Physiology & Behavior 102 (2011) 42–50 Contents lists available at ScienceDirect Physiology & Behavior journal homepage: www.elsevier.com/locate/phb Orexins in the midline thalamus are involved in the expression of conditioned place aversion to morphine withdrawal Yonghui Li a, Huiying Wang b, Keke Qi b, Xiaoyu Chen c,SaLic, Nan Sui b, Gilbert J. Kirouac c,d,⁎ a Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences, Beijing, China and Faculty of Dentistry, University of Manitoba, Winnipeg, Manitoba, Canada b Behavioral Pharmacology Laboratory, Institute of Psychology, Chinese Academy of Sciences, Beijing, China c Department of Oral Biology, Faculty of Dentistry, University of Manitoba, Winnipeg, Manitoba, Canada d Department of Psychiatry, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada article info abstract Article history: Previous studies have implicated the bed nucleus of the stria terminalis, central nucleus of the amygdala and Received 19 July 2010 the shell of the nucleus accumbens (collectively called the extended amygdala) as playing an important role in Received in revised form 1 October 2010 mediating the aversive emotion associated with opioid withdrawal. The paraventricular nucleus of the Accepted 8 October 2010 thalamus (PVT) provides a very dense input to the extended amygdala, and the PVT is densely innervated by orexin neurons, which appear to be involved in producing some of the physical and emotional effects Keywords: associated with morphine withdrawal. In the present study, we confirm that the PVT is densely innervated by Orexin orexin fibers, whereas the regions of the extended amygdala associated with the effects of morphine Hypocretin Morphine withdrawal withdrawal are poorly innervated. Microinjections of the orexin-1 receptor (OX1R) antagonist SB334867 or Thalamus the orexin-2 receptor (OX2R) antagonist TCSOX229 at doses of 5.0 or 15.0 μg into the PVT region did not affect Extended amygdala the acquisition of the conditioned place aversion (CPA) nor the physical effects produced by naloxone- Conditioned place aversion precipitated morphine withdrawal. In contrast, microinjections of TCSOX229 (15.0 μg) in the PVT region significantly attenuated the expression of naloxone-induced CPA while microinjections of SB334867 at the same dose had no effect. The results from these experiments indicate a role for OX2R in the PVT on the expression of CPA associated with morphine withdrawal. Orexins may mediate the aversive effects of morphine withdrawal by engaging the extended amygdala indirectly through the action of orexins on the PVT. © 2010 Elsevier Inc. All rights reserved. 1. Introduction Neuropeptides called orexins have received attention for their importance in regulating behaviors related to drug reward [3,4]. Several Opioid drugs like heroin and morphine produce euphoric effects studies have also shown that orexins may be involved in the withdrawal that can lead to chronic drug use and dependence. In contrast, effects of morphine. First, morphine withdrawal was found to increase abstinence from opiates in dependent individuals produces negative the expression of c-Fos gene or protein in orexin neurons as well as to physiological (nausea, diarrhea, vomiting, tachycardia and sweating) increase orexin mRNA level in the hypothalamus [5–7].Second,orexin and emotional (irritability, anxiety, and dysphoria) states that are knock-out mice display fewer jumps, tremor and diarrhea in naloxone- extremely unpleasant to the drug user [1]. While the physiological precipitated morphine withdrawal [7]. Third, systemic administration of effects produced by opiate withdrawal dissipate over several days, the the orexin-1 receptor (OX1R) antagonist SB334867 attenuated beha- unpleasant emotional effects produced by opioids remain for weeks viors associated with morphine withdrawal in mice, an effect that or months and represent a major factor contributing to relapse [1,2]. appeared to be mediated by a decrease in the activity of neurons in the Consequently, there has been significant interest in identifying the shell of the nucleus accumbens [6]. The same paper reported that only a brain mechanisms that contribute to the negative emotional states few orexin neurons in the hypothalamus innervate the nucleus produced by abstinence of opioid and other addictive drugs with the accumbens, suggesting that orexins influence neurotransmission in hope that novel pharmacological targets can be identified to prevent this area of the striatum via an indirect projection [6]. relapse. The paraventricular nucleus of the midline thalamus (PVT) is densely innervated by orexin fibers [8,9] and is a major source of glutamatergic afferents to the shell of the nucleus accumbens, dorsolateral