ad libitum. The mice were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) after pretreatment with atropine sulfate (0.5 mg/kg, i.p.) and placed in a stereotaxic instrument (Narishige). Two 26-gauge stainless-steel guide cannulae were implanted bilaterally 1 mm above the LHb. The coordinates were based on the atlas of Paxinos and Franklin (2001): AP, -1.58 mm from bregma; ML, 1.5 mm from midline; DV, 2.6 mm from skull surface with a 20˚ lateral angle. The cannulae were secured to the skull with two anchoring screws and dental cement. Wire plugs (28-gauge) were inserted into the cannulae to prevent occlusion. After surgery, animals were housed individually and were allowed to recover for more than a week. For microinjection, 32-gauge injection needles were
inserted into the cannulae. The injection needles were connected to a 1 μl microsyringe (Hamilton) by polyethylene tubing and controlled by a syringe pump (Harvard Apparatus). After the injection, the needles were left in place for another 1 min. 2.5. Conditioned place aversion The conditioned place aversion (CPA) test was conducted according to procedures described previously [16], with some modification. CPA was conducted with a three-compartment place conditioning apparatus (Med Associates). Preconditioning test (pre-test): On day 0, the mice were given a Pre-test. The animals were placed in the middle neutral area and were allowed to freely access both sides of the apparatus for 15 min. Mice with a strong preference (60%) for any compartment were discarded. Before conditioning, morphine dependence was induced by twice daily i.p. injections of morphine at 9:00 A.M. and 5:00 P.M. Chronic morphine treatment: The morphine dose was progressively increased by 10 mg/kg increments from 10 mg/kg on day 1 to 40 mg/kg on day 4, and this dose was maintained on day 5 and 6. Conditioning: On day 5, 2 h after 40 mg/kg morphine administration, mice were confined to either compartment for 20 min immediately after the i.p. injection of naloxone (1 mg/kg). On day 6, mice were confined to the opposite compartment for 20 min after the i.p. injection of saline. Postconditioning test (post-test): The post-test was conducted 24 h after conditioning on day 7. The mice were allowed to freely explore three compartments for 15 min and CPA score was calculated as difference between the time spent in the saline-paired compartment and the time spent in the naloxone-paired compartment (time in the naloxone-paired compartment minus time in the saline-paired compartment). After the testing sessions each mouse was deeply anaesthetized and 200 nl of a 4% methylene-blue solution was microinjected into the LHb to verify the injection sites under light microscope (MODEL BX53F, OLYMPUS, Tokyo, Japan), photographs were taken and analyzed using software OLYMPUS cellSens Entry (OLYMPUS, Tokyo, Japan). To observe the effect of inactivation of the LHb by TTX on naloxone-precipitated CPA, mice received
bilaterally microinjection of TTX (5 ng/200 nl/side) into the LHb 30 min before the administration of naloxone (1 mg/kg, i.p.). The control mice received bilaterally microinjection of saline solution (200 nl/side). In order to observe the effect of 1-[N,O-bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosy]-4-phenylpiperazine (KN-62) (Sigma Aldrich), a specific CaMK II inhibitor, on naloxone-precipitated CPA, mice received bilaterally microinjection of KN-62 (20 nmol/200 nl/side) into the LHb 30 min prior to each morphine treatment. The control mice received bilaterally microinjection of 0.1% Dimethyl Sulphoxide (DMSO) in saline solution (200 nl/side). 2.6.
Western blot Two hours after morphine administration on day 5, the mice were sacrificed.
