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Prostaglandin E2 has antinociceptive effect through EP1 receptor in the ventromedial hypothalamus in rats
Pain 83 (1999) 221±227 www.elsevier.nl/locate/pain
Prostaglandin E2 has antinociceptive effect through EP1 receptor in the ventromedial hypothalamus in rats Masako Hosoi a, Takakazu Oka a,b, Michie Abe b, Tetsuro Hori b,*, Hiroshi Yamamoto a, Kazunori Mine a, Chiharu Kubo a a
Department of Psychosomatic Medicine, Kyushu University Faculty of Medicine, Fukuoka 812-8582, Japan b Department of Physiology, Kyushu University Faculty of Medicine, Fukuoka 812-8582, Japan Received 20 August 1998; received in revised form 1 March 1999; accepted 21 May 1999
Abstract The effects of microinjection of prostaglandin E2 (PGE2) (50 fg±50 ng/0.2 ml) into the ventromedial hypothalamus (VMH) on nociception were studied using a hot-plate test in rats. Microinjection of PGE2 (5±500 pg and 50 ng/0.2 ml) into the VMH signi®cantly prolonged the pawwithdrawal latency on a hot plate 5 and 10 min after injection, respectively. Maximal prolongation was obtained 5 min after the injection of PGE2 at 5 pg. Subsequently, to determine whether the PGE2 receptor subtype EP1 is involved in the PGE2-induced antinociceptive effect in the VMH, we observed the changes in nociception after intraVMH microinjection of SC19220, an EP1 receptor antagonist, and 17-phenyl-v trinor PGE2, an EP1 receptor agonist. Simultaneous injection of SC19220 (150 ng) with PGE2 (500 pg) into the VMH blocked the PGE2induced prolongation of the paw-withdrawal latency. Moreover, an intraVMH microinjection of 17-phenyl-v -trinor PGE2 (500 pg) prolonged it. These results indicate that PGE2 in the VMH has antinociceptive effect through its actions on EP1 receptors in rats. q 1999 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: Prostaglandin E2; 17-phenyl-v -trinor PGE2; EP1-receptor; Analgesia; Ventromedial hypothalamus; Dorsomedial hypothalamus
1. Introduction The ventromedial hypothalamus (VMH) is involved in various functions such as affective-defensive (aversive, aggressive or escape/¯ight) behaviors (Kruk et al., 1984; Fuchs et al., 1985; Hilton and Redfern, 1986; Yardley and Hilton, 1986; Pott et al., 1987), food intake and energy balance (Shimazu et al., 1966; McGowan et al., 1992) and female reproductive behavior (for review see Micevych and Ulibarri, 1992). Emerging evidence has shown that the VMH may play a role in nociception. There is a direct connection from this nucleus to the periaqueductal grey matter (PAG) which constitutes the descending inhibitory control of nociception (Dostrovsky et al., 1983). Recent studies (Bester et al., 1995) have revealed that the VMH is a major nucleus in a newly proposed ascending nociceptive pathway, the spino(trigemino)parabrachiohypothalamic pathway. Moreover, it is known that electrical stimulation of the VMH induces analgesia (Rhodes and Liebeskind, 1978; Culhane and Carstens, 1988; Duysens et al., 1989) and its * Corresponding author. Tel.: 181-92-642-6085; fax: 181-92-6426093;. E-mail address: [email protected] (T. Hori)
lesioning induces hyperalgesia (Vidal and Jacob, 1980). We have observed that intraVMH microinjection of interleukin1b (IL-1b ) produces an analgesia in rats, which is blocked by a cyclooxygenase inhibitor, indicating the obligatory role of prostanoids synthesis (Oka et al., 1995). Since IL-1b speci®cally increases the release of prostaglandin E2 (PGE2) from rat hypothalamic explants (Navarra et al., 1992), we hypothesized that PGE2 in the VMH has analgesic effect. Intracerebroventricular (i.c.v.) injection of PGE2 has biphasic effect on nociception, i.e. i.c.v. injection of PGE2 at high doses induces analgesia while that of PGE2 at low doses induces hyperalgesia (Oka et al., 1994, 1997c). Our behavioral and electrophysiological studies in rats indicate that PGE2 (i.c.v.)-induced analgesia is mediated by PGE2 receptor subtype EP1 and that PGE2 (i.c.v.)-induced hyperalgesia is mediated by EP3 receptors (Oka et al., 1994, 1997a,c). Moreover, the sites of action of PGE2 to produce thermal hyperalgesia through EP3 receptors were found to be located in the preoptic hypothalamus and the diagonal band of Broca in rats (Hosoi et al., 1997). However, the precise sites in the brain where PGE2 induces analgesia still remain obscure.
