- Email: [email protected]
The α1 adrenoceptors in ventrolateral orbital cortex contribute to the expression of morphine-induced behavioral sensitization in rats
Accepted Manuscript Title: The ␣1 adrenoceptors in ventrolateral orbital cortex contribute to the expression of morphine-induced behavioral sensitization in rats Author: Lai Wei Yuan-Mei Zhu Yu-Xiang Zhang Feng Liang Teng Li Hong-Yu Gao Fu-Quan Huo Chun-Xia Yan PII: DOI: Reference:
S0304-3940(15)30220-2 http://dx.doi.org/doi:10.1016/j.neulet.2015.10.057 NSL 31623
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
8-8-2015 9-10-2015 22-10-2015
Please cite this article as: Lai Wei, Yuan-Mei Zhu, Yu-Xiang Zhang, Feng Liang, Teng Li, Hong-Yu Gao, Fu-Quan Huo, Chun-Xia Yan, The rmalpha1 adrenoceptors in ventrolateral orbital cortex contribute to the expression of morphine-induced behavioral sensitization in rats, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2015.10.057 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.
The α1 adrenoceptors in ventrolateral orbital cortex contribute to the expression of morphine-induced behavioral sensitization in rats Lai Wei1,4, Yuan-Mei Zhu1, Yu-Xiang Zhang1, Feng Liang1, Teng Li1, Hong-Yu Gao1, Fu-Quan Huo2,3* [email protected], Chun-Xia Yan1,3* [email protected] 1
College of Forensic Medicine, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi
710061, China. 2
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Xi’an
Jiaotong University Health Science Center, Xi’an, Shaanxi 710061, China. 3
Key Laboratory of Environment and Genes Related to Diseases (Xi’an Jiaotong University),
Ministry of Education, China. 4
Division of Forensic Medicine, School of Basic Medical Sciences, Hubei University of Medicine,
Shiyan, Hubei 442000, China. *
Corresponding authors at: College of Forensic Medicine, Xi’an Jiaotong University Health
Science Center, Xi’an, Shaanxi 710061, China. Tel: +86 29 82655117; Fax: +86 29 82655472
1
Highlights The VLO is a novel anatomical substrate mediating morphine-induced behavioral sensitization. Blocking α1 adrenoceptors in the VLO suppressed the expression of morphine-induced behavioral sensitization. Morphine-induced behavioral sensitization may be associated with activated ERK in the VLO.
Abstract The aim of the present study was to investigate the effect of microinjection of benoxathian, selective α1 adrenoceptor antagonist, into the ventrolateral orbital cortex (VLO) on morphine-induced behavioral sensitization and its underlying molecular mechanism in rats. A single morphine treatment protocol was used in establishing the behavioral sensitization model. The effect of bilateral intra-VLO benoxathian injection on locomotor activity was examined and the protein expression levels of α1 adrenoceptors and activation of extracellular signal-regulated kinase (ERK) in the VLO were detected after locomotor test. The results showed that a single injection of morphine could induce behavioral sensitization by a low challenge dosage of morphine after a 7-day drug free period. Benoxathian significantly suppressed the expression but not the development of morphine-induced behavioral sensitization. Morphine treatment significantly elicited ERK phosphorylation and downregulated the expression level of α1 adrenoceptors in the VLO. In addition, intra-VLO 2
benoxathian injection enhanced the expression levels of α1 adrenoceptors and phosphorylated ERK. These results suggest that α1 adrenoceptors in the VLO are involved in regulating the expression of morphine-induced behavioral sensitization. The effect of decreased locomotor activity by blocking α1 adrenoceptors might be associated with activation of ERK in the VLO.
Abbreviations α1-AR: α1 adrenoceptor Beno: benoxathian ERK: extracellular signal-regulated kinase fmi: forceps minor corpus callosum i.p.: intraperitoneally m.i.: microinjection min: minute(s) Mor: morphine NAc: nucleus accumbens OFC: orbitofrontal cortex p-ERK: phosphorylated ERK PFC: prefrontal cortex Sal: saline VLO: ventrolateral orbital cortex VTA: ventral tegmental area. 3
Keywords: Morphine; α1 adrenoceptor; Ventrolateral orbital cortex; Extracellular signal-regulated kinase; Behavioral sensitization
4
1. Introduction Behavioral sensitization, which is thought to play an important role in certain aspects of drug addiction such as compulsive drug seeking, is the result of an abundance of neuroplastic changes occurring within the brain circuitry involved in motivational behavior and neurochemical effects [5, 25]. A single or repeated drug exposure can cause long-lasting behavioral sensitization which is reflected by an increase in locomotor activity [11, 24]. Evidence from molecular, behavioral, and computational levels of analysis suggests that addiction represents a pathological usurpation of the neural processes of learning and memory [10]. The disruption of drug-related memories may help to prevent addiction [28]. Our laboratory studies demonstrate that the ventrolateral orbital cortex (VLO), a major subdivision of orbitofrontal cortex (OFC), is involved in stress-related memory formation and antinociception [22, 31]. Anatomic studies indicate that the OFC has reciprocal fibers connection with nucleus accumbens (NAc) and hippocampus, and receives input from dopaminergic terminals originating from the ventral tegmental area (VTA) as crucial brain regions associated with opioid addiction [2, 21]. These results imply that the VLO may be a candidate anatomical substrate involving in opioid addiction. Furthermore, recent studies have indicated that norepinephrine (NE) in the prefrontal cortex plays a critical role in the rewarding effects of opiates [26]. The VLO receives norepinephrinergic terminal input from the pons, and adrenoceptors, including α1 and α2, are expressed in the VLO [4, 32]. These results suggest that NE and their receptors in the VLO may be involved in opioid 5
addiction. Alteration of α1 adrenoceptors by the administration of agonists or antagonists affects the locomotor sensitization to the stimulant effect of ethanol consumption, suggesting that the normal functioning of the noradrenergic system is essential to development and expression of behavioral sensitization [12, 27]. The aim of the present study is to investigate the effects of microinjection of a selective α1 adrenoceptor antagonist, benoxathian, into the VLO on morphine-induced behavioral sensitization in rats. Evidence suggests that extracellular signal-regulated kinase (ERK) is involved in behavioral sensitization and the development of neuroplasticity relating to the addictive properties of drug abuse [3, 19, 23]. ERK could be activated by α1 adrenoceptor, a Gq-protein coupled receptor, via phospholipase C (PLC) / protein kinase C (PKC) signal pathways [9]. However, the effect of ERK in the VLO on morphine-induced behavioral sensitization is not yet clear. Therefore, we further examined the expression levels of α1 adrenoceptor and ERK activation in the VLO in an
attempt
to
gain
insight
into
the
molecular
mechanism
underlying
morphine-induced behavioral sensitization. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats weighing between 250 and 300 g were provided by the Medical Experimental Animal Center of Xi’an Jiaotong University, Shaanxi Province, China. Animals were housed in a colony room with controlled ambient temperature (25 ± 2 °C), humidity (50 ± 10%) and under a 12/12 h light-dark cycle 6
