Neurochemistry International 57 (2010) 749–755 Contents lists available at ScienceDirect Neurochemistry International journal homepage: www.elsevier.com/locate/neuint Activation of serotonin 1A receptors in ventrolateral orbital cortex depresses persistent nociception: A presynaptic inhibition mechanism Fu-Quan Huo a, Fen-Sheng Huang a, Bo-Chang Lv b, Tao Chen b, Jie Feng a, Chao-Ling Qu a, Jing-Shi Tang a,*, Yun-Qing Li b,** a Department of Physiology and Pathophysiology, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an Jiaotong University School of Medicine, Xi’an 710061, China b Department of Anatomy, Histology and Embryology and K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi’an 710032, China ARTICLE INFO ABSTRACT Article history: The present study examined the effect of serotonin 1A (5-HT1A) receptor activation in the ventrolateral Received 26 May 2010 orbital cortex (VLO) upon formalin-evoked flinching behavior and spinal Fos expression, and further Received in revised form 24 July 2010 determined whether activation of 5-HT1A receptors affected the spontaneous GABAergic miniature Accepted 11 August 2010 inhibitory postsynaptic currents (mIPSCs) in rat VLO slice by pharmacologically separated neurons to Available online 8 September 2010 understand the possible mechanism underlying this effect. Microinjection of the 5-HT1A receptors agonist 8-OH-DPAT (8-hydro-2-(di-n-propylamino) tetralin) into the VLO depressed the formalin- Keywords: evoked nociceptive behavior flinching response and the Fos expression in the lumbar spinal cord dorsal, 5-HT1A receptor which was antagonized by pre-treatment with 5-HT receptors antagonist NAN-190 (1-(2- Fos 1A mIPSCs methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide). Furthermore, application of Ventrolateral orbital cortex 8-OH-DPAT into VLO slice inhibited GABAergic mIPSC frequency in a dose-dependent manner without Antinociception effects on amplitude of the GABAergic mIPSCs, this effect was blocked by NAN-190. These results provide Formalin test evidence for the involvement of 5-HT1A receptors in VLO in the modulation of persistent inflammatory nociception, and suggest that a presynaptic inhibition of the GABA release may contribute to the 5-HT1A receptor-mediated descending antinociception. ß 2010 Elsevier Ltd. All rights reserved. 1. Introduction Zhang et al., 1995, 1998b, 1999). However, electrically or chemically induced activation of the VLO depresses tail flick and jaw-opening Previous studies in our laboratory have demonstrated that reflexes. These antinociceptive effects are eliminated by lesion or electrolytic lesions or microinjection of g-aminobutyric acid (GABA) functional blocking of the periaqueductal gray (PAG) (Y.Q. Zhang into the ventrolateral orbital cortex (VLO) eliminates antinocicep- et al., 1997; S. Zhang et al., 1997; Zhang et al., 1998a). These data tive effects induced by peripheral electrical stimulation, or by suggest that the VLO is involved in an endogenous analgesic system activation of the thalamic nucleus submedius (Sm) (Lu et al., 1996; consisting of a spinal/medulla cord-Sm-VLO-PAG-spinal/medulla cord loop (Tang et al., 2009). Morphological studies have indicated that the VLO receives the ascending projections of serotonin (5-hydroxytryptamine, 5- Abbreviations: 5-HT, 5-hydroxytryptamine, serotonin; 8-OH-DPAT, 8-hydro-2-(di- HT)-ergic terminals from the dorsal raphe nucleus (Huo et al., n-propylamino) tetralin; ABC, avidin–biotin-peroxidase; ACSF, artificial cerebro- 2009; Li et al., 1993; Matsuzaki et al., 1993), and 5-HT receptors spinal fluid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DAB, 3,3-diaminoben- are distributed in the VLO (Barnes and Sharp, 1999; Hossein et zidine tetrahydrochloride; DL-AP5, DL-2-amino-5-phosphovaleric acid; F 13640, (3-chloro-4-fluoro-phenyl)-[4-fluoro-4-[[(5-methyl-pyridin-2-ylmethyl)-amino]- al., 1996). Behavioral studies also demonstrated that the 5-HT1A methyl]piperidin-1-yl]-methadone; GABA, g-aminobutyric acid; mIPSCs, minia- receptors are implicated in mediating the VLO-evoked descend- ture inhibitory postsynaptic currents; NAN-190, 1-(2-methoxyphenyl)-4-[4-(2- ing antinociception in the physiological pain model (tail flick phthalimido)butyl]piperazine hydrobromide; NGS, normal goat serum; PAG, test) (Qu et al., 2008; Huo et al., 2008). However, it is unknown, periaqueductal gray; PB, phosphate buffer; PBS, phosphate-buffered saline; Sm, thalamic nucleus submedius; TTX, tetrodotoxin; VLO, ventrolateral orbital cortex; under persistent inflammatory pain state, whether the 5-HT1A WAY-100635, {N-2-[4-(2-methoxyphenyl-1-piperazinyl]ethyl]-N-2-pyridinylcy- receptors also are involved. The formalin injected into rat clohexane carboxamide}. hindpaw evoked nociceptive behavior and spinal Fos expression * Corresponding author. Tel.: +86 29 82655172; fax: +86 29 82656364. has been widely used in studying the persistent inflammatory ** Corresponding author. Tel.: +86 29 84774501; fax: +86 29 83283229. painprocessesanditsmodulation(Abbadie et al., 1997; E-mail addresses: [email protected] (J.