0304-3959/99/$20.00 q 1999 International Association for the Study of Pain. Published by Elsevier Science B.V. PII: S 0304-395 9(99)00105-0
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Taken the above ®ndings together, we hypothesized that PGE2 in the VMH induces analgesia through EP1 receptors. To verify this hypothesis, we microinjected PGE2 and its related substances into the VMH and observed the changes in nociceptive behavior by a hot-plate test in rats. Some of these ®ndings have previously been reported in an abstract form (Oka et al., 1997b). 2. Materials and methods 2.1. Subjects Male Wistar rats (Kyudo, Tosu, Japan), weighing between 300 and 350 g on the experimental day, were used. They were housed two per cage directly on bedding in the colony which was maintained at an ambient temperature of 23 ^ 18C on a 12-h light/12-h dark cycle with lights on at 08:00. Food and water were available ad libitum. 2.2. Implantation of the microinjection cannula Under pentobarbital sodium anesthesia (50 mg/kg, i.p.), rats were stereotaxically implanted with a 23-gauge stainless steel cannula containing a 30-gauge stainless steel wire stylet of the same length into the VMH. The tip of the cannula was unilaterally placed 1.0 mm above the VMH according to the brain atlas of Paxinos and Watson (1986). The cannula was ®xed to the skull with acrylic dental cement. After surgery, the animals received a prophylactic injection of an antibiotic (sulfamethoxide, 100 mg/rat, i.p.). They were then returned to the colony and housed individually. During a recovery period of at least 7 days, the animals were transported to the experimental room and placed on an unheated (25 ^ 0:18C) aluminium plate which was to be used for the hot-plate test for about 10 min daily. The animals were also placed in an acrylic box (24 cm in length, 17 cm in width and 12 cm in depth) where microinjection was to be done to become accustomed to the experimental procedures. 2.3. Drugs PGE2 and 17-phenyl-v -trinor PGE2 (an EP1 receptor agonist) were purchased from Sigma, St Louis, MO, USA and Cayman Chemicals, Ann Arbor, MI, USA, respectively. SC-19220 (an EP1 receptor antagonist) was a generous gift from Dr R.A. Marks (Searle). PGE2 was dissolved in physiological saline. 17-Phenyl-v -trinor PGE2 and SC19220 were dissolved in 99.5% ethanol and dimethyl sulfoxide (DMSO), respectively. They were stored at 2808C and diluted with physiological saline before use. Each drug was injected in a volume of 0.2 ml into the VMH. The same saline dilution of either ethanol or DMSO as the maximal dose of the test solutions was used as the vehicle of each drug.
2.4. Experimental procedures The effects on nociception of a microinjection of PGE2 and its related substances into the VMH were determined using the hot-plate test. Each rat was placed on an aluminium hot plate at 50 ^ 0:18C and the time until the animal showed the ®rst avoidance response (i.e. withdrawing the hindpaw) was recorded. The paw-withdrawal latency was determined three times at 10-min intervals before the microinjection and the average of the three values was taken as the baseline latency. Only the rats consistently exhibiting latencies between 11 and 34 s (the three values did not differ from each other by more than 25%) were used in the experiment. The rats were divided into different groups (n 6±12/ group). The control animals received intraVMH microinjection of the vehicles. The PGE2-treated groups were injected with PGE2 at different doses (50 fg± 50 ng) into the VMH. The 17-phenyl-v -trinor PGE2-treated group was given at a dose of 500 pg. Both the control and PGE2-treated groups were subdivided into groups which were co-injected either with SC19220 (150 ng) or DMSO. For the intraVMH microinjection, the animal was transferred to the acrylic box and the cannula was opened. Then, a 30-gauge injection needle, which was connected to a 1.0 ml Hamilton microsyringe, was inserted into the cannula 1.0 mm beyond its tip. All drug injections were performed using a solution volume of 0.2 ml at an injection rate of 0.1 ml per min. After injection, the injection needle was replaced by the stylet and the animal was returned to its home cage until used for further testing. The completion of the microinjection was determined to be time zero. Paw-withdrawal latency was then measured 5, 10, 15 and 30 min after the microinjection. All experiments were performed between 10:00 h and 17:00 h in a laboratory room kept at 23 ^ 0.18C. 2.5. Histology After completion of the experiments, the animals received a microinjection of pontamine sky blue acetate (0.2 ml) through the cannulas. Then, the animals, under deep anesthesia with a large dose of pentobarbital sodium (i.p.), were perfused transcardially with 3.7% neutral formaldehyde and the brain was removed. The brain was stored in 3.7% formaldehyde for at least 24 h. Each brain was then sectioned coronally into 120 mm serial sections on a freezing microtome and the sections were stained with neutral red. The distribution of the microinjected dye in the brain was veri®ed microscopically. The maximal diameter of the spread of injected solution was ,0.6 mm. 2.6. Statistical analyses The data were presented as the mean ^ SEM. Analysis of variance (ANOVA) followed by Duncan's new multiple range test or Student's t-test for unmatched data was used to determine any statistical differences between the values
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the saline group (P , 0:01) and the PGE2 (500 pg) group and the saline group (P , 0:05). 3.2. Inhibitory effect of microinjection of SC19220 into the VMH on PGE2-induced prolongation of paw-withdrawal latency
Fig. 1. The effects of microinjection of PGE2 into the VMH on paw-withdrawal latency on a hot-plate. The paw-withdrawal latency was assessed 5, 10, 15 and 30 min after injection. Six groups of rats were microinjected with PGE2 at 50 fg (n 12), 500 fg (n 7), 5 pg (n 8), 500 pg (n 7), 50 ng (n 10) or physiological saline (n 11). Each column represents the mean ^ SEM. The symbols adjacent to columns represent the level of signi®cance (one-way ANOVA followed by Duncan's new multiple range test) when compared with the saline-injected controls. **P , 0:01, *P , 0:05.