(lights on at 07:00) with access to food and water ad libitum. The experimental protocols were approved by the Institutional Animal Care Committee of Xi'an Jiaotong University. All efforts were made to minimize the number of rats used and their suffering in the experiments. 2.2. Bilateral intracerebral guide cannula placement Rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally, i.p.), and the head was immobilized in a stereotaxic frame. A small craniotomy was performed just above the VLO. Stainless steel guide cannulas (0.8 mm in diameter) were stereotaxically inserted bilaterally, with the tip 2.0 mm dorsal to the VLO at the following coordinates: + 3.2 mm anterior to bregma, ± 2.0 mm lateral, and + 2.6 mm below the cortical surface [20, 31], followed by attachment to the skull with two microscrews and dental cement. Once the rats recovered from anesthesia, they were administered sodium penicillin (0.2 million units/day for 3 days, i.p.) to prevent wound and intracerebral infections. The rats were carefully nursed and fed in clean cages. After surgery, animals were housed separately in cages and allowed to recover for 7 days before behavioral procedures. 2.3. Intracerebral microinjection of drugs Morphine hydrochloride was obtained from First Pharmaceutical Factory of Shenyang (China) and administered by i.p. route (10 or 5 mg/kg). Benoxathian hydrochloride was purchased from Sigma-Aldrich (Sigma Chemical, St. Louis, MO, USA) and microinjected at a final concentration of 2 μg/0.5 μl. All drugs were dissolved in physiological saline and freshly prepared before the experiments. The 7
effective drug doses were based on previous reports and our preliminary experiments [8, 15, 24]. Equal volumes of saline served as the vehicle control. Rats were randomly divided into 4 groups (16 rats/group): saline-saline group (intracerebral microinjection of saline 30 min prior to initial saline injection, i.p.); benoxathian-saline group (intracerebral microinjection of benoxathian 30 min prior to initial saline injection, i.p.); saline-morphine group (intracerebral microinjection of saline 30 min prior to initial
morphine
injection,
i.p.);
benoxathian-morphine
group
(intracerebral
microinjection of benoxathian 30 min prior to initial morphine injection, i.p.), as shown in Fig. 1A. Microsyringe was inserted into the VLO, with the tip extending 2.0 mm beyond the end of the guide cannula (total depth 4.6 mm) from the brain surface. Benoxathian was slowly infused bilaterally through a 1.0-μl Hamilton syringe at a constant speed over 1 minute to make sure the drugs completely diffuse from the tips. Saline of equal volume was injected into the VLO as vehicle control. 2.4. The locomotor activity test The locomotor activity tests were conducted in a quiet, dimly lit room between 8:00 am and 12:00 am. An animal locomotor activity measurement system consisting of four testing boxes (60 × 60 × 40 cm3, length × width × height) with a video camera was used to test individual rats. The interior bottoms of the testing boxes were painted black, whereas the interior sides were painted white. The boxes were set in an isolated dark room, and four standard laboratory lamps were used for illumination. After the experimental animals were immediately placed in the boxes, their locomotor activities were recorded by the video camera and analyzed off line using a computer by the 8
video-tracking software (SMART, Panlab SL, Barcelona, Spain). Horizontal trajectories (reflection of locomotor activity) of the rats were recorded and analyzed to determine their travelled distance. 2.5. Protein extraction and western blotting After behavior test, eight rats in each group were decapitated. The VLO were dissected and removed according to the rat brain atlas [20] and homogenized at 4 °C in a RIPA buffer with a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). A BCA protein assay kit (Pierce, Rockland, IL, USA) was used to determine total protein levels for each sample. Equal amounts of protein for each group were separated on a 12% SDS-PAGE gel and transferred to PVDF membranes (Millipore Corporation, Bedford, MA, USA). The membranes were blocked in 5 % (w/v) non-fat milk in Tris-buffered saline (pH 7.5) containing 0.05% Tween-20 for 1 h at room temperature and then incubated with the following primary antibodies overnight at 4 °C: alpha 1 adrenergic receptor (ab3462, Abcam, Cambridge, UK ) at 1:1000, p-ERK 1/2, ERK 1/2 (Cell Signaling Technology, Danvers, MA, USA) at 1:1000, β-actin (Santa Cruz, California, USA) at 1:1000. The membranes were washed and incubated with secondary antibodies: goat anti-rabbit or anti-mouse IgG horse-radish peroxidase (HRP)-conjugated secondary antibody (Bioworld, Dublin, OH, USA) at 1:10,000 dilution. Color development was performed with an enhanced chemiluminescence plus detection kit (Millipore Corporation, Bedford, MA, USA). The band intensities were analyzed using the Quantity One software (Bio-Rad,
9
Hercules, USA) to calculate the target protein versus the loading control (β-actin) for each protein. 2.6. Histology The injection sites were histologically identified or visually confirmed after the behavioral tests as previous reported [32]. At the end of the test, the drug injection sites were marked by injecting Pontamine Sky Blue dye. Under deep anesthesia, the rats were intracardially perfused and fixed. The brains were cut and stained with Cresyl Violet. The injection sites were histologically identified to be within the VLO, as shown in Fig. 1B. 2.7. Data analysis All values were expressed as mean ± SEM. The data of locomotor activity were analyzed by Student’s t-test, one-way ANOVA and two-way repeated measures ANOVA, followed by a Bonferroni post hoc analysis for multiple comparisons. For western blotting, the difference of the protein levels in the VLO was determined by one-way ANOVA. The protein levels of the control group were set at 100% and all data were normalized to loading control. The data analyses were performed using software GraphPad Prism 5.0. Values of P < 0.05 were considered statistically significant. 3. Results 3.1. The effect of microinjection of benoxathian alone into the VLO on locomotor activity in rats.
10
Microinjection of a selective α1 adrenoceptor antagonist, benoxathian (2 μg/0.5 μl, n=7), alone into the VLO had no effect on the accumulated distance during 240 min, except for a transient reduction at 20 min compared to saline-injected group (P < 0.05) (Fig. 2A). Two-way repeated-measures ANOVA analyses revealed, a significant main effect of time (F(23, 276) = 29.46, P < 0.0001), but no effect of group (F(1, 12) = 0.2357, P > 0.05), nor their interaction (F(23, 276) = 1.405, P > 0.05). 3.2. The effect of intra-VLO injection of benoxathian on the development and expression of morphine-induced behavioral sensitization in rats. On day 1, two-way repeated-measures ANOVA revealed significant main effects of time (F(23, 598) = 10.97, P < 0.0001), group (F(3, 26) = 33.17, P < 0.0001), as well as their interaction (F(69, 598) = 5.673, P < 0.0001). As shown in Fig. 2B, morphine (10 mg/kg) significantly increased the total distance (P < 0.001, Saline-Morphine group vs. Saline-Saline group, and P < 0.001, Benoxathian-Morphine group vs. Benoxathian-Saline group). Microinjection of benoxathian (2 μg/0.5 μl) had no statistically significant effect on the total distance of rats in 240 min (P > 0.05, Benoxathian-Saline
group
vs.
