Distinct effect of orphanin FQ in nucleus raphe magnus and nucleus reticularis gigantocellularis on the rat tail flick reflex

Neuroscience Letters 306 (2001) 69±72

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Distinct effect of orphanin FQ in nucleus raphe magnus and nucleus reticularis gigantocellularis on the rat tail ¯ick re¯ex Zhi-Lan Yang, Yu-Qiu Zhang, Gen-Cheng Wu* State Key Laboratory of Medical Neurobiology, Department of Neurobiology, Medical Center of Fudan University (The former Shanghai Medical University), 138 Yi Xue Yuan Road, Shanghai, 200032, China Received 26 January 2001; received in revised form 25 April 2001; accepted 25 April 2001

Abstract The aim of the present study is to investigate the effects of orphanin FQ (OFQ) microinjected into the nucleus raphe magnus (NRM) and the nucleus reticularis gigantocellularis (NGC) on pain modulation. The tail-¯ick latency (TFL) was used as a behavioral index of nociceptive responsiveness. The result showed microinjection of OFQ into the NRM signi®cantly increased the TFL, whereas microinjection of OFQ into the NGC decreased the TFL, suggesting the analgesic effect of OFQ in the NRM and the hyperalgesic effect of OFQ in the NGC. As there are three classes of putative pain modulating neurons in the rostral ventromedial medulla (RVM), the hyperalgesic or analgesic effect of OFQ in the RVM might depend upon the different class of the neurons being acted. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: Orphanin FQ; Nucleus raphe magnus; Nucleus reticularis gigantocellularis; Analgesia; Hyperalgesia

The newly discovered neuropeptide orphanin FQ/nociceptin (OFQ/NOC) has been identi®ed as an endogenous ligand of the opioid receptor-like (ORL1) receptor [6,10]. Increasing evidence indicates that OFQ plays an important role in pain modulation, although its effects on the nociceptive response remain controversial [7,11]. Morphological studies have established that OFQ-containing neurons and ®bers as well as ORL-1 receptor are widely distributed along all parts of pain circuitry, including the ascending and descending pain pathways, such as the periaqueductal gray (PAG), the rostral ventromedial medulla (RVM), locus coeruleus and the dorsal horn of the spinal cord [3,8]. RVM includes the NRM, the adjacent ventral nucleus reticularis gigantocellularis(NGC) and gigantocellularis pars alpha (NGCa). It plays a crucial role in descending pain modulation [17]. Heinricher et al. reported that local infusion of OFQ within the RVM suppressed the ®ring of all three classes of the RVM neurons, Off-cell, On-cell and Neutral-cell [4]. It would seem necessary to characterize the action of OFQ in different nucleus which involve in descending pain modulation. Our previous work have demonstrated that microinjection of OFQ into the PAG facilitated the * Corresponding author. Tel.: 186-21-64041900 ext. 2397; fax: 186-21-64174579. E-mail address: [email protected] (G.-C. Wu).

tail-¯ick (TF) re¯ex and nociceptive responses of spinal dorsal horn wide dynamic range neurons evoked by noxious subcutaneous electrical stimulation in rats [14,16]. The present study was designed to investigate the effects of OFQ microinjected into the NRM and the NGC on pain modulation in rats. Male Sprague±Dawley rats (220±240 g) were supplied by the Experimental Animal Center, Medical Center of Fudan University. The treatment of the rats conformed to the guidelines of the International Association for the Study of Pain [18]. Heptadecapeptide OFQ was supplied by the Shanghai Institute of Biochemistry, Chinese Academy of Science. It was synthesized by an ABI 430A Peptide Synthesizer, manually using Rink Amide MBHA resin and Fmoc strategy, and was sequentially puri®ed on columns of Sephadex G-10 and HPLC. Its amino acid composition was consistent with documents/the theoretical value. The drugs were dissolved in sterilized normal saline (NS). Since OFQ is rich in alkaline amino acid, the solution as well as NS was added with arrowhead double-headed proteinase inhibitor (1 g/l, the product of Shanghai Institute of Biochemistry), which was reported to be able to inhibit trypsin, chymotrypsin and kallikrein for preventing from proteolysis after injection [15]. Implantation of the injection cannulae into the NRM or the NGC was performed stereotaxically under sodium pentobarbital anesthesia (30 mg/kg, i.p.). A stainless steel