of the vehicle control group and those of the drug-treated groups. P , 0:05 was regarded as statistically signi®cant.
3. Results 3.1. Effects of microinjections of PGE2 into the VMH on nociception Since our previous study (Oka et al., 1995) demonstrated that, among several nuclei in the hypothalamus, the VMH was the most sensitive site to microinjected IL-1b to elicit hypoalgesia, we investigated the effects of microinjection of PGE2 (50 fg±50 ng/0.2 ml) into the VMH on the paw-withdrawal latency on a hot plate. Saline-treated control rats responded to the hot plate with fairly constant latencies between 20:5^1:2 s and 22:2^1:6 s during the observation period of 30 min, which did not differ signi®cantly from the corresponding baseline latency (20.4^1.1 s). In comparison with the saline-injected controls, intraVMH injection of PGE2 at 5 and 500 pg showed statistically signi®cant prolongation of the paw-withdrawal latency 5 min after injection, producing a maximal response at 5 pg (Fig. 1). When the amount of PGE2 was increased to 50 ng, the paw-withdrawal latency was prolonged signi®cantly 10 min, but not 5 min, after injection. Microinjection of PGE2 at doses of 50 and 500 fg did not change the paw-withdrawal latency. Fig. 2 shows individual data of the maximal % changes in the paw-withdrawal latency from the corresponding baseline latency, i.e. (the most prolonged latency 2 baseline latency) £ 100/baseline latency. The maximal % changes after intraVMH injection of saline and PGE2 at 5 pg and 500 pg were 19:9 ^ 3:6%(n 11), 87:3 ^ 15:8% (n 8) and 67.7 ^ 19.2% (n 7), respectively. There are statistically signi®cant differences between the PGE2 (5 pg) group and
To investigate whether EP1 receptors are involved in the PGE2-induced prolongation of paw-withdrawal latency, we microinjected PGE2 (500 pg) or physiological saline with either SC19220 (150 ng), an EP1 receptor antagonist, or its vehicle (DMSO) into the VMH (total volume, 0.2 ml) and then observed the changes in nociception. Both the vehicle (saline/DMSO)-treated control rats and saline/SC19220treated rats responded to the hot-plate with fairly constant latencies, which were not signi®cantly different from each other and from the corresponding baseline latencies (Fig. 3). An intraVMH injection of PGE2/DMSO signi®cantly prolonged the paw-withdrawal latency, whereas that of PGE2/SC19220 did not. 3.3. Effects of microinjection of 17-phenyl-v -trinor PGE2 into the VMH on nociception To further con®rm the involvement of EP1 receptors in the PGE2-induced antinociception, we examined the effects of microinjection of 17-phenyl-v -trinor PGE2, an EP1 receptor agonist, at 500 pg on nociceptive behavior. In our previous studies, the potency of i.c.v. injected 17-phenyl-v trinor PGE2 to produce hypoalgesia is 10±50 times less than that of PGE2 to produce the equivalent hypoalgesic effect (Oka et al., 1994, 1997a). Therefore, we investigated the
Fig. 2. The maximal % increases in the paw-withdrawal latency from the baseline latency after intraVMH microinjection of drugs and their vehicles. Each dot represents the maximal % increase in the paw-withdrawal latency from each baseline latency ((maximal latency 2 baseline latency) £ 100/ baseline latency) after intraVMH injection of saline (n 11), PGE2 at 5 pg (n 8) and 500 pg (n 7), vehicle of 17-phenyl-v -trinor PGE2 (n 6) and 17-phenyl-v -trinor PGE2 at 500 pg (n 4). P represents the level of signi®cance of difference (PGE2 vs. saline, one-way ANOVA followed by Duncan's new multiple range test; 17-phenyl-v -trinor PGE2 vs. vehicle, Students's t-test for unmatched data).