Saline-Saline
group,
and
P
>
0.05,
Benoxathian-Morphine group vs. Saline-Morphine group). On day 8, two-way repeated-measures ANOVA revealed significant main effects of time (F(23, 575) = 16.18, P < 0.0001), group (F(3, 25) = 17.15, P < 0.0001), as well as their interaction (F(69, 575) = 2.977, P < 0.0001). As shown in Fig. 2C, morphine (5 mg/kg) significantly increased the total distance (P < 0.01, Saline-Morphine group vs. Saline-Saline group). Microinjection of benoxathian (2 μg/0.5 μl) significantly 11
decreased the total distance of rats in 240 min (P < 0.05, Benoxathian-Morphine group vs. Saline-Morphine group). 3.3. Effect of intra-VLO injection of benoxathian on the protein levels of α1 adrenoceptors and ERK in the VLO The challenge dose of morphine exposure on day 8 significantly decreased α1 adrenoceptor expression compared with Saline-Saline group (P < 0.001). Microinjection benoxathian into the VLO significantly increased the protein levels of α1 adrenoceptor in Benoxathian-Morphine group compared with Saline-Morphine group (P < 0.001). However, there was no significant effect between Benoxathian-Saline group and Saline-Saline group in the VLO (P > 0.05) (Fig. 3A). The expression of p-ERK in the VLO was significantly enhanced on day 8 (Saline-Morphine group vs. Saline-Saline group, P < 0.05). The p-ERK levels were significantly higher in rats with microinjection of benoxathian into the VLO compared to rats administered saline (P < 0.01). The total ERK expression level was not changed in all groups (Fig. 3B). 4. Discussion Previous studies have indicated that a single exposure to morphine could evoke long-lasting behavioral sensitization in rodents [11, 24]. The present study demonstrated that a single i.p. injection of morphine, as reported previously, was sufficient to induce behavioral sensitization in rats. Animals exposed to morphine showed an initial inhibition of locomotion lasting approximately 60 min and then a robust increase of locomotion from 60 min to 240 min. Though the total travelled 12
distance in 240 min had a decreased tendency after bilateral intra-VLO injection of benoxathian, the statistic analysis indicated that there was no significant effect in the development phase of morphine-induced behavioral sensitization. However, in the expression phase of morphine-induced behavioral sensitization in 240 min, the total travelled distance was reduced significantly by bilateral intra-VLO injection of benoxathian, whereas benoxathian applying alone to the VLO did not influence the basal locomotion. Drouin et al. previously found that local administration of α1 adrenergic antagonist in the prefrontal cortex (PFC) or loss of α1b-adrenergic receptor expression in knock-out mice could reduce the expression, but not the development, of behavioral sensitization by acute morphine administration in mice [6, 7]. Our present results in rats are in line with Drouin’s findings in mice. It has been established that behavioral sensitization is relevant to drug addiction, which has been used extensively as a promising animal model to evaluate the key features of addiction, including relapse, drug-seeking and drug- taking behaviors [30]. To the best of our knowledge, the present results provide evidence for the first time that the VLO may participate in opioid addiction and α1 adrenoceptors in the VLO are involved in mediating morphine-induced behavioral sensitization. In addition, western blotting results from the present study showed that the expression levels of α1 adrenoceptors in the VLO were significantly decreased in Saline-Morphine group compared with Saline-Saline group, whereas there was a significant increase of α1 adrenoceptors levels in Benoxathian-Morphine group compared with Saline-Morphine group during the expression of morphine-induced 13
behavioral sensitization. What is the reason of the changes of α1 adrenoceptors in the VLO? Previous studies have demonstrated that systemic administration of morphine results in increased synthesis and catabolism of dopamine as well as selectively depressed norepinephrine cell activity in striatum and limbic structures of the rats [13, 18]. It has been well characterized that α1 adrenoceptors are expressed throughout the mesocorticolimbic system and these receptors in the PFC play important roles in dopamine release as well as behavioral responses to drug abuse. Dopamine, which has been shown to activate α1 adrenoceptor under some conditions, could be acting as an α1 adrenoceptor ligand in the NAc core [14, 17]. Biochemical, electrophysiological and behavioral data have also indicated crosstalk between dopamine receptors and α1 adrenoceptors signaling in many brain areas, including the PFC [16]. On the other hand, morphine exposure induced inhibition of neural progenitor cell proliferation and increased active caspase-3 expression to induce apoptosis in distinct anatomical regions known to be important for sensory (cortex) and emotional memory processing (amygdala) [1, 29]. So we could speculate that morphine might, directly or indirectly crosstalk with dopamine receptors, depress norepinephrine cell activity and disrupt the α1 adrenoceptors in Saline-Morphine group while benoxathian prevents this process and induces the α1 adrenoceptors compensatory increase. Further experiments are needed to fully address the potential mechanism in future. Recent studies indicated that NE played a key role in rewarding effects of opiates in PFC [26]. Morphological studies have demonstrated that the VLO receives norepinephrinergic terminals input from the pons, and α1 adrenoceptors are widely 14
expressed in the VLO [4, 32]. Meanwhile, α1 adrenoceptors can activate ERK via PLC or Ca2+ influx [9]. ERK has been shown to participate in behavioral sensitization and the development of neuroplasticity related to the addictive properties of drug abuse [19, 23]. Therefore, we speculate that morphine could impact ERK activation by α1 adrenoceptor receptors in the VLO. Our results showed that morphine induced the accumulation of p-ERK and benoxathian further augmented the effect of morphine in the VLO during the expression phase of morphine-induced behavioral sensitization, while benoxathian did not affect the levels of p-ERK protein. These findings support our hypothesis, though there was some difference with the Valjent et al. report that activation of ERK contributed to the development instead of the expression phase of cocaine sensitized activity [23]. The discrepancy possibly owes to different drug treatment, specificity of brain areas and animal species. In light of this literature, our present findings indicate that ERK could mediate the locomotor activity on the expression of sensitization suppressed by benoxathian. The present results further confirm that α1 adrenoceptors play an important role in morphine-induced behavioral sensitization, possibly via the ERK signaling pathway. In conclusion, α1 adrenoceptors in the VLO are involved in regulating the expression but not the development phase of morphine-induced behavioral sensitization. The effect of decreased locomotor activity by blocking α1 adrenoceptors might be associated with activation of ERK in the VLO. Conflict of interest The authors have no conflicts of interest to declare. 15
Acknowledgment The authors wish to thank Dr. Devin Barry for expert help in revising the manuscript. This study was funded by the National Natural Science Foundation of China (No. 30800334, 81371230, 81172903, 81472820, 81430048), China Postdoctoral Science Foundation (No. 2014M560760), Natural Science Basic Research Plan in Shaanxi Province of China (No. 2015JM3122) and the Fundamental Research Funds for the Central Universities (Projects of International Cooperation and Exchanges).
16
References [1]
D. Bajic, K.G. Commons, S.G. Soriano, Morphine-enhanced apoptosis in selective brain regions of neonatal rats, Int J Dev Neurosci 31 (2013) 258-266.
[2]
F.M. Benes, S.L. Vincent, R. Molloy, Dopamine-immunoreactive axon varicosities form nonrandom contacts with GABA-immunoreactive neurons of rat medial prefrontal cortex, Synapse 15 (1993) 285-295.
[3]
M.T. Berhow, N. Hiroi, E.J. Nestler, Regulation of ERK (extracellular signal regulated kinase), part of the neurotrophin signal transduction cascade, in the rat mesolimbic dopamine system by chronic exposure to morphine or cocaine, J Neurosci 16 (1996) 4707-4715.
[4]
H.E. Day, S. Campeau, S.J. Watson, Jr., H. Akil, Distribution of alpha 1a-, alpha 1b- and alpha 1d-adrenergic receptor mRNA in the rat brain and spinal cord, J Chem Neuroanat 13 (1997) 115-139.
[5]
T.J. De Vries, A.N. Schoffelmeer, R. Binnekade, A.H. Mulder, L.J. Vanderschuren, Drug-induced reinstatement of heroin- and cocaine-seeking behaviour following long-term extinction is associated with expression of behavioural sensitization, Eur J Neurosci 10 (1998) 3565-3571.
[6]
C. Drouin, G. Blanc, F. Trovero, J. Glowinski, J.P. Tassin, Cortical alpha 1-adrenergic regulation of acute and sensitized morphine locomotor effects, Neuroreport 12 (2001) 3483-3486.
17
[7]
C. Drouin, L. Darracq, F. Trovero, G. Blanc, J. Glowinski, S. Cotecchia, J.P. Tassin, Alpha1b-adrenergic receptors control locomotor and rewarding effects of psychostimulants and opiates, J Neurosci 22 (2002) 2873-2884.
[8]
R.E. Grahn, S.E. Hammack, M.J. Will, K.A. O'Connor, T. Deak, P.D. Sparks, L.R. Watkins, S.F. Maier, Blockade of alpha1 adrenoreceptors in the dorsal raphe nucleus prevents enhanced conditioned fear and impaired escape performance following uncontrollable stressor exposure in rats, Behav Brain Res 134 (2002) 387-392.