bed nucleus of the stria terminalis and the central nucleus of the amygdala [10–13]. ⁎ Corresponding author. Tel.: +1 204 977 5696; fax: +1 204 789 3913. These areas of the basal forebrain are collectively called the extended E-mail address: [email protected] (G.J. Kirouac). amygdala [14–17] and represent an anatomical macrostructure important 0031-9384/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2010.10.006 Y. Li et al. / Physiology & Behavior 102 (2011) 42–50 43 for mediating both the physical and emotional effects of morphine containing 5% normal donkey serum, 0.3% Triton X-100 and 0.1% sodium withdrawal [6,18–21]. In fact, the entire extended amygdala is a large azide followed by incubation in rabbit antiserum against OXA (1:3000, and complex area of the basal forebrain involved in the integration of diluted in pre-incubation solution; Chemicon, Temacula, CA; catalogue the motivational and physiological responses associated with emotions #AB3704) or goat antiserum against OXB overnight (1:500, diluted in [22–24]. Subregions of the extended amygdala including the shell of the pre-incubation solution; Santa Cruz Biotech Inc., Santa Cruz, CA; nucleus accumbens, lateral bed nucleus of the stria terminalis and the catalogue No. sc 8071). The sections were then rinsed for 3×10 min central nucleus of the amygdala have been identified as being especially in PBS, followed by placing the sections in either biotinylated donkey important for mediating the different effects of morphine withdrawal anti-rabbit or donkey anti-goat (1:500; Jackson Immunoresearch, West [6,18,19,21,25,26]. However, it is questionable if the release of orexins in Grove, PA) antisera for 2 h. Then sections were rinsed thoroughly and the extended amygdala mediates the effect of morphine withdrawal incubated with an avidin-biotin complex (ABC) solution (Elite ABC kit; because most of the critical areas of the extended amygdala activated by Vector Laboratories, Burlingame, CA). After three more rinsing steps, morphine withdrawal appear to be poorly innervated by orexin fibers sections were reacted with 3,3 diaminobenzidine tetrachloride (DAB) [6,27]. As such, the PVT, with its dense projections to those areas of the with nickel intensification (Vector DAB Kit, Vector Laboratories extended amygdala activated by morphine withdrawal, represents a Burlingame, CA) to produce a black reaction product. The DAB reaction potential site where orexins could act to produce the physiological and was terminated by rinsing the sections with PBS and the sections were emotional effects associated with morphine withdrawal. mounted on slides, air dried and cover-slipped with Fluka DPX The present study was done to determine if blockade of orexin mountant. Sections of the forebrain were examined and photographed receptors in the PVT with antagonists for the OX1R or the orexin-2 under magnification using an Olympus BX51 microscope equipped with receptors (OX2R) attenuated the symptoms associated with morphine a digital camera (SPOT RT Slicer, Diagnostic Instruments Inc, Sterling withdrawal. This was done by examining the effects of orexin receptor Heights, MI). Calibration bars were inserted using SPOT software antagonists on the acute physical symptoms and conditioned place (Version 3.2, Diagnostic Instruments) and images were transferred to aversion (CPA) produced by naloxone-induced precipitated morphine Adobe Photoshop 5.5 to optimize light and contrast levels. withdrawal. The CPA paradigm involves Pavlovian conditioning where the negative emotional state an animal experiences during morphine 2.3. Behavioral experiments withdrawal is paired with the particular environment where the emotional state occurs [28]. Re-exposure of the animal to the paired 2.3.1. Surgery environment leads to the experience of a negative emotional state and Rats were anesthetized with equithesin (0.3 ml/100 g, i.p.) and the avoidance of that environment [28]. In this case, CPA can be used to placed in a Stoelting stereotaxic frame. Stainless steel guide cannula investigate the neural mechanisms involved in the expression of (23 gauge, Plastics One, Roanoke, VA, USA) were unilaterally negative emotions link to opioid withdrawal. We also compared the implanted into the posterior aspect of the PVT (3.1 mm posterior to density of orexin-A (OXA) and orexin-B (OXB) fibers in the PVT and the bregma, 1.3 mm lateral to the midline, and 4.0 mm ventral to the extended amgydala to re-examine the extent of orexin innervation of skull, at 10˚ angle, with the incisor bar set at 3.3 mm below intraaural key areas of the forebrain linked to the negative emotional and aversive line). The guide cannula was secured with three small screws attached state associated