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effect of 17-phenyl-v -trinor PGE2 at 500 pg. The paw-withdrawal latency was prolonged signi®cantly 5 min after the intraVMH injection of 17-phenyl-v -trinor PGE2, when compared with those at any time after injection of its vehicle (Fig. 4). The maximal % changes in the paw-withdrawal latency from the corresponding baseline latency after intraVMH injection of 17-phenyl-v -trinor PGE2 at 500 pg (53:5 ^ 8:4%, n 4) is also greater than that after injection of its vehicle (16:7 ^ 4:5%, n 6) (P , 0:01). The individual data are plotted in Fig. 2. We have observed no changes in the skin temperatures of the pad and tail as well as the colonic temperature after intraVMH injection of 17-phenyl-v -trinor PGE2 at 500 pg and even at 100 ng in the present and our previous studies (Oka et al., 1997). 3.4. The location of brain sites where the microinjection of PGE2 produced antinociceptive effect Fig. 5 is a schematic representation of the localization of responsive and non-responsive sites to microinjected saline (Fig. 5A left), PGE2 at 5 pg (Fig. 5B, left) and at 500 pg (Fig. 5B, right), vehicle of 17-phenyl-v -trinor PGE2 (Fig. 5C left), and 17-phenyl-v -trinor PGE2 at 500 pg (Fig. 5C right). The sites were plotted on the coronal sections according to the magnitude of the responses, expressed as the percentage of the maximal increase in the paw-withdrawal latency to the baseline latency. The microinjection of PGE2 at 5 pg and 500 pg and 17-phenyl-v -trinor PGE2 at 500 pg into the VMH, in all but one case, prolonged the paw-withdrawal latency (see individual data in Fig. 2). On the other hand, the maximal change did not exceed 37% after intraVMH injection of saline (19:9 ^ 3:6%) and the vehicle of 17-phenyl-v -trinor PGE2 (16:7 ^ 4:6%). Within the VMH, the microinjection of PGE2 at 5 pg into the dorsomedial part of the VMH tended to produce larger increase in the paw-withdrawal latency than that into the ventrolateral
Fig. 3. The effects of microinjection of SC19220 (150 ng) into the VMH on PGE2 (500 pg)-induced prolongation of the paw-withdrawal latency on a hot-plate. Four groups of rats were microinjected with physiological saline/ DMSO (n 8), physiological saline/SC19220 (n 10), PGE2/DMSO (n 10), and PGE2/SC19220 (n 5). The symbol adjacent to a column represents the level of signi®cance (one-way ANOVA followed by Duncan's new multiple range test) when compared with the physiological saline/DMSO-injected controls. *P , 0:05.
Fig. 4. The effects of microinjection of 17-phenyl-v -trinor PGE2 (17-ptPGE2; 500 pg) into the VMH on paw-withdrawal latency on a hot-plate. Two groups of rats were microinjected with 17-phenyl-v -trinor PGE2 (n 4) and vehicle (n 6). Each columns represents the mean ^ SEM. The symbol adjacent to a column represents the level of signi®cance (Student's t-test for unmatched data) when compared with the vehicleinjected control. **P , 0:01.
part. Furthermore, PGE2 at 5 pg (n 3) and 500 pg (n 3) microinjected into the dorsomedial hypothalamus (DMH) also showed an increase in the paw-withdrawal latency in all cases by 62:4 ^ 35:3% and 49:7 ^ 14:7%, respectively. In contrast, intraDMH injection of saline (n 2) did not increase the latency.