[9]
C. Hague, P.J. Gonzalez-Cabrera, W.B. Jeffries, P.W. Abel, Relationship between alpha(1)-adrenergic receptor-induced contraction and extracellular signal-regulated kinase activation in the bovine inferior alveolar artery, J Pharmacol Exp Ther 303 (2002) 403-411.
[10]
S.E. Hyman, Addiction: a disease of learning and memory, Am J Psychiatry 162 (2005) 1414-1422.
[11]
L. Jing, M. Zhang, J.X. Li, P. Huang, Q. Liu, Y.L. Li, H. Liang, J.H. Liang, Comparison of single versus repeated methamphetamine injection induced behavioral sensitization in mice, Neurosci Lett 560 (2014) 103-106.
[12]
A.K. Kim, M.L. Souza-Formigoni, Alpha1-adrenergic drugs affect the development and expression of ethanol-induced behavioral sensitization, Behav Brain Res 256 (2013) 646-654.
[13]
J. Korf, B.S. Bunney, G.K. Aghajanian, Noradrenergic neurons: Morphine inhibition of spontaneous activity, Eur J Pharmacol 25 (1974) 165-169. 18
[14]
Y. Lin, D. Quartermain, A.J. Dunn, D. Weinshenker, E.A. Stone, Possible dopaminergic stimulation of locus coeruleus α1-adrenoceptors involved in behavioral activation, Synapse 62 (2008) 516-523.
[15]
Q. Liu, M. Zhang, W.J. Qin, Y.T. Wang, Y.L. Li, L. Jing, J.X. Li, A.J. Lawrence, J.H. Liang, Septal nuclei critically mediate the development of behavioral sensitization to a single morphine injection in rats, Brain Res 1454 (2012) 90-99.
[16]
D.A. Mitrano, J.F. Pare, Y. Smith, D. Weinshenker, D1-dopamine and alpha1-adrenergic receptors co-localize in dendrites of the rat prefrontal cortex, Neuroscience 258 (2014) 90-100.
[17]
D.A. Mitrano, J.P. Schroeder, Y. Smith, J.J. Cortright, N. Bubula, P. Vezina, D. Weinshenker, alpha-1 Adrenergic receptors are localized on presynaptic elements in the nucleus accumbens and regulate mesolimbic dopamine transmission, Neuropsychopharmacology 37 (2012) 2161-2172.
[18]
P. Moleman, C.F. van Valkenburg, J.A. vd Krogt, Effects of morphine on dopamine metabolism in rat striatum and limbic structures in relation to the activity of dopaminergic neurones, Naunyn Schmiedebergs Arch Pharmacol 327 (1984) 208-213.
[19]
D.L. Muller, E.M. Unterwald, In vivo regulation of extracellular signal-regulated protein kinase (ERK) and protein kinase B (Akt) phosphorylation by acute and chronic morphine, J Pharmacol Exp Ther 310 (2004) 774-782. 19
[20]
Paxinos. G, Watson. C, The Rat Brain in Stereotaxic Coordinates, 2th Edition, (1986).
[21]
D.R. Ramirez, L.M. Savage, Differential involvement of the basolateral amygdala, orbitofrontal cortex, and nucleus accumbens core in the acquisition and use of reward expectancies, Behav Neurosci 121 (2007) 896-906.
[22]
J.S. Tang, C.L. Qu, F.Q. Huo, The thalamic nucleus submedius and ventrolateral orbital cortex are involved in nociceptive modulation: a novel pain modulation pathway, Prog Neurobiol 89 (2009) 383-389.
[23]
E. Valjent, J.C. Corvol, J.M. Trzaskos, J.A. Girault, D. Herve, Role of the ERK pathway in psychostimulant-induced locomotor sensitization, BMC Neurosci 7 (2006) 20.
[24]
L.J. Vanderschuren, T.J. De Vries, G. Wardeh, F.A. Hogenboom, A.N. Schoffelmeer, A single exposure to morphine induces long-lasting behavioural and neurochemical sensitization in rats, Eur J Neurosci 14 (2001) 1533-1538.
[25]
L.J. Vanderschuren, P.W. Kalivas, Alterations in dopaminergic and glutamatergic transmission in the induction and expression of behavioral sensitization: a critical review of preclinical studies, Psychopharmacology (Berl) 151 (2000) 99-120.
[26]
R. Ventura, A. Alcaro, S. Puglisi-Allegra, Prefrontal cortical norepinephrine release is critical for morphine-induced reward, reinstatement and dopamine release in the nucleus accumbens, Cereb Cortex 15 (2005) 1877-1886.
20
[27]
T.L. Verplaetse, D.D. Rasmussen, J.C. Froehlich, C.L. Czachowski, Effects of prazosin, an alpha1-adrenergic receptor antagonist, on the seeking and intake of alcohol and sucrose in alcohol-preferring (P) rats, Alcohol Clin Exp Res 36 (2012) 881-886.
[28]
C. von der Goltz, F. Kiefer, Learning and memory in the aetiopathogenesis of addiction: future implications for therapy?, Eur Arch Psychiatry Clin Neurosci 259 Suppl 2 (2009) S183-187.
[29]
D. Willner, A. Cohen-Yeshurun, A. Avidan, V. Ozersky, E. Shohami, R.R. Leker, Short term morphine exposure in vitro alters proliferation and differentiation of neural progenitor cells and promotes apoptosis via mu receptors, PLoS One 9 (2014) e103043.
[30]
R.A. Wise, M.A. Bozarth, A psychomotor stimulant theory of addiction, Psychol Rev 94 (1987) 469-492.
[31]
Y. Zhao, B. Xing, Y.H. Dang, C.L. Qu, F. Zhu, C.X. Yan, Microinjection of valproic acid into the ventrolateral orbital cortex enhances stress-related memory formation, PLoS One 8 (2013) e52698.
[32]
J.X. Zhu, F.Y. Xu, W.J. Xu, Y. Zhao, C.L. Qu, J.S. Tang, D.M. Barry, J.Q. Du, F.Q. Huo, The role of alpha(2) adrenoceptor in mediating noradrenaline action in the ventrolateral orbital cortex on allodynia following spared nerve injury, Exp Neurol 248 (2013) 381-386.
21
Figure Captions Fig. 1. The experimental schedule of morphine-induced behavioral sensitization (A) and photomicrograph example of injection sites in the bilateral VLO (B). Abbreviations: Beno, benoxathian; fmi, forceps minor corpos callosum; i.p., intraperitoneally; m.i., microinjection; min, minute. Mor, morphine; Sal, saline. Scale bar = 500 μm.
22
Fig. 2. Effect of intra-VLO injection of benoxathian on morphine-induced behavioral sensitization. (A) Effect of intra-VLO injection of benoxathian alone on the total distance of 240 min with traveled distance within every 10 min (line chart)) and the total distance (bar graph) (n = 7). *P < 0.05, vs. Sal-Sal group. (B) Effect of intra-VLO injection of benoxathian on the development of morphine-induced behavioral sensitization in 240 min every 10 min (line chart)) and the total distance (bar graph) (n = 7-8). *P < 0.05, **P < 0.01, ***P < 0.001, vs. Sal-Sal group; ^P < 0.05; ^^P < 0.01; ^^^P < 0.001, vs. Beno-Sal group. (C) Effect of intra-VLO injection of benoxathian on the expression of morphine-induced behavioral sensitization in 240 min every 10 min (line chart) and the total distance (bar graph) (n = 6-8). *P < 0.05, **P < 0.01, ***P < 0.001, vs. Sal-Sal group; #P < 0.05; ##P < 0.01; ###P < 0.001, vs. Sal-Mor group. Abbreviations: α1-AR, α1 adrenoceptor; Beno, benoxathian; Mor, morphine; Sal, saline.
