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Morphine microinjections into the rat nucleus submedius depress nociceptive behavior in the formalin test
Neuroscience Letters 328 (2002) 141–144 www.elsevier.com/locate/neulet
Morphine microinjections into the rat nucleus submedius depress nociceptive behavior in the formalin test Zhi-Jie Yang, Jing-Shi Tang*, Hong Jia Department of Physiology, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, People’s Republic of China Received 7 March 2002; received in revised form 30 April 2002; accepted 6 May 2002
Abstract Our previous studies have indicated that the thalamic nucleus submedius (Sm) is involved in modulation of nociception and plays an important role in an endogenous analgesic system (a feedback loop) consisting of spinal cord–Sm– ventrolateral orbital cortex–periaqueductal gray–spinal cord. To investigate whether opioids are involved in this antinociception pathway, the effects of microinjection of morphine and naloxone into the Sm on the nociceptive behavior (agitation) evoked in the formalin test were investigated in the awake rat using an automated movement detection system. The results indicate that a unilateral microinjection of morphine (5 mg, 0.5 ml) into the Sm suppresses the formalin-induced agitation response, but does not influence spontaneous motor activity, and that the morphine-induced depression can be reversed by microinjection of the opioid receptor antagonist naloxone (1.0 mg, 0.5 ml) into the same Sm site. The results suggest that opioid receptors in the Sm may be involved in the Sm-mediated depression of persistent inflammatory pain. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nucleus submedius; Morphine; Naloxone; Nociceptive modulation; Formalin test; Rat
Our previous studies have indicated [16–19] that activation of the thalamic nucleus submedius (Sm) by electrical or chemical stimulation can depress the rat tail-flick reflex, the jaw-opening reflex and the nociceptive responses of neurons in the spinal dorsal horn and that these antinociceptive effects can be attenuated or eliminated by lesion or inhibition of the ventrolateral orbital cortex (VLO) or the periaqueductal gray (PAG). These findings suggest that the Sm may be involved not only in nociception, as reported previously [2,8,14], but also in modulation of nociception in an endogenous analgesic system (a feedback loop) consisting of a spinal cord–Sm–VLO–PAG–spinal cord loop. There is evidence that a discrete population of the axon terminals in the Sm arising from spinal dorsal horn lamina I neurons are immunoreactive for enkephalin [11], and that mu- and kappa3-opioid receptors are located in this nucleus [1,10]. A recent study in our laboratory has demonstrated that morphine application into the Sm depresses the rat tail-flick reflex [3]. These observations suggest an opioid link in Sm-mediated antinociception. The aim of the present study was to explore whether morphine application into the * Corresponding author. Tel.: 186-29-527-5172; fax: 186-29526-7364. E-mail address: [email protected] (J.-S. Tang).
Sm could depress the nociceptive behavioral responses (agitation) elicited in the formalin test, a test widely used in studies on persistent inflammatory-mediated pain, and to determine whether this effect could be antagonized by administration of the opioid receptor antagonist naloxone into the Sm. The experiments were performed on 42 adult Sprague– Dawley rats weighing 220–350 g of either sex provided by the Medical Experimental Animal Center of Shaanxi Province. Under sodium pentobarbital (50 mg/kg, intraperitoneally) anesthesia, the head was positioned in a stereotaxic frame with the incisor bar 3 mm below the horizontal zero. A small craniotomy was performed, and a guide cannula was stereotaxically placed at a position 2 mm dorsal to the Sm and fixed on the skull with three microscrews and dental cement. The experiments were performed according to the guidelines of the International Association for the Study of Pain. To determine the effect of microinjection of morphine into the Sm on the agitation responses elicited in the formalin test 7 days after animal surgery was performed, a needle attached to a 1 ml Hamilton syringe was inserted via the guide cannula into the Sm (2.3–2.8 mm posterior to Bregma, 0.6–0.9 mm lateral, 6.0–7.0 mm from the cortical surface) [12], and morphine hydrochloride (5 mg, 0.5 ml, dissolved in