4. Discussion 4.1. Hypoalgesia induced by an intraVMH microinjection of PGE2 The present study demonstrated that microinjection of PGE2 and 17-phenyl-v -trinor PGE2 into the VMH prolonged paw-withdrawal latency on a hot plate. It has been known that PGE2 and 17-phenyl-v -trinor PGE2 induces hyperthermia when they are administered into the cerebroventricle (Oka and Hori, 1994) and the preoptic area and the neighboring basal forebrain (Oka et al., 1997). However, the prolongation of the paw-withdrawal latency was not caused secondarily by changes in body temperature, because an intraVMH microinjection of 17-phenyl-v -trinor PGE2 at 500 pg in the present study and even at 100 ng in the previous study (Oka et al., 1997) did not change the colonic temperature. The awaking action of PGE2 is also unlikely to explain the prolonged paw-withdrawal latency. It was shown that the decrease of sleep during intracerebral infusion of PGE2 was accompanied by a rise in body temperature in the rat (Matsumura et al., 1989), whereas a nonpyrogenic dose of the EP1 agonist prolonged the paw-withdrawal latency in the present study. Furthermore, our electrophysiological studies (Oka et al., 1997a,c) have demonstrated that i.c.v. injections of PGE2 at 0.1±1 nmol (35.3±353 ng) and 17-phenyl-v -trinor PGE2 at 1±10 nmol suppressed the nociceptive responses of wide dynamic range neurons in the trigeminal nucleus caudalis in the rat,
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Fig. 5. The location of the brain sites microinjected with saline (A, left), PGE2 at 5 pg (B, left) and 500 pg (B, right), vehicle of 17-phenyl-v -trinor PGE2 (C, left) and 17-phenyl-v -trinor PGE2 at 500 pg (C, right) according to the magnitude of the produced nociceptive responses. The magnitude of hypoalgesic responses was expressed as the percentage of maximal change in the paw-withdrawal latency to the baseline latency. DMH, dorsomedial hypothalamus; f, fornix; LH, lateral hypothalamic area; OX, optic tract; VMH, ventromedial hypothalamus; 3V, third ventricle. Bar 1 mm. The numbers above individual frontal sections indicate the distances from bregma (adapted from Paxinos and Watson, 1986).
with a similar time course (5±15 min after injection) as that of the changes in behavioral nociceptive responses in the present study. We have previously demonstrated that IL-1b produces the behavioral analgesia in a prostanoids-dependent way only when IL-1b was administered into the VMH (Oka et al., 1995). These ®ndings suggest that the prolonged paw-withdrawal latency observed in the present study re¯ects hypoalgesia. 4.2. The EP1 receptors mediation of the PGE2-induced hypoalgesia in the VMH The present study revealed that intraVMH microinjection of 17-phenyl-v -trinor PGE2 (an EP1 agonist) produced hypoalgesia. This is in agreement with our previous study which demonstrated that i.c.v. injection of 17-phenyl-v trinor PGE2 induces hypoalgesic effect both behaviorally (Oka et al., 1994) and electrophysiologically (Oka et al., 1997a). Furthermore, intraVMH microinjection of SC19220 (an EP1 receptor antagonist) blocked the PGE2induced hypoalgesia in the present study. These ®ndings suggest that the brain PGE2-induced hypoalgesia is mediated by EP1 receptors in the VMH. Although PGE2 binding is found in the VMH (Matsumura et al., 1992), an in situ hybridization showed that EP1 recep-
tor transcripts are observed in nerve cells only in the paraventricular nucleus and the supraoptic nucleus in the hypothalamus of mice (Batshake et al., 1995). Further study is required to determine whether or not the VMH actually have EP1 receptors in the rat. The hypoalgesia after intraVMH injection of PGE2 in the present study is in good contrast with the hyperalgesia after its injection into the preoptic hypothalamus and the neighboring basal forebrain (for review see Hori et al., 1998; Oka and Hori, 1999). First, hypoalgesia was observed by larger amounts of PGE2 at 330 ng injected i.c.v. (Oka et al., 1994) and at 5 pg±50 ng injected into the VMH in the present study, whereas hyperalgesia was caused by only smaller doses of PGE2 at 3.3 pg±3.3 ng injected i.c.v. (Oka et al., 1994) and 5±50 fg injected into the preoptic hypothalamus (Hosoi et al., 1997). Second, the hypoalgesic response is rapid and short-lasting, i.e. peaking at 5 min after i.c.v. injection (Oka et al., 1994) and 5±10 min after intraVMH injection in the present study, whereas the hyperalgesic response is long-lasting, i.e. 5±60 min (peak time, 15 min) after i.c.v. injection of PGE2 (Oka et al., 1994) and 15 min after intrapreoptic injection (Hosoi et al., 1997). Finally, the hypoalgesia and hyperalgesia caused by PGE2 is mediated by its actions on EP1 and EP3 receptors in the brain, respectively (Oka et al., 1994; Hosoi et al., 1997). Thus, the oppo-