23
24
Fig. 3. Effect of intra-VLO injection of benoxathian on the protein levels of α1 adrenoceptor (A) and p-ERK/ERK (B) in the VLO (n = 8). *P < 0.05, ***P < 0.001 vs. Sal-Sal group,
##
P < 0.01,
###
P < 0.001, vs. Saline-Mor group,
Beno-Sal group.
25
^^^
P < 0.001, vs.
S0304-3940(15)30220-2 http://dx.doi.org/doi:10.1016/j.neulet.2015.10.057 NSL 31623
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
8-8-2015 9-10-2015 22-10-2015
Please cite this article as: Lai Wei, Yuan-Mei Zhu, Yu-Xiang Zhang, Feng Liang, Teng Li, Hong-Yu Gao, Fu-Quan Huo, Chun-Xia Yan, The rmalpha1 adrenoceptors in ventrolateral orbital cortex contribute to the expression of morphine-induced behavioral sensitization in rats, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2015.10.057 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.
The α1 adrenoceptors in ventrolateral orbital cortex contribute to the expression of morphine-induced behavioral sensitization in rats Lai Wei1,4, Yuan-Mei Zhu1, Yu-Xiang Zhang1, Feng Liang1, Teng Li1, Hong-Yu Gao1, Fu-Quan Huo2,3* [email protected], Chun-Xia Yan1,3* [email protected] 1
College of Forensic Medicine, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi
710061, China. 2
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Xi’an
Jiaotong University Health Science Center, Xi’an, Shaanxi 710061, China. 3
Key Laboratory of Environment and Genes Related to Diseases (Xi’an Jiaotong University),
Ministry of Education, China. 4
Division of Forensic Medicine, School of Basic Medical Sciences, Hubei University of Medicine,
Shiyan, Hubei 442000, China. *
Corresponding authors at: College of Forensic Medicine, Xi’an Jiaotong University Health
Science Center, Xi’an, Shaanxi 710061, China. Tel: +86 29 82655117; Fax: +86 29 82655472
1
Highlights The VLO is a novel anatomical substrate mediating morphine-induced behavioral sensitization. Blocking α1 adrenoceptors in the VLO suppressed the expression of morphine-induced behavioral sensitization. Morphine-induced behavioral sensitization may be associated with activated ERK in the VLO.
Abstract The aim of the present study was to investigate the effect of microinjection of benoxathian, selective α1 adrenoceptor antagonist, into the ventrolateral orbital cortex (VLO) on morphine-induced behavioral sensitization and its underlying molecular mechanism in rats. A single morphine treatment protocol was used in establishing the behavioral sensitization model. The effect of bilateral intra-VLO benoxathian injection on locomotor activity was examined and the protein expression levels of α1 adrenoceptors and activation of extracellular signal-regulated kinase (ERK) in the VLO were detected after locomotor test. The results showed that a single injection of morphine could induce behavioral sensitization by a low challenge dosage of morphine after a 7-day drug free period. Benoxathian significantly suppressed the expression but not the development of morphine-induced behavioral sensitization. Morphine treatment significantly elicited ERK phosphorylation and downregulated the expression level of α1 adrenoceptors in the VLO. In addition, intra-VLO 2
benoxathian injection enhanced the expression levels of α1 adrenoceptors and phosphorylated ERK. These results suggest that α1 adrenoceptors in the VLO are involved in regulating the expression of morphine-induced behavioral sensitization. The effect of decreased locomotor activity by blocking α1 adrenoceptors might be associated with activation of ERK in the VLO.
Abbreviations α1-AR: α1 adrenoceptor Beno: benoxathian ERK: extracellular signal-regulated kinase fmi: forceps minor corpus callosum i.p.: intraperitoneally m.i.: microinjection min: minute(s) Mor: morphine NAc: nucleus accumbens OFC: orbitofrontal cortex p-ERK: phosphorylated ERK PFC: prefrontal cortex Sal: saline VLO: ventrolateral orbital cortex VTA: ventral tegmental area. 3
Keywords: Morphine; α1 adrenoceptor; Ventrolateral orbital cortex; Extracellular signal-regulated kinase; Behavioral sensitization
4
1. Introduction Behavioral sensitization, which is thought to play an important role in certain aspects of drug addiction such as compulsive drug seeking, is the result of an abundance of neuroplastic changes occurring within the brain circuitry involved in motivational behavior and neurochemical effects [5, 25]. A single or repeated drug exposure can cause long-lasting behavioral sensitization which is reflected by an increase in locomotor activity [11, 24]. Evidence from molecular, behavioral, and computational levels of analysis suggests that addiction represents a pathological usurpation of the neural processes of learning and memory [10]. The disruption of drug-related memories may help to prevent addiction [28]. Our laboratory studies demonstrate that the ventrolateral orbital cortex (VLO), a major subdivision of orbitofrontal cortex (OFC), is involved in stress-related memory formation and antinociception [22, 31]. Anatomic studies indicate that the OFC has reciprocal fibers connection with nucleus accumbens (NAc) and hippocampus, and receives input from dopaminergic terminals originating from the ventral tegmental area (VTA) as crucial brain regions associated with opioid addiction [2, 21]. These results imply that the VLO may be a candidate anatomical substrate involving in opioid addiction. Furthermore, recent studies have indicated that norepinephrine (NE) in the prefrontal cortex plays a critical role in the rewarding effects of opiates [26]. The VLO receives norepinephrinergic terminal input from the pons, and adrenoceptors, including α1 and α2, are expressed in the VLO [4, 32]. These results suggest that NE and their receptors in the VLO may be involved in opioid 5
addiction. Alteration of α1 adrenoceptors by the administration of agonists or antagonists affects the locomotor sensitization to the stimulant effect of ethanol consumption, suggesting that the normal functioning of the noradrenergic system is essential to development and expression of behavioral sensitization [12, 27]. The aim of the present study is to investigate the effects of microinjection of a selective α1 adrenoceptor antagonist, benoxathian, into the VLO on morphine-induced behavioral sensitization in rats. Evidence suggests that extracellular signal-regulated kinase (ERK) is involved in behavioral sensitization and the development of neuroplasticity relating to the addictive properties of drug abuse [3, 19, 23]. ERK could be activated by α1 adrenoceptor, a Gq-protein coupled receptor, via phospholipase C (PLC) / protein kinase C (PKC) signal pathways [9]. However, the effect of ERK in the VLO on morphine-induced behavioral sensitization is not yet clear. Therefore, we further examined the expression levels of α1 adrenoceptor and ERK activation in the VLO in an
attempt
to
gain
insight
into
the
molecular
mechanism
underlying
morphine-induced behavioral sensitization. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats weighing between 250 and 300 g were provided by the Medical Experimental Animal Center of Xi’an Jiaotong University, Shaanxi Province, China. Animals were housed in a colony room with controlled ambient temperature (25 ± 2 °C), humidity (50 ± 10%) and under a 12/12 h light-dark cycle 6