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 51 4- 1
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Z.-J. Yang et al. / Neuroscience Letters 328 (2002) 141–144
0.9% saline; Shenyang) was slowly injected into the Sm over 2 min. Five minutes after injection of morphine, formalin (5%, 50 ml) was injected subcutaneously into the rat’s hindpaw pad either contralateral or ipsilateral to the intracerebral injection site. The animal was placed immediately in a polycarbonate box (H, 16 cm; L, 16 cm; W, 10 cm) placed on a spring balance (2.0 kg). Nociceptive responses elicited by formalin injection such as licking, flinching, shaking, rearing, clutching and favoring the affected paw, induced movements of the balance that were transformed into electrical signals via an electromagnetic transducer. The electrical signals were amplified, filtered and displayed on an oscilloscope and fed into a window discriminator and a computer system that allowed quantitative recording of the number of agitation events and construction of time histograms over a 65 min observation period. In control experiments, 0.5 ml of 0.9% saline was injected into the Sm. In addition, the effects of morphine microinjection on spontaneous motor activity (no formalin injection) were also observed to determine the possible effect of morphine on movements. To determine if microinjection of naloxone into the Sm could reverse the inhibition produced by morphine, naloxone hydrochloride (1.0 mg, 0.5 ml, dissolved in 0.9% saline; Sigma) was slowly injected into the same Sm site immediately after morphine injection into the Sm and the changes of the agitation responses elicited by formalin were reexamined. A period of at least 7 days elapsed before testing the same animal again. At the end of the experiment, the drug injection site was marked by injection of Pontamine Sky Blue dye (0.5 ml, 2% in 0.5 M sodium acetate acid). Under deep anesthesia, the animal was perfused transcardially with 0.9% saline followed by 10% formalin. Transverse brain sections were processed with conventional histological methods. The locations of injection sites in the Sm were reconstructed. Data were analyzed for statistical significance (P , 0:05) by two-way analysis of variance (ANOVA) with a pairwise multiple comparison assessed with Tukey’s procedure. Subcutaneous injection of formalin into the rat hindpaw elicited a typical two phase nociceptive behavioral response (agitation) clearly quantified by the automated movement detection system, consisting of an early response lasting about 5 min followed by a 5–10 min period of decreased activity and then a late response lasting about 50 min as shown in Fig. 1a. During the 0–65 min observation period, the mean rate of agitation responses was 3.0 ^ 0.5 Hz (n ¼ 11). Microinjection of saline (0.5 ml) into the Sm did not significantly influence the formalin-induced movements, as shown in Fig. 1b,c (P . 0:05; n ¼ 11). In contrast, a unilateral microinjection of morphine into the Sm markedly attenuated the agitation responses (decrease in mean rate to 1.8 ^ 0.3 Hz; n ¼ 16). The difference was statistically significant as compared with that (4.9 ^ 0.5 Hz; n ¼ 16) obtained by injection of saline into the Sm (P , 0:001), as shown in Fig. 2. The locations of the
morphine injection sites in the Sm region are indicated in Fig. 2e. Most (88%; 14/16) of the injection sites within the Sm were effective in inhibiting the formalin responses, whereas the five sites more than 0.5 mm dorsal to the Sm were ineffective. The three sites adjacent to the dorsal Sm border and one site within the dorsal hypothalamic area were also effective. Microinjection of morphine into the Sm had no significant effect on spontaneous motor activity
Fig. 1. Histograms and graph showing formalin-evoked agitation and lack of effect of saline microinjections into Sm. Parts (a) and (b) show mean activity counts in 2 s bins over a 4000 s (67 min) observation period. A 50 ml subcutaneous injection of 5% formalin into the hindpaw was made at time 0. In (a), the effects of formalin injection (n ¼ 11) are displayed, and in (b), formalin injection in association with 0.9% saline injection into the Sm (n ¼ 11). Part (c) plots the data shown in (a) and (b) in 5 min periods. Two-way ANOVA followed by a multiple comparison test indicated that saline injection into the Sm did not influence the agitation responses elicited by formalin (P . 0:05).