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site effects of PGE2 on nociception are caused by its concentration dependent actions on different types EP receptors in different sites in the hypothalamus. 4.3. Possible mechanisms that PGE2 in the VMH produces hypoalgesia There are several lines of evidence that the VMH is involved in nociception. First, electrical stimulation of the VMH induces hypoalgesia (Rhodes and Liebeskind, 1978; Culhane and Carstens, 1988; Duysens et al., 1989) and its lesioning induces hyperalgesia (Vidal and Jacob, 1980). Second, the VMH receives nociceptive inputs from the mesencephalic parabrachial area (mPB) via spino(trigemino)parabrachiohypothalamic pathway (Bester et al., 1995). A recent study using a sensitive and selective anterograde axonal tracer, Phaseolus vulgaris-leucoagglutinin, has shown that the mPB region projects much densely to almost the entire dorsomedial subdivision of the ipsilateral VMH nucleus. The DMH receives more diffuse but less dense projections from the parabrachial area compared with the VMH (Bester et al., 1997). It is interesting that PGE2 (5 pg)induced analgesic effect was pronounced in the dorsomedial subdivision of the VMH and it was also observed in the DMH in the present study. On the other hand, the VMH projects to the PAG which exerts a descending inhibitory control on the spinal and trigeminal nociceptive transmission (Mokha et al., 1987; Hori et al., 1998). Therefore, it is possible to suggest that PGE2-induced analgesic effect in the VMH is mediated by its actions on the spinoparabrachiohypothalamic pathway and/or by in¯uencing the activity of the descending inhibitory system of nociception. Our previous studies have suggested that IL-1, IL-6 and TNFa in the hypothalamus may modulate pain, at least in part, by local synthesis of PGE2, although the involvement of the other mediators such as the other arachidonate metabolites and nitric oxide remains to be elucidated. Our ®ndings, together with the present one, show that small amounts of IL-1b induce hyperalgesia via an action of PGE2 on EP3 receptors in the preoptic hypothalamic area and large amounts of IL-1b produce analgesia via an action of PGE2 on EP1 receptors in the VMH area (Oka et al., 1994, 1995, 1997a,c; Hosoi et al., 1997). The hyperalgesia produced by IL-1b and PGE2 at non-pyrogenic doses may explain systemic decreases in nociceptive threshold clinically observed in the early phase of infectious diseases and thereby alarming the infection before the development of sickness symptoms such as fever. When the disease becomes intense, the nociception is switched to analgesia with the development of typical sickness symptoms. This may re¯ect the changes in the strategy of the host for ®ghting microbial invasion (Oka and Hori, 1999). Acknowledgements We thank to Dr R.A. Marks (Searle) for kind supply of
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M. Hosoi et al. / Pain 83 (1999) 221±227 Interleukin-1b and interleukin-6 speci®cally increase the release of prostaglandin E2 from rat hypothalamic explants in vitro. Neuroendocrinology 1992;56:61±68. Oka T, Hori T. EP1-receptor mediation of prostaglandin E2-induced hyperthermia in rats. Am J Physiol 1994;267:R289±R294. Oka T, Aou S, Hori T. Intracerebroventricular injection of prostaglandin E2 induces thermal hyperalgesia in rats: the possible involvement of EP3 receptors. Brain Res 1994;663:287±292. Oka T, Oka K, Hosoi M, Aou S, Hori T. The opposing effects of interleukin-1b microinjected into the preoptic hypothalamus and the ventromedial hypothalamus on nociceptive behavior in rats. Brain Res 1995;700:271±278. Oka K, Oka T, Hori T. Prostaglandin E2 may induce hyperthermia through EP1 receptor in the anterior wall of the third ventricle and neighboring preoptic regions. Brain Res 1997;767:92±99. Oka T, Hori T, Hosoi M, Oka K, Abe M, Kubo C. Biphasic modulation in the trigeminal nociceptive neuronal responses by the intracerebroventricular prostaglandin E2 may be mediated through different EP receptors subtypes in rats. Brain Res 1997a;771:278±284. Oka T, Hosoi M, Abe M, Oka K, Imanishi T, Hori T. The opposing effects of prostaglandin E2 microinjected into the preoptic hypothalamus and
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the ventromedial hypothalamus on nociception in rats. 27th Soc. Neurosci Abstr 1997b;23-1:160. Oka T, Hosoi M, Oka K, Hori T. Biphasic alteration in the trigeminal nociceptive neuronal responses after intracerebroventricular injection of prostaglandin E2 in rats. Brain Res 1997c;749:354±357. Oka T, Hori T. Brain cytokines and pain. In: Watkins LR, Maier SF, editors. Cytokines and Pain, Basel: Birkhauser Verlag, 1999. pp. 183±203. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. Academic Press, 1986. Pott CB, Kramer SZ, Siegel A. Central gray modulation of affective defense is differentially sensitive to naloxone. Physiol Behav 1987;40:207±213. Rhodes DL, Liebeskind JC. Analgesia from rostral brain stem stimulation in the rat. Brain Res 1978;143:521±532. Shimazu T, Fukuda A, Ban T. Reciprocal in¯uences of the ventromedial and lateral hypothalamic nuclei on blood glucose level and liver glycogen content. Nature 1966;210:1178±1179. Vidal C, Jacob J. The effect of medial hypothalamus lesions on pain control. Brain Res 1980;199:89±100. Yardley CP, Hilton SM. The hypothalamic and brainstem areas from which the cardiovascular and behavioural components of the defence reaction are elicited in the rat. J Autonom Nerv Syst 1986;15:227±244.