(lights on at 07:00) with access to food and water ad libitum. The experimental protocols were approved by the Institutional Animal Care Committee of Xi'an Jiaotong University. All efforts were made to minimize the number of rats used and their suffering in the experiments. 2.2. Bilateral intracerebral guide cannula placement Rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally, i.p.), and the head was immobilized in a stereotaxic frame. A small craniotomy was performed just above the VLO. Stainless steel guide cannulas (0.8 mm in diameter) were stereotaxically inserted bilaterally, with the tip 2.0 mm dorsal to the VLO at the following coordinates: + 3.2 mm anterior to bregma, ± 2.0 mm lateral, and + 2.6 mm below the cortical surface [20, 31], followed by attachment to the skull with two microscrews and dental cement. Once the rats recovered from anesthesia, they were administered sodium penicillin (0.2 million units/day for 3 days, i.p.) to prevent wound and intracerebral infections. The rats were carefully nursed and fed in clean cages. After surgery, animals were housed separately in cages and allowed to recover for 7 days before behavioral procedures. 2.3. Intracerebral microinjection of drugs Morphine hydrochloride was obtained from First Pharmaceutical Factory of Shenyang (China) and administered by i.p. route (10 or 5 mg/kg). Benoxathian hydrochloride was purchased from Sigma-Aldrich (Sigma Chemical, St. Louis, MO, USA) and microinjected at a final concentration of 2 μg/0.5 μl. All drugs were dissolved in physiological saline and freshly prepared before the experiments. The 7
effective drug doses were based on previous reports and our preliminary experiments [8, 15, 24]. Equal volumes of saline served as the vehicle control. Rats were randomly divided into 4 groups (16 rats/group): saline-saline group (intracerebral microinjection of saline 30 min prior to initial saline injection, i.p.); benoxathian-saline group (intracerebral microinjection of benoxathian 30 min prior to initial saline injection, i.p.); saline-morphine group (intracerebral microinjection of saline 30 min prior to initial
morphine
injection,
i.p.);
benoxathian-morphine
group
(intracerebral
microinjection of benoxathian 30 min prior to initial morphine injection, i.p.), as shown in Fig. 1A. Microsyringe was inserted into the VLO, with the tip extending 2.0 mm beyond the end of the guide cannula (total depth 4.6 mm) from the brain surface. Benoxathian was slowly infused bilaterally through a 1.0-μl Hamilton syringe at a constant speed over 1 minute to make sure the drugs completely diffuse from the tips. Saline of equal volume was injected into the VLO as vehicle control. 2.4. The locomotor activity test The locomotor activity tests were conducted in a quiet, dimly lit room between 8:00 am and 12:00 am. An animal locomotor activity measurement system consisting of four testing boxes (60 × 60 × 40 cm3, length × width × height) with a video camera was used to test individual rats. The interior bottoms of the testing boxes were painted black, whereas the interior sides were painted white. The boxes were set in an isolated dark room, and four standard laboratory lamps were used for illumination. After the experimental animals were immediately placed in the boxes, their locomotor activities were recorded by the video camera and analyzed off line using a computer by the 8
video-tracking software (SMART, Panlab SL, Barcelona, Spain). Horizontal trajectories (reflection of locomotor activity) of the rats were recorded and analyzed to determine their travelled distance. 2.5. Protein extraction and western blotting After behavior test, eight rats in each group were decapitated. The VLO were dissected and removed according to the rat brain atlas [20] and homogenized at 4 °C in a RIPA buffer with a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). A BCA protein assay kit (Pierce, Rockland, IL, USA) was used to determine total protein levels for each sample. Equal amounts of protein for each group were separated on a 12% SDS-PAGE gel and transferred to PVDF membranes (Millipore Corporation, Bedford, MA, USA). The membranes were blocked in 5 % (w/v) non-fat milk in Tris-buffered saline (pH 7.5) containing 0.05% Tween-20 for 1 h at room temperature and then incubated with the following primary antibodies overnight at 4 °C: alpha 1 adrenergic receptor (ab3462, Abcam, Cambridge, UK ) at 1:1000, p-ERK 1/2, ERK 1/2 (Cell Signaling Technology, Danvers, MA, USA) at 1:1000, β-actin (Santa Cruz, California, USA) at 1:1000. The membranes were washed and incubated with secondary antibodies: goat anti-rabbit or anti-mouse IgG horse-radish peroxidase (HRP)-conjugated secondary antibody (Bioworld, Dublin, OH, USA) at 1:10,000 dilution. Color development was performed with an enhanced chemiluminescence plus detection kit (Millipore Corporation, Bedford, MA, USA). The band intensities were analyzed using the Quantity One software (Bio-Rad,
9
Hercules, USA) to calculate the target protein versus the loading control (β-actin) for each protein. 2.6. Histology The injection sites were histologically identified or visually confirmed after the behavioral tests as previous reported [32]. At the end of the test, the drug injection sites were marked by injecting Pontamine Sky Blue dye. Under deep anesthesia, the rats were intracardially perfused and fixed. The brains were cut and stained with Cresyl Violet. The injection sites were histologically identified to be within the VLO, as shown in Fig. 1B. 2.7. Data analysis All values were expressed as mean ± SEM. The data of locomotor activity were analyzed by Student’s t-test, one-way ANOVA and two-way repeated measures ANOVA, followed by a Bonferroni post hoc analysis for multiple comparisons. For western blotting, the difference of the protein levels in the VLO was determined by one-way ANOVA. The protein levels of the control group were set at 100% and all data were normalized to loading control. The data analyses were performed using software GraphPad Prism 5.0. Values of P < 0.05 were considered statistically significant. 3. Results 3.1. The effect of microinjection of benoxathian alone into the VLO on locomotor activity in rats.
10
Microinjection of a selective α1 adrenoceptor antagonist, benoxathian (2 μg/0.5 μl, n=7), alone into the VLO had no effect on the accumulated distance during 240 min, except for a transient reduction at 20 min compared to saline-injected group (P < 0.05) (Fig. 2A). Two-way repeated-measures ANOVA analyses revealed, a significant main effect of time (F(23, 276) = 29.46, P < 0.0001), but no effect of group (F(1, 12) = 0.2357, P > 0.05), nor their interaction (F(23, 276) = 1.405, P > 0.05). 3.2. The effect of intra-VLO injection of benoxathian on the development and expression of morphine-induced behavioral sensitization in rats. On day 1, two-way repeated-measures ANOVA revealed significant main effects of time (F(23, 598) = 10.97, P < 0.0001), group (F(3, 26) = 33.17, P < 0.0001), as well as their interaction (F(69, 598) = 5.673, P < 0.0001). As shown in Fig. 2B, morphine (10 mg/kg) significantly increased the total distance (P < 0.001, Saline-Morphine group vs. Saline-Saline group, and P < 0.001, Benoxathian-Morphine group vs. Benoxathian-Saline group). Microinjection of benoxathian (2 μg/0.5 μl) had no statistically significant effect on the total distance of rats in 240 min (P > 0.05, Benoxathian-Saline
group
vs.