Z.-J. Yang et al. / Neuroscience Letters 328 (2002) 141–144
143
Fig. 2. The time histograms (2 s bins) in the top half of the figure show the inhibitory effect of morphine (5 mg, 0.5 ml) microinjection into the Sm on the formalin-evoked agitation responses (b), but lack of effect with saline (a). This inhibition produced by morphine was reversed by naloxone (1.0 mg, 0.5 ml) injection into the same site (c); (d), plot comparing the effects shown in (a–c) (5 min periods); (e), diagram showing the locations of morphine injection sites in the Sm region and whether they produced an effect. The two-way ANOVAs followed by a multiple comparison test indicated that the differences between the effects of morphine and saline injections, and after morphine and morphine plus naloxone injections into the Sm were statistically significant (P , 0:001), but not significant (P . 0:05) between those after morphine plus naloxone and saline injection. CM, central medial thalamic nucleus; DA, hypothalamic dorsal area; mt, mammillothalamic tract; Mor, morphine; Nal, naloxone; Re, reunions thalamic nucleus; Rh, rhomboid thalamic nucleus; Smd, dorsal nucleus submedius; Smv, ventral nucleus submedius.
(1.41 ^ 0.1 Hz, n ¼ 6 with morphine vs. 1.76 ^ 0.2 Hz, n ¼ 6 with saline, P . 0:05). Microinjection of naloxone into the Sm immediately after morphine was injected into the same site significantly reversed the morphine-evoked inhibition of the agitation responses elicited by formalin (mean number of events, 5.0 ^ 0.9 Hz; n ¼ 8). The difference was significant as compared with that from application of morphine only (P , 0:001), but not significant between the saline and naloxone applications (P . 0:05; also, see Fig. 2). The present study has demonstrated that a unilateral administration of morphine into the Sm in the awake rat depresses the agitation responses elicited by formalin but does not influence spontaneous motor activity, and this antinociception can be reversed by microinjection of the opioid receptor antagonist naloxone into the same site. Since the second phase of nociceptive behavior elicited by formalin injection is considered to be a persistent inflammatory
hyperalgesic response, the results of this study suggest that opioid peptides and their receptors may be involved in the descending modulation of the inflammatory hyperalgesia through activation of the Sm–VLO–PAG pathway. As the direct action of morphine on central neurons is thought to produce hyperpolarization of the cell membrane rather than depolarization [6,15], it has been presumed that morphine-evoked excitatory effects may result from inhibition of inhibitory interneurons [6,15]. There is evidence, at least in the cat [5], that about one-fourth of the neurons in the Sm are gamma-aminobutyric acid (GABA) immunoreactive, and these GABAergic neurons are probably local circuit neurons. Therefore, it is possible that the inhibitory effect elicited by morphine injection into the Sm on the agitation responses elicited by formalin injection might be due to inhibition of GABAergic interneurons leading to increased activity of Sm output neurons projecting to the VLO and in turn to activation of the VLO–PAG–brainstem
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Z.-J. Yang et al. / Neuroscience Letters 328 (2002) 141–144
descending inhibitory system and depression of the nociceptive inputs at the spinal level. The present study used the automated movement detection system developed in our laboratory to quantify the agitation responses elicited by subcutaneous injection of formalin into the rat’s hindpaw. It has been demonstrated [9] that the agitation responses recorded using this system reveal the typical two phases of nociceptive behavior that have been observed and quantified using manual scoring methods [4,13], and that systemic morphine significantly depresses the agitation responses and this effect can be antagonized by systemic naloxone. These recent results suggest that this simple automated system for quantifying the nociceptive behavior in the formalin test is a valid measure and comparable with that reported using a computer-driven, dynamic-force automatic detection system [7]. Histological reconstruction in this study indicated that all but two of the morphine injection sites within the Sm and those that were above but very close to the dorsal border of the Sm were effective in inhibiting the agitation responses elicited by formalin. However, those sites far from the Sm (dorsal to the Sm over 0.5 mm) had no effect despite the fact that opioid receptors (mu, delta and kappa3 receptors) are also located in these thalamic regions [1,10]. These results suggest that the morphine-evoked inhibition of the agitation responses following injections into the Sm and surrounding regions of the thalamus may be specific to an action on the Sm. Since morphine is not a selective opioid receptor agonist, and naloxone is not a selective opioid receptor antagonist, further studies using selective opioid receptor agonists and antagonists are required to determine which opioid receptors are involved in mediating the antinociceptive effects. The authors wish to thank Professor J.O. Dostrovsky for help in revising the manuscript. The project was supported by the National Nature Science Foundation of China (No. 39920016) and the Doctorate Foundation of Xi’an Jiaotong University (DFXJTU 2001-5). [1] Cheng, J., Roques, B.P., Gacel, G.A., Huang, E. and Pasternak, G.W., Kappa 3 opiate receptor binding in the mouse and rat, Eur. J. Pharmacol., 226 (1992) 15–20. [2] Craig, A.D. and Burton, K., Spinal and medullary lamina I projection to nucleus submedius in medial thalamus: a possible pain center, J. Neurophysiol., 45 (1981) 443–465. [3] Dong, Y.F., Tang, J.S., Yuan, B. and Jia, H., Morphine applied to the thalamic nucleus submedius produces a naloxone reversible antinociceptive effect in the rat, Neurosci. Lett., 271 (1999) 17–20.