Treated and control LHb were initially microdissected and stored at −80 °C until processing. To detect total protein level of α-CaMKⅡ and β-CaMKⅡ in the LHb, tissue was homogenized in ice-cold RIPA lysis buffer (Sigma-Aldrich). Proteins were quantified using Bradford assay (Bio-Rad Laboratories) and 20 μg per sample were loaded on 10% SDS-PAGE gel and transferred to nitrocellulose membrane using Trans-Blot Turbo Transfer System (Bio-Rad Laboratories) to perform western blot analysis. The blots were blocked by 5% BSA at room temperature and then incubated with a goat anti-α-CaMKⅡ (1:800, Santa Cruz) or a goat anti-β-CaMKⅡ (1:800, Santa Cruz) or a goat anti-β-actin (1:1500, Sigma-Aldrich). Following extensive washing, membranes were incubated with donkey anti-goat horseradish peroxydase-conjugated secondary antibody (1:3000, Santa Cruz) for 1 h at room temperature. The washings were repeated, and the blots were visualized with a chemiluminescence system (Bio-Rad Laboratories). The densities of the CaMKII bands were quantified by scanning densitometry with the Bio-Rad imaging densitometer. The immunoreactivity of α-CaMKⅡand β-CaMKⅡwas normalized to that of β-actin. To detect post-synaptic membrane proteins level of phospho-GluA1 (Ser845) and phospho-GluA1 (Ser831) in the LHb, The following method was used. Briefly, the LHb from mice were gathered, resuspended in the solution of 0.32 M sucrose, 4
mM HEPES (pH 7.4) with protease/phosphatase inhibitors (Sigma-Aldrich) and homogenized. With utilization of centrifugations, lysis, and sucrose gradient, a postsynaptic density (PSD) pellet was made. LHb membrane proteins from the PSD pellet were extracted using the ProteoExtract Transmembrane Protein Extraction Kit (Millipore). Then 20 µg of each lysate was separated on SDS-PAGE gels and transferred to nitrocellulose membranes (Bio-Rad Laboratories) using a semi-dry electrotransfer system (Bio-Rad Laboratories). The blots were blocked by 5% BSA and then the blots were incubated overnight at 4 °C with primary antibodies, followed by washes and incubation with appropriate secondary antibodies, and visualized with a chemiluminescence system. We used the following primary antibodies: goat antiSer845 (1:800, Santa Cruz), goat anti-Ser831 (1:800, Santa Cruz) and rabbit anti-β-actin (1:1500, Sigma-Aldrich). Each experiment was repeated three times. Each sample is from at least 3 mice. 2.7. Data analysis Numerical data were expressed as mean ± s.e.m. Off-line data analysis was performed using the Mini Analysis Program (Synaptosoft), Clampfit (Axon Instruments), SigmaPlot (Systat Software), and Origin (Microcal Software). Spontaneous firing was analyzed using the Event detection function of Clampfit. Statistical significance was determined using Student’s t-test for comparisons between two groups. In the patch-clamp studies, n refers to the number of cells. Every cell was from a different slice, and a group of cells in each experiment was from at least five animals. Two-way ANOVAs were performed on the data from CPA with the between-subjects factors treatment (different drugs) and within-subjects factors test condition (pre-test and post-test). All post-hoc comparisons were made using Tukey’s test. Results with p < 0.05 were accepted as being statistically significant. 3. Results 3.1. The effect of inactivation of the LHb by TTX on naloxone-precipitated CPA in morphine-dependent mice
We observed the effect of TTX on naloxone-precipitated CPA in mice. TTX were locally injected into the LHb. A schematic of the experimental design for CPA and drug application is shown in Fig. 1A. As shown in Fig. 1B, two-way ANOVAs conducted on the CPA score revealed that there was a significant interaction of treatment (F(2,30) = 6.26; p = 0.005) and test condition (F(1,30) = 50.73; p < 0.001). Post-hoc analysis showed that after CPA training, the morphine + saline + naloxone group (n = 6 mice) exhibited significant CPA compared with the morphine + saline + saline group (n = 6 mice, p < 0.05), but in groups receiving TTX (morphine + TTX + naloxone group) (n = 6 mice), naloxone-precipitated CPA was absent (p < 0.01). These results suggest that intra-LHb injection of TTX abolishes naloxone-precipitated CPA. 3.2. The effect of the intra-lateral habenular administration of selective CaMKⅡ inhibitor KN-62 on naloxone-precipitated CPA in morphine-dependent mice We also observed the effect of KN-62 on naloxone-precipitated CPA in morphine-dependent mice. KN-62 was locally injected into the LHb. A schematic of the experimental design for CPA and drug application is shown in Fig. 1C. As shown in Fig. 1D, two-way ANOVAs conducted on the CPA score revealed that there was a significant interaction of treatment (F(2,30) = 8.32; p = 0.001) and test condition (F(1,30) = 71.92; p < 0.001). Post-hoc analysis showed that after CPA training, the morphine + DMSO + naloxone group (n = 6 mice) exhibited significant CPA compared with the morphine + DMSO + saline group (n = 6 mice, p < 0.05), but in groups receiving KN-62 (morphine + KN-62 + naloxone) (n = 6 mice), morphine-induced CPA was absent (p < 0.01). These results suggest that intra-LHb injection of KN-62 abolishes naloxone-precipitated CPA. For all the CPA tests, injection sites were verified under light microscope (Fig. 1E); the injection sites used for data analysis are shown in Fig. 1F. 3.3. The effect of chronic morphine treatment on the protein levels of α-CaMKⅡ and β-CaMKⅡ in the LHb
The effect of chronic morphine treatment on the protein levels of CaMKII in the LHb was detected by western blot analysis. The results indicated that the basal expression of β-CaMKⅡ (28.52% of β-actin) is relatively low to that of α-CaMKⅡ (76.85% of β-actin) (As shown Fig. 2A and 2B). After five consecutive days of morphine treatment, the α (167.73 ± 9.07% of control, n=3, p<0.05, Student’s t test) and β isoforms (333.73 ± 8.17% of control, n=3, p<0.01, Student’s t test) were significantly upregulated (As shown in Fig. 2A and 2B). Notably, β isoform showed more increase than α isoform (333.73 ± 8.17% of control for β isoform VS 167.73 ± 9.07% of control for α isoform). 3.4. The effect of chronic morphine treatment on neuronal spontaneous firing in the LHb To investigate the effect of chronic morphine treatment on the activity of the LHb, the spontaneous action potential (AP) of the lateral habenular neurons were recorded by using in vitro whole-cell patch-clamp recording. There were only three cells which had spontaneous firing in twenty neurons recorded in five saline-treated mice. However, there were ten cells which had spontaneous firing in twenty neurons recorded in six chronic morphine-treated mice. Furthermore, the average of firing frequency in chronic morphine-treated mice (n=10, 2.60 ± 0.29 Hz) was much higher than that in saline-treated mice (n=3, 0.92 ± 0.32 Hz) (p<0.05, student’s t test) (As shown in Fig. 3). The above results suggest that chronic morphine treatment induces lateral habenular hyperactivity. 3.5.