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Z.-L. Yang et al. / Neuroscience Letters 306 (2001) 69±72

guide cannula of 0.5 mm outer diameter was placed at a position 1 mm dorsal to the NRM or the NGC and ®xed on the skull with dental cement. After surgery, the animals were returned to the cages and housed individually and allowed to recover for 3±4 days before testing. Experiments were performed at room temperature (22 ^ 18C). The tail-¯ick latency (TFL) was used as a behavioral index of nociceptive responsiveness. During the test, rats were lightly restrained in a wood holder. The distal part of the tail was immersed into 508C water and the TFL was measured. Baseline TFL was de®ned as the mean of three determinations performed at 5 min intervals before drug injection. Only those rats with the baseline TFL within the range of 5±8 s were used for further studies. Drug was then injected slowly over 30 s (0.2 ml) through an injection cannula (0.26 mm outer diameter) that protruded approach the NRM (P 11.3 mm, R 0.2 mm, H 8.5 mm) or the NGC (P 11.6 mm, R 0.6 mm, H 7.0 mm) area, and the injection cannula remained in place an additional 1-min to minimize back¯ow of the drug up the cannula track. The testing of TF re¯ex commenced at the termination of injection and was repeated at 10-min intervals throughout the 60-min period observed. A cut-off time (15 s) was used to avoid tail hurt. At the end of each experiment, the microinjection site was marked by injecting pontamine sky blue (0.2 ml) and then the animals were perfused transcardially with saline followed by 10% formalin under deep anesthesia. The brain was removed and ®xed in fresh formalin for 3±7 days. Sections were cut at 100 mm with a freezing microtome and stained with Neutral Red to determine the location of microinjection on the basis of the atlas of Paxinos and Watson [9]. Data were expressed as mean ^ SEM. Statistical signi®cance was determined using analysis of variance (ANOVA) and t-test. A probability level of 0.05 indicated signi®cance. The stability of the hot water-evoked TF re¯ex was ®rst tested, showing that the baseline TFL was stable for a period of 2±3 h examined. Microinjection of NS (0.2 ml) into the unilateral NRM or NGC had no effect on the TF re¯ex. No signi®cant differences were found between and after injection of NS (KW-ANOVA, NRM: H ˆ 12:028, P ˆ 0:212, n ˆ 8; NGC: H ˆ 7:808, P ˆ 0:554, n ˆ 6) (Fig. 1A,B). The effects of OFQ injected into the unilateral NRM in four different doses (0.02, 0.2, 2.0 and 5.0 mg, dissolved in 0.2 ml NS) on TF re¯ex were examined. As shown in Fig. 1A, 0.02 mg OFQ had no obvious change of TFL (KWANOVA, H ˆ 16:435, P ˆ 0:058, n ˆ 7). Doses of 0.2, 2.0 and 5.0 mg OFQ signi®cantly increased the TFL (P , 0:05 to P , 0:001, either compared with the baseline value or those obtained from NS control, KW-ANOVA, n ˆ 7±10). The effect was dose-dependent, and the peak increased TFL occurred at 20±40 min after administration (Fig. 1A). Note that OFQ applied to the NGC and the structure in the ventral to the NRM (Fig. 2A) had no effect on TF re¯ex in four tests. Similar to the NRM, a low dose (0.02 mg) of OFQ was injected unilateral into NGC without any effect on TF re¯ex

(ANOVA, F ˆ 0:708, P ˆ 0:699, n ˆ 7). Doses of 0.2, 2.0 and 5.0 mg OFQ dose-dependently decreased the TFL. As shown in Fig. 1B, the decreases of the TFL were statistically signi®cant as compared with either the baseline value or those obtained from the NS control experiments (P , 0:05 to P , 0:001, ANOVA, n ˆ 7). The maximal facilitation was usually observed within 30-min after OFQ injection, and thereafter, the TFL gradually returned to the baseline level. In addition, two injection sites were located in the structure of the medial to the NGC (Fig. 2B), and they were ineffective to the TF re¯ex. The RVM, including the NRM and the NGC as well as the NGCa, projects directly to the trigeminal nucleus caudalis and spinal cord dorsal horn [17]. Through these descending projections, the RVM plays a crucial role in pain modulation. The high level expression of OFQ and ORL1

Fig. 1. (A) Effect of microinjection of OFQ into the NRM on TFL in rats. NS and 0.02 mg OFQ had no effect on TFL (P . 0:05). 0.2 mg, 2.0 mg and 5.0 mg OFQ increased the TFL (*P , 0:05, **P , 0:01, ***P , 0:001 vs. NS). Data were expressed as the mean ^ SEM. (B) Effect of microinjection of orphanin FQ (OFQ) into the NGC on TFL in rats. NS and 0.02 mg OFQ had no effect on TFL (P . 0:05). 0.2 mg, 2.0 mg and 5.0 mg OFQ shortened the TFL (*P , 0:05, **P , 0:01 vs. NS). Data were expressed as the mean ^ SEM. NS: normal saline.