[4] Dubuisson, D. and Dennis, S.G., The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brainstem stimulation in rats and cats, Pain, 4 (1977) 161–164. [5] Ericson, A.C., Craig, A.D. and Blomqvist, A., GABA-like immunoreactivity in the thalamic nucleus submedius of the cat, Neuroscience, 76 (1997) 491–502. [6] Heinricher, M.M., Morgan, M.M. and Fields, H.L., Direct and indirect action of morphine on medullary neurons that modulate nociception, Neuroscience, 48 (1992) 533–543. [7] Jett, M.F. and Michelson, S., The formalin test in rat: validation of an automated system, Pain, 64 (1996) 19–25. [8] Kawakita, K., Dostrovsky, J.O., Tang, J.S. and Chiang, C.Y., Response of neurons in the rat thalamic nucleus submedius to cutaneous, muscle and visceral nociceptive stimuli, Pain, 55 (1993) 327–338. [9] Li, Y., Yuan, B. and Tang, J.S., A simple automated detecting system for formalin test in rats, Chin. J. Neurosci., 17 (2001) 145–148. [10] Mansour, A., Khachaturian, H., Lewis, M.E., Akil, H. and Watson, S.J., Autoradiographic differentiation of m-, d-, kopioid receptors in the rat forebrain and midbrain, J. Neurosci., 7 (1987) 2445–2464. [11] Miletic, V. and Coffield, J.A., Enkephalin-like immunoreactivity in the nucleus submedius of the cat and rat thalamus, Somatosens. Res., 5 (1988) 325–334. [12] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd Edition, Academic Press, Sydney, 1986. [13] Watson, G.S., Sufka, K.J. and Coderre, T.J., Optimal scoring strategies and weights for the formalin test in rats, Pain, 70 (1997) 53–58. [14] Yoshida, A., Dostrovsky, J.O., Sessle, B.J. and Chiang, C.Y., Trigeminal projections to the nucleus submedius of the thalamus in the rat, J. Comp. Neurol., 307 (1991) 609– 625. [15] Zagon, A., Meng, X. and Fields, H.L., Intrinsic membrane characteristics distinguish two subsets of nociceptive modulatory neurons in rat RVM, J. Neurophysiol., 78 (1997) 2848–2858. [16] Zhang, Y.Q., Tang, J.S., Yuan, B. and Jia, H., Inhibitory effects of electrical stimulation of thalamic nucleus submedius area on the rat tail flick reflex, Brain Res., 696 (1995) 205–212. [17] Zhang, Y.Q., Tang, J.S. and Yuan, B., Inhibitory effects of electrical stimulation of thalamic nucleus submedius on the nociceptive responses of spinal dorsal horn neurons in the rat, Brain Res., 737 (1996) 16–24. [18] Zhang, S., Tang, J.S., Yuan, B. and Jia, H., Inhibitory effects of glutamate-induced activation of thalamic nucleus submedius are mediated by ventrolateral orbital cortex and periaqueductal gray in rats, Eur. J. Pain, 2 (1998) 153– 163. [19] Zhang, S., Tang, J.S., Yuan, B. and Jia, H., Electricallyevoked inhibitory effects of the nucleus submedius on the jaw-opening reflex are mediated by ventrolateral orbital cortex and periaqueductal gray matter in the rat, Neuroscience, 92 (1999) 867–875.