Prostaglandin E2 has antinociceptive effect through EP1 receptor in the ventromedial hypothalamus in rats Masako Hosoi a, Takakazu Oka a,b, Michie Abe b, Tetsuro Hori b,*, Hiroshi Yamamoto a, Kazunori Mine a, Chiharu Kubo a a
Department of Psychosomatic Medicine, Kyushu University Faculty of Medicine, Fukuoka 812-8582, Japan b Department of Physiology, Kyushu University Faculty of Medicine, Fukuoka 812-8582, Japan Received 20 August 1998; received in revised form 1 March 1999; accepted 21 May 1999
Abstract The effects of microinjection of prostaglandin E2 (PGE2) (50 fg±50 ng/0.2 ml) into the ventromedial hypothalamus (VMH) on nociception were studied using a hot-plate test in rats. Microinjection of PGE2 (5±500 pg and 50 ng/0.2 ml) into the VMH signi®cantly prolonged the pawwithdrawal latency on a hot plate 5 and 10 min after injection, respectively. Maximal prolongation was obtained 5 min after the injection of PGE2 at 5 pg. Subsequently, to determine whether the PGE2 receptor subtype EP1 is involved in the PGE2-induced antinociceptive effect in the VMH, we observed the changes in nociception after intraVMH microinjection of SC19220, an EP1 receptor antagonist, and 17-phenyl-v trinor PGE2, an EP1 receptor agonist. Simultaneous injection of SC19220 (150 ng) with PGE2 (500 pg) into the VMH blocked the PGE2induced prolongation of the paw-withdrawal latency. Moreover, an intraVMH microinjection of 17-phenyl-v -trinor PGE2 (500 pg) prolonged it. These results indicate that PGE2 in the VMH has antinociceptive effect through its actions on EP1 receptors in rats. q 1999 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: Prostaglandin E2; 17-phenyl-v -trinor PGE2; EP1-receptor; Analgesia; Ventromedial hypothalamus; Dorsomedial hypothalamus
1. Introduction The ventromedial hypothalamus (VMH) is involved in various functions such as affective-defensive (aversive, aggressive or escape/¯ight) behaviors (Kruk et al., 1984; Fuchs et al., 1985; Hilton and Redfern, 1986; Yardley and Hilton, 1986; Pott et al., 1987), food intake and energy balance (Shimazu et al., 1966; McGowan et al., 1992) and female reproductive behavior (for review see Micevych and Ulibarri, 1992). Emerging evidence has shown that the VMH may play a role in nociception. There is a direct connection from this nucleus to the periaqueductal grey matter (PAG) which constitutes the descending inhibitory control of nociception (Dostrovsky et al., 1983). Recent studies (Bester et al., 1995) have revealed that the VMH is a major nucleus in a newly proposed ascending nociceptive pathway, the spino(trigemino)parabrachiohypothalamic pathway. Moreover, it is known that electrical stimulation of the VMH induces analgesia (Rhodes and Liebeskind, 1978; Culhane and Carstens, 1988; Duysens et al., 1989) and its * Corresponding author. Tel.: 181-92-642-6085; fax: 181-92-6426093;. E-mail address: [email protected] (T. Hori)
lesioning induces hyperalgesia (Vidal and Jacob, 1980). We have observed that intraVMH microinjection of interleukin1b (IL-1b ) produces an analgesia in rats, which is blocked by a cyclooxygenase inhibitor, indicating the obligatory role of prostanoids synthesis (Oka et al., 1995). Since IL-1b speci®cally increases the release of prostaglandin E2 (PGE2) from rat hypothalamic explants (Navarra et al., 1992), we hypothesized that PGE2 in the VMH has analgesic effect. Intracerebroventricular (i.c.v.) injection of PGE2 has biphasic effect on nociception, i.e. i.c.v. injection of PGE2 at high doses induces analgesia while that of PGE2 at low doses induces hyperalgesia (Oka et al., 1994, 1997c). Our behavioral and electrophysiological studies in rats indicate that PGE2 (i.c.v.)-induced analgesia is mediated by PGE2 receptor subtype EP1 and that PGE2 (i.c.v.)-induced hyperalgesia is mediated by EP3 receptors (Oka et al., 1994, 1997a,c). Moreover, the sites of action of PGE2 to produce thermal hyperalgesia through EP3 receptors were found to be located in the preoptic hypothalamus and the diagonal band of Broca in rats (Hosoi et al., 1997). However, the precise sites in the brain where PGE2 induces analgesia still remain obscure.