The effect of chronic morphine treatment on the protein levels of
phospho-GluA1 (Ser831) and phospho-GluA1 (Ser845) in the LHb The effect of chronic morphine treatment on the protein levels of Ser831 and Ser845 in the LHb was detected by western blot analysis. After five consecutive days of morphine treatment, no any significant change of the protein levels of Ser831 and Ser845 in the LHb (As shown in Fig. 2C and 2D) was observed, which is different from cocaine-driven change reported previously [17].
4. Discussion Considering aversive emotional state driven by drugs abuse is one of the most important reasons contribute to addiction [1] and the LHb is the negative emotional center [18], so we addressed that LHb may be implicated in chronic morphine treatment-induced withdrawal syndromes. Despite the fact that the LHb play important roles in cocaine [3, 4] and alcohol addiction [5], there are no evidences showing the LHb is crucial for morphine withdrawal syndromes. We found that the inactivation of LHb by microinjection of TTX completely eliminated the naloxone-challenge induced withdrawal manifestations in morphine-dependent mice as evidenced by no aversion for naloxone-paired compartment in the morphine+TTX +naloxone group compared with the morphine+saline +naloxone group. This result indicates that LHb is a crucial brain region implicated in naloxone-precipitated withdrawal syndrome in morphine-dependent mice, which is in agree with the finding that LHb in mice is crucial for cocaine-evoked negative symptoms [17]. These results strongly suggest that LHb is a vital brain region mediating drug-driven aversive behaviors. Then, what could be the molecular mechanisms underlie LHb-mediating naloxone-precipitated CPA? Accumulating evidences show that acute and chronic morphine can regulate activity and expression of CaMKII in different brain regions such as hippocampus [11, 12], frontal cortex [11], piriform cortex [11] and amygdala [11]. Furthermore, microinjection of KN-62 into hippocampus decreased withdrawal signs induced by naloxone [10]. These results suggest that CaMK II is an important candidate for morphine addiction. However, there is no evidence showing CaMKII in the LHb play important roles in naloxone-precipitated withdrawal syndrome. In present study, we found that dependence-inducing morphine treatment induced strong overexpression of both α and β isoform of CaMKII in the LHb. More important, microinjection of KN-62 into the LHb completely abolished morphine withdrawal syndrome. These results suggest that upregulated CaMKII in the LHb is one of the most important substrates underlying aversive effects of chronic morphine treatment.