Morphine microinjections into the rat nucleus submedius depress nociceptive behavior in the formalin test Zhi-Jie Yang, Jing-Shi Tang*, Hong Jia Department of Physiology, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, People’s Republic of China Received 7 March 2002; received in revised form 30 April 2002; accepted 6 May 2002
Abstract Our previous studies have indicated that the thalamic nucleus submedius (Sm) is involved in modulation of nociception and plays an important role in an endogenous analgesic system (a feedback loop) consisting of spinal cord–Sm– ventrolateral orbital cortex–periaqueductal gray–spinal cord. To investigate whether opioids are involved in this antinociception pathway, the effects of microinjection of morphine and naloxone into the Sm on the nociceptive behavior (agitation) evoked in the formalin test were investigated in the awake rat using an automated movement detection system. The results indicate that a unilateral microinjection of morphine (5 mg, 0.5 ml) into the Sm suppresses the formalin-induced agitation response, but does not influence spontaneous motor activity, and that the morphine-induced depression can be reversed by microinjection of the opioid receptor antagonist naloxone (1.0 mg, 0.5 ml) into the same Sm site. The results suggest that opioid receptors in the Sm may be involved in the Sm-mediated depression of persistent inflammatory pain. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nucleus submedius; Morphine; Naloxone; Nociceptive modulation; Formalin test; Rat
Our previous studies have indicated [16–19] that activation of the thalamic nucleus submedius (Sm) by electrical or chemical stimulation can depress the rat tail-flick reflex, the jaw-opening reflex and the nociceptive responses of neurons in the spinal dorsal horn and that these antinociceptive effects can be attenuated or eliminated by lesion or inhibition of the ventrolateral orbital cortex (VLO) or the periaqueductal gray (PAG). These findings suggest that the Sm may be involved not only in nociception, as reported previously [2,8,14], but also in modulation of nociception in an endogenous analgesic system (a feedback loop) consisting of a spinal cord–Sm–VLO–PAG–spinal cord loop. There is evidence that a discrete population of the axon terminals in the Sm arising from spinal dorsal horn lamina I neurons are immunoreactive for enkephalin [11], and that mu- and kappa3-opioid receptors are located in this nucleus [1,10]. A recent study in our laboratory has demonstrated that morphine application into the Sm depresses the rat tail-flick reflex [3]. These observations suggest an opioid link in Sm-mediated antinociception. The aim of the present study was to explore whether morphine application into the * Corresponding author. Tel.: 186-29-527-5172; fax: 186-29526-7364. E-mail address: [email protected] (J.-S. Tang).
Sm could depress the nociceptive behavioral responses (agitation) elicited in the formalin test, a test widely used in studies on persistent inflammatory-mediated pain, and to determine whether this effect could be antagonized by administration of the opioid receptor antagonist naloxone into the Sm. The experiments were performed on 42 adult Sprague– Dawley rats weighing 220–350 g of either sex provided by the Medical Experimental Animal Center of Shaanxi Province. Under sodium pentobarbital (50 mg/kg, intraperitoneally) anesthesia, the head was positioned in a stereotaxic frame with the incisor bar 3 mm below the horizontal zero. A small craniotomy was performed, and a guide cannula was stereotaxically placed at a position 2 mm dorsal to the Sm and fixed on the skull with three microscrews and dental cement. The experiments were performed according to the guidelines of the International Association for the Study of Pain. To determine the effect of microinjection of morphine into the Sm on the agitation responses elicited in the formalin test 7 days after animal surgery was performed, a needle attached to a 1 ml Hamilton syringe was inserted via the guide cannula into the Sm (2.3–2.8 mm posterior to Bregma, 0.6–0.9 mm lateral, 6.0–7.0 mm from the cortical surface) [12], and morphine hydrochloride (5 mg, 0.5 ml, dissolved in
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 51 4- 1
142
Z.-J. Yang et al. / Neuroscience Letters 328 (2002) 141–144