-S. Tang), [email protected] (Y.-Q. Li). Buritova et al., 2005; Chapman and Besson, 1997; Harris, 0197-0186/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2010.08.011 750 F.-Q. Huo et al. / Neurochemistry International 57 (2010) 749–755 1998; Jinks et al., 2002; Okuda et al., 2001; Watanabe et al., were transcardially perfused with 100 ml of 0.01 M phosphate-buffered saline 2003). The aim of the present study was to examine whether (PBS, pH 7.4), followed by 500 ml of 4% (w/v) paraformaldehyde and 15% (v/v) saturated picric acid in 0.1 M phosphate buffer (PB, pH 7.4). The brain and spinal microinjection of 5-HT1A receptor agonist into the VLO could cord were immediately removed and placed into the fresh fixative. Subsequently, depress the formalin-evoked nociceptive behavior (paw flinch- the brain and lumbar spinal cord were placed in 30% sucrose solution/0.1 M PB (pH ing response) and the Fos expression in the spinal dorsal horn. 7.4) as a cryprotectant overnight at 4 8C. The brain was serially cut into 50-mm thick coronal sections on a freezing microtome. The sections were mounted and Since 5-HT1A receptors are coupled to inhibitory G-proteins and stained with Cresyl Violet. The injection sites were plotted on photographs of the their activation results in hyperpolarization of cell membrane coronal sections modified from the Paxinos and Watson’s atlas (1986).The and inhibits neuronal activity (Aghajanian, 1995; Albert et al., locations of drug injection sites were verified histologically to be within the VLO. 1997), it has been suggested that the excitatory effect of 5-HT1A An example is shown in Fig. 1. receptors on neurons may be produced by inhibiting an The lumbar spinal cords were cut in 30-mm serial sections on a freezing inhibitory action of the GABAergic interneuron on the projection microtome (Kryostat 1720, Leitz, Mannheim, Germany). The L4-5 spinal cord was isolated. Lumbar spinal cord sections were serially collected into three separate neurons (Koyama et al., 1999, 2002; Susana et al., 2003). To dishes containing 0.01 M PBS (pH 7.4), according to numerical order. All sections provide evidence for this suggestion, this study also examined were carefully washed with 0.01 M PBS and immunohistochemically processed as the influence of 5-HT1A receptor agonist application into the VLO free-floating sections. slice on spontaneous GABAergic miniature inhibitory postsyn- The sections in the first dish were used for immunohistochemistry staining of Fos using the avidin–biotin-peroxidase (ABC) method. Briefly, the sections were aptic currents (mIPSCs) by using patch clamp technique. washed in 0.01 M PBS (pH 7.4) and incubated sequentially with: (1) rabbit antiserum against Fos polyclonal antibody (ab7963, 1:500 dilution; Abcam, 2. Materials and methods Cambridge, MA) in 0.01 M PBS containing 5% normal goat serum (NGS), 0.3% Triton X-100, 0.05% NaN3, and 0.25% carrageenan (PBS-NGS, pH 7.4) for 48–72 h at 4 8C; 2.1. Part I: animal experiment (2) biotinylated goat anti-rabbit IgG (1:200 dilution; Vector, Burlingame, CA) in 2.1.1. Animal preparation PBS-NGS overnight at 4 8C; and (3) ABC Elite complex (Vector: 1:100) in 0.01 M Male Sprague–Dawley rats (220–250 g) were provided by the Experimental PBS (pH 7.4) containing 0.3% Triton X-100 (PBS-X) for 2 h at room temperature. Animal Center of the Fourth Military Medical University (Xi’an, China). The Bound peroxidase was visualized by incubation with 0.05% 3, 3-diaminobenzidine experimental protocol was approved by the Institutional Animal Care Committee tetrahydrochloride (DAB; Dojin, Kumamoto, Japan) and 0.003% H2O2 in 0.05 M of the University. According to the ethical guidelines of the International Association Tris–HCl buffer (pH 7.6) for 20–30 min. The sections were rinsed at least three for the Study of Pain (Zimmermann, 1983), all efforts were made to minimize the times in 0.01 M PBS (pH 7.4), for at least 10 min, after all incubations. The sections number of animals used, as well as to minimize distress to the animals. The animals were then mounted onto gelatin-coated glass slides, air-dried, dehydrated and were anesthetized with sodium pentobarbital (50 mg kgÀ1, intraperitoneally) and the cleaned, coverslipped with DPX, and observed under light microscope. Micro- head was immobilized in a stereotaxic frame. A small craniotomy was performed just photographs were taken with a digital camera (DP-70; Olympus) attached to a above the VLO. A stainless steel guide cannula (0.8 mm in diameter) was microscope. stereotaxically inserted, with its tip 2.0 mm dorsal to the VLO, at the following Sections in the second dish were mounted onto gelatin-coated glass slides and coordinates: 3.2–3.7 mm anterior to bregma, 2.0–2.5 mm lateral, 4.0–5.0 mm below processed for Nissl staining.