Z.-L. Yang et al. / Neuroscience Letters 306 (2001) 69±72

Fig. 2. Histological reconstructions of OFQ microinjected into right lateral NRM (A) and NGC (B) sites. Abbreviations: NRM, nucleus raphe magnus; NGC, nucleus reticularis gigantocellularis; NGCa, gigantocellularis pars alpha; sp5, spinal trigeminal tr; VII, facial nucleus; py, pyramidal tr. X Effective ,W Ineffective.

receptor in NRM and NGC suggested its involvement in nociceptive processing [3,8]. Neurons in the RVM are heterogeneous in term of their pharmacology and physiology. Three classes of putative pain modulating neurons were identi®ed in this regions [2]. On-cells are de®ned by a sudden increase in ®ring just before the occurrence of nociceptive re¯exes. Off-cells characteristically display an abrupt pause in ®ring just before the occurrence of nociceptive re¯exes. The third class of cells, Neutral-cells, show no response to noxious stimulation. Analgesia and hyperalgesia may be involved in the different pain-modulating neurons. It has been demonstrated that supraspinal actions of opioids could activate a nociceptive modulating out¯ow that exerts an antinociceptive effect [5]. In the RVM, opioids directly inhibits On-cells and indirectly activated Off-cells. It is likely that opioids inhibited a subset of GABAergic On-cells leading to disinhibition of Off-cells that contact with On-cells [12]. OFQ has been identi®ed as a endogenous ligand of ORL1 receptor. In spite of its structural homology with the endogenous opioid peptides, OFQ does not show appreciable binding to either m, d or k receptors [6,10]. Concerning its pain-modulating effect, some studies have demonstrated

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that supraspinal OFQ induced hyperalgesia and antagonism to opioid antinociception [7]. Thus far, all studies about the effect mechanism of the ORL-1 receptor would support the concept that the receptor is inhibitory, since ORL-1 receptor activation was reported to inhibit adenylate cyclase [6,10], to increase an inwardly rectifying potassium conductance and inhibited calcium channel currents [1,13]. Heinricher and his colleagues demonstrated that local infusion of OFQ within the RVM inhibits all three classes of the RVM neuron [4]. Thus, OFQ would be expected to reduce antinociceptive actions generated by increased the RVM Off-cell activity to produce hyperalgesia, whereas reduce nociceptive action generated by increased RVM On-cell activity to produce analgesia. The effects of OFQ in the RVM might depend upon the class of the neurons being acted. The present study showed that microinjection OFQ into the NRM signi®cantly increased the TFL whereas microinjection OFQ into the NGC signi®cantly decreased the TFL, suggesting the analgesia effect of OFQ in the NRM and the hyperalgesia effect of OFQ in the NGC. The possible explanation is that OFQ in the NRM may directly inhibit On-cells containing ORL-1 receptor or act on GABAergic neurons containing ORL-1 receptor in the NRM to inhibit GABA release, thus disinhibiting Off-cells to produced analgesia; Whereas OFQ in the NGC may directly inhibit Off-cells containing ORL-1 receptor or indirectly inhibit opioids release via acting on opioidergic neurons containing ORL-1 receptor in the NGC, thus disinhibiting On-cells and inhibiting Off-cells to produced hyperalgesia. Recent studies in our laboratory revealed that there were a few co-existence neurons of b-endophin, leu-enkephalin and dynorphin with ORL1 mRNA in the RVM and OFQ inhibited opioids release (unpublished data). However, complete understanding of the role of OFQ will require development of an antagonist that will allow acute block of the actions of the peptide in speci®c brain regions, with parallel analysis of behavioral effects and underlying neuronal circuits. Clearly, further experiments will be required to address this question. This study was supported by grants from the National Natural Science Foundation of China (39970925 and 30070948), and the Research Fund for the Doctoral Program of Higher Education (9835). [1] Connor, M., Yeo, A. and Henderson, G., The effect of nociceptin on Ca2 1 channel current and intracellular Ca2 1 in the SH-SY5Y human neuroblastoma cell line, Br. J. Pharmacol., 118 (1996) 205±207. [2] Fields, H.L., Heinricher, M.M. and Mason, P., Neurotransmitters in nociceptive modulatory circuits, Annu. Rev. Neurosci., 14 (1991) 219±245. [3] Harrison, L.M. and Grandy, D.K., Opiate modulating properties of nociceptin/orphanin FQ, Peptides, 21 (2000) 151±172. [4] Heinricher, M.M., McGaraughty, S. and Grandy, D.K., Circuitry underlying antiopioid actions of Orphanin FQ in the rostral ventromedial medulla, J. Neurophysiol., 78 (1997) 3351±3358.

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