0.9% saline; Shenyang) was slowly injected into the Sm over 2 min. Five minutes after injection of morphine, formalin (5%, 50 ml) was injected subcutaneously into the rat’s hindpaw pad either contralateral or ipsilateral to the intracerebral injection site. The animal was placed immediately in a polycarbonate box (H, 16 cm; L, 16 cm; W, 10 cm) placed on a spring balance (2.0 kg). Nociceptive responses elicited by formalin injection such as licking, flinching, shaking, rearing, clutching and favoring the affected paw, induced movements of the balance that were transformed into electrical signals via an electromagnetic transducer. The electrical signals were amplified, filtered and displayed on an oscilloscope and fed into a window discriminator and a computer system that allowed quantitative recording of the number of agitation events and construction of time histograms over a 65 min observation period. In control experiments, 0.5 ml of 0.9% saline was injected into the Sm. In addition, the effects of morphine microinjection on spontaneous motor activity (no formalin injection) were also observed to determine the possible effect of morphine on movements. To determine if microinjection of naloxone into the Sm could reverse the inhibition produced by morphine, naloxone hydrochloride (1.0 mg, 0.5 ml, dissolved in 0.9% saline; Sigma) was slowly injected into the same Sm site immediately after morphine injection into the Sm and the changes of the agitation responses elicited by formalin were reexamined. A period of at least 7 days elapsed before testing the same animal again. At the end of the experiment, the drug injection site was marked by injection of Pontamine Sky Blue dye (0.5 ml, 2% in 0.5 M sodium acetate acid). Under deep anesthesia, the animal was perfused transcardially with 0.9% saline followed by 10% formalin. Transverse brain sections were processed with conventional histological methods. The locations of injection sites in the Sm were reconstructed. Data were analyzed for statistical significance (P , 0:05) by two-way analysis of variance (ANOVA) with a pairwise multiple comparison assessed with Tukey’s procedure. Subcutaneous injection of formalin into the rat hindpaw elicited a typical two phase nociceptive behavioral response (agitation) clearly quantified by the automated movement detection system, consisting of an early response lasting about 5 min followed by a 5–10 min period of decreased activity and then a late response lasting about 50 min as shown in Fig. 1a. During the 0–65 min observation period, the mean rate of agitation responses was 3.0 ^ 0.5 Hz (n ¼ 11). Microinjection of saline (0.5 ml) into the Sm did not significantly influence the formalin-induced movements, as shown in Fig. 1b,c (P . 0:05; n ¼ 11). In contrast, a unilateral microinjection of morphine into the Sm markedly attenuated the agitation responses (decrease in mean rate to 1.8 ^ 0.3 Hz; n ¼ 16). The difference was statistically significant as compared with that (4.9 ^ 0.5 Hz; n ¼ 16) obtained by injection of saline into the Sm (P , 0:001), as shown in Fig. 2. The locations of the
morphine injection sites in the Sm region are indicated in Fig. 2e. Most (88%; 14/16) of the injection sites within the Sm were effective in inhibiting the formalin responses, whereas the five sites more than 0.5 mm dorsal to the Sm were ineffective. The three sites adjacent to the dorsal Sm border and one site within the dorsal hypothalamic area were also effective. Microinjection of morphine into the Sm had no significant effect on spontaneous motor activity
Fig. 1. Histograms and graph showing formalin-evoked agitation and lack of effect of saline microinjections into Sm. Parts (a) and (b) show mean activity counts in 2 s bins over a 4000 s (67 min) observation period. A 50 ml subcutaneous injection of 5% formalin into the hindpaw was made at time 0. In (a), the effects of formalin injection (n ¼ 11) are displayed, and in (b), formalin injection in association with 0.9% saline injection into the Sm (n ¼ 11). Part (c) plots the data shown in (a) and (b) in 5 min periods. Two-way ANOVA followed by a multiple comparison test indicated that saline injection into the Sm did not influence the agitation responses elicited by formalin (P . 0:05).
Z.-J. Yang et al. / Neuroscience Letters 328 (2002) 141–144
143
Fig. 2. The time histograms (2 s bins) in the top half of the figure show the inhibitory effect of morphine (5 mg, 0.5 ml) microinjection into the Sm on the formalin-evoked agitation responses (b), but lack of effect with saline (a). This inhibition produced by morphine was reversed by naloxone (1.0 mg, 0.5 ml) injection into the same site (c); (d), plot comparing the effects shown in (a–c) (5 min periods); (e), diagram showing the locations of morphine injection sites in the Sm region and whether they produced an effect. The two-way ANOVAs followed by a multiple comparison test indicated that the differences between the effects of morphine and saline injections, and after morphine and morphine plus naloxone injections into the Sm were statistically significant (P , 0:001), but not significant (P . 0:05) between those after morphine plus naloxone and saline injection. CM, central medial thalamic nucleus; DA, hypothalamic dorsal area; mt, mammillothalamic tract; Mor, morphine; Nal, naloxone; Re, reunions thalamic nucleus; Rh, rhomboid thalamic nucleus; Smd, dorsal nucleus submedius; Smv, ventral nucleus submedius.