Saline-Saline
group,
and
P
>
0.05,
Benoxathian-Morphine group vs. Saline-Morphine group). On day 8, two-way repeated-measures ANOVA revealed significant main effects of time (F(23, 575) = 16.18, P < 0.0001), group (F(3, 25) = 17.15, P < 0.0001), as well as their interaction (F(69, 575) = 2.977, P < 0.0001). As shown in Fig. 2C, morphine (5 mg/kg) significantly increased the total distance (P < 0.01, Saline-Morphine group vs. Saline-Saline group). Microinjection of benoxathian (2 μg/0.5 μl) significantly 11
decreased the total distance of rats in 240 min (P < 0.05, Benoxathian-Morphine group vs. Saline-Morphine group). 3.3. Effect of intra-VLO injection of benoxathian on the protein levels of α1 adrenoceptors and ERK in the VLO The challenge dose of morphine exposure on day 8 significantly decreased α1 adrenoceptor expression compared with Saline-Saline group (P < 0.001). Microinjection benoxathian into the VLO significantly increased the protein levels of α1 adrenoceptor in Benoxathian-Morphine group compared with Saline-Morphine group (P < 0.001). However, there was no significant effect between Benoxathian-Saline group and Saline-Saline group in the VLO (P > 0.05) (Fig. 3A). The expression of p-ERK in the VLO was significantly enhanced on day 8 (Saline-Morphine group vs. Saline-Saline group, P < 0.05). The p-ERK levels were significantly higher in rats with microinjection of benoxathian into the VLO compared to rats administered saline (P < 0.01). The total ERK expression level was not changed in all groups (Fig. 3B). 4. Discussion Previous studies have indicated that a single exposure to morphine could evoke long-lasting behavioral sensitization in rodents [11, 24]. The present study demonstrated that a single i.p. injection of morphine, as reported previously, was sufficient to induce behavioral sensitization in rats. Animals exposed to morphine showed an initial inhibition of locomotion lasting approximately 60 min and then a robust increase of locomotion from 60 min to 240 min. Though the total travelled 12
distance in 240 min had a decreased tendency after bilateral intra-VLO injection of benoxathian, the statistic analysis indicated that there was no significant effect in the development phase of morphine-induced behavioral sensitization. However, in the expression phase of morphine-induced behavioral sensitization in 240 min, the total travelled distance was reduced significantly by bilateral intra-VLO injection of benoxathian, whereas benoxathian applying alone to the VLO did not influence the basal locomotion. Drouin et al. previously found that local administration of α1 adrenergic antagonist in the prefrontal cortex (PFC) or loss of α1b-adrenergic receptor expression in knock-out mice could reduce the expression, but not the development, of behavioral sensitization by acute morphine administration in mice [6, 7]. Our present results in rats are in line with Drouin’s findings in mice. It has been established that behavioral sensitization is relevant to drug addiction, which has been used extensively as a promising animal model to evaluate the key features of addiction, including relapse, drug-seeking and drug- taking behaviors [30]. To the best of our knowledge, the present results provide evidence for the first time that the VLO may participate in opioid addiction and α1 adrenoceptors in the VLO are involved in mediating morphine-induced behavioral sensitization. In addition, western blotting results from the present study showed that the expression levels of α1 adrenoceptors in the VLO were significantly decreased in Saline-Morphine group compared with Saline-Saline group, whereas there was a significant increase of α1 adrenoceptors levels in Benoxathian-Morphine group compared with Saline-Morphine group during the expression of morphine-induced 13
behavioral sensitization. What is the reason of the changes of α1 adrenoceptors in the VLO? Previous studies have demonstrated that systemic administration of morphine results in increased synthesis and catabolism of dopamine as well as selectively depressed norepinephrine cell activity in striatum and limbic structures of the rats [13, 18]. It has been well characterized that α1 adrenoceptors are expressed throughout the mesocorticolimbic system and these receptors in the PFC play important roles in dopamine release as well as behavioral responses to drug abuse. Dopamine, which has been shown to activate α1 adrenoceptor under some conditions, could be acting as an α1 adrenoceptor ligand in the NAc core [14, 17]. Biochemical, electrophysiological and behavioral data have also indicated crosstalk between dopamine receptors and α1 adrenoceptors signaling in many brain areas, including the PFC [16]. On the other hand, morphine exposure induced inhibition of neural progenitor cell proliferation and increased active caspase-3 expression to induce apoptosis in distinct anatomical regions known to be important for sensory (cortex) and emotional memory processing (amygdala) [1, 29]. So we could speculate that morphine might, directly or indirectly crosstalk with dopamine receptors, depress norepinephrine cell activity and disrupt the α1 adrenoceptors in Saline-Morphine group while benoxathian prevents this process and induces the α1 adrenoceptors compensatory increase. Further experiments are needed to fully address the potential mechanism in future. Recent studies indicated that NE played a key role in rewarding effects of opiates in PFC [26]. Morphological studies have demonstrated that the VLO receives norepinephrinergic terminals input from the pons, and α1 adrenoceptors are widely 14
expressed in the VLO [4, 32]. Meanwhile, α1 adrenoceptors can activate ERK via PLC or Ca2+ influx [9]. ERK has been shown to participate in behavioral sensitization and the development of neuroplasticity related to the addictive properties of drug abuse [19, 23]. Therefore, we speculate that morphine could impact ERK activation by α1 adrenoceptor receptors in the VLO. Our results showed that morphine induced the accumulation of p-ERK and benoxathian further augmented the effect of morphine in the VLO during the expression phase of morphine-induced behavioral sensitization, while benoxathian did not affect the levels of p-ERK protein. These findings support our hypothesis, though there was some difference with the Valjent et al. report that activation of ERK contributed to the development instead of the expression phase of cocaine sensitized activity [23]. The discrepancy possibly owes to different drug treatment, specificity of brain areas and animal species. In light of this literature, our present findings indicate that ERK could mediate the locomotor activity on the expression of sensitization suppressed by benoxathian. The present results further confirm that α1 adrenoceptors play an important role in morphine-induced behavioral sensitization, possibly via the ERK signaling pathway. In conclusion, α1 adrenoceptors in the VLO are involved in regulating the expression but not the development phase of morphine-induced behavioral sensitization. The effect of decreased locomotor activity by blocking α1 adrenoceptors might be associated with activation of ERK in the VLO. Conflict of interest The authors have no conflicts of interest to declare. 15
Acknowledgment The authors wish to thank Dr. Devin Barry for expert help in revising the manuscript. This study was funded by the National Natural Science Foundation of China (No. 30800334, 81371230, 81172903, 81472820, 81430048), China Postdoctoral Science Foundation (No. 2014M560760), Natural Science Basic Research Plan in Shaanxi Province of China (No. 2015JM3122) and the Fundamental Research Funds for the Central Universities (Projects of International Cooperation and Exchanges).
16
References [1]
D. Bajic, K.G. Commons, S.G. Soriano, Morphine-enhanced apoptosis in selective brain regions of neonatal rats, Int J Dev Neurosci 31 (2013) 258-266.
[2]
F.M. Benes, S.L. Vincent, R. Molloy, Dopamine-immunoreactive axon varicosities form nonrandom contacts with GABA-immunoreactive neurons of rat medial prefrontal cortex, Synapse 15 (1993) 285-295.
[3]
M.T. Berhow, N. Hiroi, E.J. Nestler, Regulation of ERK (extracellular signal regulated kinase), part of the neurotrophin signal transduction cascade, in the rat mesolimbic dopamine system by chronic exposure to morphine or cocaine, J Neurosci 16 (1996) 4707-4715.
[4]
H.E. Day, S. Campeau, S.J. Watson, Jr., H. Akil, Distribution of alpha 1a-, alpha 1b- and alpha 1d-adrenergic receptor mRNA in the rat brain and spinal cord, J Chem Neuroanat 13 (1997) 115-139.
[5]
T.J. De Vries, A.N. Schoffelmeer, R. Binnekade, A.H. Mulder, L.J. Vanderschuren, Drug-induced reinstatement of heroin- and cocaine-seeking behaviour following long-term extinction is associated with expression of behavioural sensitization, Eur J Neurosci 10 (1998) 3565-3571.
[6]
C. Drouin, G. Blanc, F. Trovero, J. Glowinski, J.P. Tassin, Cortical alpha 1-adrenergic regulation of acute and sensitized morphine locomotor effects, Neuroreport 12 (2001) 3483-3486.
17
[7]
C. Drouin, L. Darracq, F. Trovero, G. Blanc, J. Glowinski, S. Cotecchia, J.P. Tassin, Alpha1b-adrenergic receptors control locomotor and rewarding effects of psychostimulants and opiates, J Neurosci 22 (2002) 2873-2884.
[8]
R.E. Grahn, S.E. Hammack, M.J. Will, K.A. O'Connor, T. Deak, P.D. Sparks, L.R. Watkins, S.F. Maier, Blockade of alpha1 adrenoreceptors in the dorsal raphe nucleus prevents enhanced conditioned fear and impaired escape performance following uncontrollable stressor exposure in rats, Behav Brain Res 134 (2002) 387-392.
[9]
C. Hague, P.J. Gonzalez-Cabrera, W.B. Jeffries, P.W. Abel, Relationship between alpha(1)-adrenergic receptor-induced contraction and extracellular signal-regulated kinase activation in the bovine inferior alveolar artery, J Pharmacol Exp Ther 303 (2002) 403-411.