Then, what could be the functional results of this upregulated expression for CaMKII? The previous evidences showed that overexpression of CaMKII in the RMTg-projecting neurons in the LHb significantly increase spontaneous firing rate of lateral habenular neurons [14] and it has been suggested that neuronal excitability is closely related to CaMKII [19]. In consistent with these researches, our results showed that chronic morphine treatment strongly upregulate neuronal firing frequency in the lateral habenular neurons. Therefore, the observed upregulated expression of CaMKII in the LHb could at least partially contribute to increased neuronal firing in the LHb. Of course, distinct LHb circuits encode different motivational signatures [2], so more researches should be done to clarify the specific projection neurons in the LHb underlie the plastic change. Then, what could be the cellular mechanism by which α- and β-CaMKII overexpression alters LHb neuron activity and function? We further studied the downstream molecular targets for upregulated CaMKII in the LHb using western blot method. It is well known that CaMKII is important for transporting of AMPA glutamate receptors to synapses [20-22] and trafficking of AMPA receptors is important for controlling neuronal excitability [23, 24]. In fact, the previous evidences showed that overexpression of CaMKII in RMTg-projecting neurons resulted in upregulated GluA1-type AMPAR in the LHb [14]. It also has been demonstrated that phosphorylation of serine 845 (Ser845) on the GluA1 C terminus in the LHb are instrumental for cocaine-driven aversive behaviors [17]. So, it is logical to hypothesize that morphine-induced hyperexcitability in the LHb could be at least partially attributed to modification of GluA1 in the LHb. However, we observed no any changes for phosphorylation of both Ser845 and Ser831 on the GluA1 C terminus in the LHb, which may suggest different mechanisms underlie morphine addiction and cocaine addiction. Our results are consistent with the idea that there are some different mechanisms between morphine and cocaine addiction [25]. Our results therefore suggest that there are some alternative mechanisms mediating morphine-induced hyperexcitability in the LHb. Considering HCNs play important roles in regulating neuronal excitability in the LHb [26] and the expression of HCNs
can be regulated by morphine [27] and CaMKII [28], more studies can be done in the future to clarify whether HCNs are the dowstream targets of CaMKII in the LHb under morphine addiction. Whenever, these results suggest that the LHb is crucial for chronic morphine treatment-mediated aversive component in the development of morphine dependence. Lateral habenular CaMKII is one of the most important candidates involved in morphine withdrawal syndromes. Notably, our results indicate that the change of β isoform was more prominent than α isoform. In addition, in consistent with our findings, other investigators reported that β-CaMKII but not α-CaMKII is a powerful regulator of lateral habenular neuronal function and a key molecular determinant regulating negative emotional states [14]. These findings could imply more important roles of β isoform in the LHb in the development of morphine addiction. Therefore, the future researches should be done to identify the isoform of CaMKII in the LHb involved in morphine-driven aversive behaviors. Inhibition of this kinase or finding out the dowstream targets of CaMKII may have some therapeutic benefit in the treatment of opiate dependence.
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Figure legends Fig. 1. The effects of inactivation of the LHb by TTX and inhibition of the lateral habenular CaMKⅡby locally administration of KN-62 on naloxone-precipitated CPA in morphine-dependent mice. (A) and (C) Schematics of the experimental design for CPA and administration of drugs. (B) Influence of intra-LHb injection of TTX on naloxone-precipitated CPA in morphine-dependent
mice.
Averaged
CPA
score
of
preconditioning
and
postconditioning in different groups (n = 6 mice in each group; *p < 0.05, i.p. injection of naloxone group compared with i.p. injection of saline group, ##p < 0.01, i.p. injection of naloxone plus intra-LHb injection of TTX group compared with i.p. injection of naloxone plus intra-LHb injection of saline group). (D) Influence of intra-LHb injection of KN-62 on naloxone-precipitated CPA in morphine-dependent mice. Averaged CPA score of preconditioning and postconditioning in different groups (n = 6 mice in each group; **p < 0.01, i.p. injection of naloxone group compared with i.p. injection of saline group, ##p < 0.01, i.p. injection of naloxone plus intra-LHb injection of KN-62 group compared with i.p. injection of naloxone plus intra-LHb injection of 0.1% DMSO in saline group). (E) Representative photograph of cannula tips terminating in the LHb under light microscope (MODEL BX53F, OLYMPUS, Tokyo, Japan), photographs were captured and analyzed using software OLYMPUS cellSens Entry (OLYMPUS, Tokyo, Japan). (F) Schematic representation of injection sites of LHb. The injection sites were outlined with thick black line.
Fig. 2. The effect of chronic morphine treatment on the protein levels of CaMKⅡand phospho-GluA1 in the LHb. (A) and (C) Representative of western blotting showing CaMK Ⅱ
and
phospho-GluA1 expression in the LHb. (B) and (D) Averaged density of the corresponding protein bands after normalization by β-actin protein in the chronic-saline-treated and chronic-morphine-treated mice. n = 3 samples in each
group; *P<0.05, **p < 0.01, chronic-morphine-treated mice compared with chronic-saline-treated mice).
Fig. 3. The effect of chronic morphine treatment on the neuronal spontaneous firing in the LHb. (A) Representative spontaneous firing traces in the LHb from chronic-saline-treated mice and chronic-morphine-treated mice. (B) Average frequency of spontaneous firing in the lateral habenular nerons from chronic-saline-treated mice and chronic-morphine-treated mice. (n = 3 cells in chronic-saline-treated mice and n=10 cells in chronic-morphine-treated mice, *p < 0.05, chronic-morphine-treated mice compared to chronic-saline-treated mice.).
Acknowledgments: This work was supported by Project of Foundation of National Natural Science of China (31200814); Project of Outstanding Young Talent, Educational Commission of Hubei Province, China (Q20121310) and Natural Science Foundation for the Youth Scholars of Hubei Provincial Department of Education, China (T201403). We gratefully thank Dr Ping Zheng at the Fudan University for his critical help in sharing the research platform.