(1.41 ^ 0.1 Hz, n ¼ 6 with morphine vs. 1.76 ^ 0.2 Hz, n ¼ 6 with saline, P . 0:05). Microinjection of naloxone into the Sm immediately after morphine was injected into the same site significantly reversed the morphine-evoked inhibition of the agitation responses elicited by formalin (mean number of events, 5.0 ^ 0.9 Hz; n ¼ 8). The difference was significant as compared with that from application of morphine only (P , 0:001), but not significant between the saline and naloxone applications (P . 0:05; also, see Fig. 2). The present study has demonstrated that a unilateral administration of morphine into the Sm in the awake rat depresses the agitation responses elicited by formalin but does not influence spontaneous motor activity, and this antinociception can be reversed by microinjection of the opioid receptor antagonist naloxone into the same site. Since the second phase of nociceptive behavior elicited by formalin injection is considered to be a persistent inflammatory
hyperalgesic response, the results of this study suggest that opioid peptides and their receptors may be involved in the descending modulation of the inflammatory hyperalgesia through activation of the Sm–VLO–PAG pathway. As the direct action of morphine on central neurons is thought to produce hyperpolarization of the cell membrane rather than depolarization [6,15], it has been presumed that morphine-evoked excitatory effects may result from inhibition of inhibitory interneurons [6,15]. There is evidence, at least in the cat [5], that about one-fourth of the neurons in the Sm are gamma-aminobutyric acid (GABA) immunoreactive, and these GABAergic neurons are probably local circuit neurons. Therefore, it is possible that the inhibitory effect elicited by morphine injection into the Sm on the agitation responses elicited by formalin injection might be due to inhibition of GABAergic interneurons leading to increased activity of Sm output neurons projecting to the VLO and in turn to activation of the VLO–PAG–brainstem
144
Z.-J. Yang et al. / Neuroscience Letters 328 (2002) 141–144
descending inhibitory system and depression of the nociceptive inputs at the spinal level. The present study used the automated movement detection system developed in our laboratory to quantify the agitation responses elicited by subcutaneous injection of formalin into the rat’s hindpaw. It has been demonstrated [9] that the agitation responses recorded using this system reveal the typical two phases of nociceptive behavior that have been observed and quantified using manual scoring methods [4,13], and that systemic morphine significantly depresses the agitation responses and this effect can be antagonized by systemic naloxone. These recent results suggest that this simple automated system for quantifying the nociceptive behavior in the formalin test is a valid measure and comparable with that reported using a computer-driven, dynamic-force automatic detection system [7]. Histological reconstruction in this study indicated that all but two of the morphine injection sites within the Sm and those that were above but very close to the dorsal border of the Sm were effective in inhibiting the agitation responses elicited by formalin. However, those sites far from the Sm (dorsal to the Sm over 0.5 mm) had no effect despite the fact that opioid receptors (mu, delta and kappa3 receptors) are also located in these thalamic regions [1,10]. These results suggest that the morphine-evoked inhibition of the agitation responses following injections into the Sm and surrounding regions of the thalamus may be specific to an action on the Sm. Since morphine is not a selective opioid receptor agonist, and naloxone is not a selective opioid receptor antagonist, further studies using selective opioid receptor agonists and antagonists are required to determine which opioid receptors are involved in mediating the antinociceptive effects. The authors wish to thank Professor J.O. Dostrovsky for help in revising the manuscript. The project was supported by the National Nature Science Foundation of China (No. 39920016) and the Doctorate Foundation of Xi’an Jiaotong University (DFXJTU 2001-5). [1] Cheng, J., Roques, B.P., Gacel, G.A., Huang, E. and Pasternak, G.W., Kappa 3 opiate receptor binding in the mouse and rat, Eur. J. Pharmacol., 226 (1992) 15–20. [2] Craig, A.D. and Burton, K., Spinal and medullary lamina I projection to nucleus submedius in medial thalamus: a possible pain center, J. Neurophysiol., 45 (1981) 443–465. [3] Dong, Y.F., Tang, J.S., Yuan, B. and Jia, H., Morphine applied to the thalamic nucleus submedius produces a naloxone reversible antinociceptive effect in the rat, Neurosci. Lett., 271 (1999) 17–20.
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