[10]
S.E. Hyman, Addiction: a disease of learning and memory, Am J Psychiatry 162 (2005) 1414-1422.
[11]
L. Jing, M. Zhang, J.X. Li, P. Huang, Q. Liu, Y.L. Li, H. Liang, J.H. Liang, Comparison of single versus repeated methamphetamine injection induced behavioral sensitization in mice, Neurosci Lett 560 (2014) 103-106.
[12]
A.K. Kim, M.L. Souza-Formigoni, Alpha1-adrenergic drugs affect the development and expression of ethanol-induced behavioral sensitization, Behav Brain Res 256 (2013) 646-654.
[13]
J. Korf, B.S. Bunney, G.K. Aghajanian, Noradrenergic neurons: Morphine inhibition of spontaneous activity, Eur J Pharmacol 25 (1974) 165-169. 18
[14]
Y. Lin, D. Quartermain, A.J. Dunn, D. Weinshenker, E.A. Stone, Possible dopaminergic stimulation of locus coeruleus α1-adrenoceptors involved in behavioral activation, Synapse 62 (2008) 516-523.
[15]
Q. Liu, M. Zhang, W.J. Qin, Y.T. Wang, Y.L. Li, L. Jing, J.X. Li, A.J. Lawrence, J.H. Liang, Septal nuclei critically mediate the development of behavioral sensitization to a single morphine injection in rats, Brain Res 1454 (2012) 90-99.
[16]
D.A. Mitrano, J.F. Pare, Y. Smith, D. Weinshenker, D1-dopamine and alpha1-adrenergic receptors co-localize in dendrites of the rat prefrontal cortex, Neuroscience 258 (2014) 90-100.
[17]
D.A. Mitrano, J.P. Schroeder, Y. Smith, J.J. Cortright, N. Bubula, P. Vezina, D. Weinshenker, alpha-1 Adrenergic receptors are localized on presynaptic elements in the nucleus accumbens and regulate mesolimbic dopamine transmission, Neuropsychopharmacology 37 (2012) 2161-2172.
[18]
P. Moleman, C.F. van Valkenburg, J.A. vd Krogt, Effects of morphine on dopamine metabolism in rat striatum and limbic structures in relation to the activity of dopaminergic neurones, Naunyn Schmiedebergs Arch Pharmacol 327 (1984) 208-213.
[19]
D.L. Muller, E.M. Unterwald, In vivo regulation of extracellular signal-regulated protein kinase (ERK) and protein kinase B (Akt) phosphorylation by acute and chronic morphine, J Pharmacol Exp Ther 310 (2004) 774-782. 19
[20]
Paxinos. G, Watson. C, The Rat Brain in Stereotaxic Coordinates, 2th Edition, (1986).
[21]
D.R. Ramirez, L.M. Savage, Differential involvement of the basolateral amygdala, orbitofrontal cortex, and nucleus accumbens core in the acquisition and use of reward expectancies, Behav Neurosci 121 (2007) 896-906.
[22]
J.S. Tang, C.L. Qu, F.Q. Huo, The thalamic nucleus submedius and ventrolateral orbital cortex are involved in nociceptive modulation: a novel pain modulation pathway, Prog Neurobiol 89 (2009) 383-389.
[23]
E. Valjent, J.C. Corvol, J.M. Trzaskos, J.A. Girault, D. Herve, Role of the ERK pathway in psychostimulant-induced locomotor sensitization, BMC Neurosci 7 (2006) 20.
[24]
L.J. Vanderschuren, T.J. De Vries, G. Wardeh, F.A. Hogenboom, A.N. Schoffelmeer, A single exposure to morphine induces long-lasting behavioural and neurochemical sensitization in rats, Eur J Neurosci 14 (2001) 1533-1538.
[25]
L.J. Vanderschuren, P.W. Kalivas, Alterations in dopaminergic and glutamatergic transmission in the induction and expression of behavioral sensitization: a critical review of preclinical studies, Psychopharmacology (Berl) 151 (2000) 99-120.
[26]
R. Ventura, A. Alcaro, S. Puglisi-Allegra, Prefrontal cortical norepinephrine release is critical for morphine-induced reward, reinstatement and dopamine release in the nucleus accumbens, Cereb Cortex 15 (2005) 1877-1886.
20
[27]
T.L. Verplaetse, D.D. Rasmussen, J.C. Froehlich, C.L. Czachowski, Effects of prazosin, an alpha1-adrenergic receptor antagonist, on the seeking and intake of alcohol and sucrose in alcohol-preferring (P) rats, Alcohol Clin Exp Res 36 (2012) 881-886.
[28]
C. von der Goltz, F. Kiefer, Learning and memory in the aetiopathogenesis of addiction: future implications for therapy?, Eur Arch Psychiatry Clin Neurosci 259 Suppl 2 (2009) S183-187.
[29]
D. Willner, A. Cohen-Yeshurun, A. Avidan, V. Ozersky, E. Shohami, R.R. Leker, Short term morphine exposure in vitro alters proliferation and differentiation of neural progenitor cells and promotes apoptosis via mu receptors, PLoS One 9 (2014) e103043.
[30]
R.A. Wise, M.A. Bozarth, A psychomotor stimulant theory of addiction, Psychol Rev 94 (1987) 469-492.
[31]
Y. Zhao, B. Xing, Y.H. Dang, C.L. Qu, F. Zhu, C.X. Yan, Microinjection of valproic acid into the ventrolateral orbital cortex enhances stress-related memory formation, PLoS One 8 (2013) e52698.
[32]
J.X. Zhu, F.Y. Xu, W.J. Xu, Y. Zhao, C.L. Qu, J.S. Tang, D.M. Barry, J.Q. Du, F.Q. Huo, The role of alpha(2) adrenoceptor in mediating noradrenaline action in the ventrolateral orbital cortex on allodynia following spared nerve injury, Exp Neurol 248 (2013) 381-386.
21
Figure Captions Fig. 1. The experimental schedule of morphine-induced behavioral sensitization (A) and photomicrograph example of injection sites in the bilateral VLO (B). Abbreviations: Beno, benoxathian; fmi, forceps minor corpos callosum; i.p., intraperitoneally; m.i., microinjection; min, minute. Mor, morphine; Sal, saline. Scale bar = 500 μm.
22
Fig. 2. Effect of intra-VLO injection of benoxathian on morphine-induced behavioral sensitization. (A) Effect of intra-VLO injection of benoxathian alone on the total distance of 240 min with traveled distance within every 10 min (line chart)) and the total distance (bar graph) (n = 7). *P < 0.05, vs. Sal-Sal group. (B) Effect of intra-VLO injection of benoxathian on the development of morphine-induced behavioral sensitization in 240 min every 10 min (line chart)) and the total distance (bar graph) (n = 7-8). *P < 0.05, **P < 0.01, ***P < 0.001, vs. Sal-Sal group; ^P < 0.05; ^^P < 0.01; ^^^P < 0.001, vs. Beno-Sal group. (C) Effect of intra-VLO injection of benoxathian on the expression of morphine-induced behavioral sensitization in 240 min every 10 min (line chart) and the total distance (bar graph) (n = 6-8). *P < 0.05, **P < 0.01, ***P < 0.001, vs. Sal-Sal group; #P < 0.05; ##P < 0.01; ###P < 0.001, vs. Sal-Mor group. Abbreviations: α1-AR, α1 adrenoceptor; Beno, benoxathian; Mor, morphine; Sal, saline.
23
24
Fig. 3. Effect of intra-VLO injection of benoxathian on the protein levels of α1 adrenoceptor (A) and p-ERK/ERK (B) in the VLO (n = 8). *P < 0.05, ***P < 0.001 vs. Sal-Sal group,
##
P < 0.01,
###
P < 0.001, vs. Saline-Mor group,
Beno-Sal group.
25
^^^
P < 0.001, vs.