0304-3959/99/$20.00 q 1999 International Association for the Study of Pain. Published by Elsevier Science B.V. PII: S 0304-395 9(99)00105-0
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Taken the above ®ndings together, we hypothesized that PGE2 in the VMH induces analgesia through EP1 receptors. To verify this hypothesis, we microinjected PGE2 and its related substances into the VMH and observed the changes in nociceptive behavior by a hot-plate test in rats. Some of these ®ndings have previously been reported in an abstract form (Oka et al., 1997b). 2. Materials and methods 2.1. Subjects Male Wistar rats (Kyudo, Tosu, Japan), weighing between 300 and 350 g on the experimental day, were used. They were housed two per cage directly on bedding in the colony which was maintained at an ambient temperature of 23 ^ 18C on a 12-h light/12-h dark cycle with lights on at 08:00. Food and water were available ad libitum. 2.2. Implantation of the microinjection cannula Under pentobarbital sodium anesthesia (50 mg/kg, i.p.), rats were stereotaxically implanted with a 23-gauge stainless steel cannula containing a 30-gauge stainless steel wire stylet of the same length into the VMH. The tip of the cannula was unilaterally placed 1.0 mm above the VMH according to the brain atlas of Paxinos and Watson (1986). The cannula was ®xed to the skull with acrylic dental cement. After surgery, the animals received a prophylactic injection of an antibiotic (sulfamethoxide, 100 mg/rat, i.p.). They were then returned to the colony and housed individually. During a recovery period of at least 7 days, the animals were transported to the experimental room and placed on an unheated (25 ^ 0:18C) aluminium plate which was to be used for the hot-plate test for about 10 min daily. The animals were also placed in an acrylic box (24 cm in length, 17 cm in width and 12 cm in depth) where microinjection was to be done to become accustomed to the experimental procedures. 2.3. Drugs PGE2 and 17-phenyl-v -trinor PGE2 (an EP1 receptor agonist) were purchased from Sigma, St Louis, MO, USA and Cayman Chemicals, Ann Arbor, MI, USA, respectively. SC-19220 (an EP1 receptor antagonist) was a generous gift from Dr R.A. Marks (Searle). PGE2 was dissolved in physiological saline. 17-Phenyl-v -trinor PGE2 and SC19220 were dissolved in 99.5% ethanol and dimethyl sulfoxide (DMSO), respectively. They were stored at 2808C and diluted with physiological saline before use. Each drug was injected in a volume of 0.2 ml into the VMH. The same saline dilution of either ethanol or DMSO as the maximal dose of the test solutions was used as the vehicle of each drug.
2.4. Experimental procedures The effects on nociception of a microinjection of PGE2 and its related substances into the VMH were determined using the hot-plate test. Each rat was placed on an aluminium hot plate at 50 ^ 0:18C and the time until the animal showed the ®rst avoidance response (i.e. withdrawing the hindpaw) was recorded. The paw-withdrawal latency was determined three times at 10-min intervals before the microinjection and the average of the three values was taken as the baseline latency. Only the rats consistently exhibiting latencies between 11 and 34 s (the three values did not differ from each other by more than 25%) were used in the experiment. The rats were divided into different groups (n 6±12/ group). The control animals received intraVMH microinjection of the vehicles. The PGE2-treated groups were injected with PGE2 at different doses (50 fg± 50 ng) into the VMH. The 17-phenyl-v -trinor PGE2-treated group was given at a dose of 500 pg. Both the control and PGE2-treated groups were subdivided into groups which were co-injected either with SC19220 (150 ng) or DMSO. For the intraVMH microinjection, the animal was transferred to the acrylic box and the cannula was opened. Then, a 30-gauge injection needle, which was connected to a 1.0 ml Hamilton microsyringe, was inserted into the cannula 1.0 mm beyond its tip. All drug injections were performed using a solution volume of 0.2 ml at an injection rate of 0.1 ml per min. After injection, the injection needle was replaced by the stylet and the animal was returned to its home cage until used for further testing. The completion of the microinjection was determined to be time zero. Paw-withdrawal latency was then measured 5, 10, 15 and 30 min after the microinjection. All experiments were performed between 10:00 h and 17:00 h in a laboratory room kept at 23 ^ 0.18C. 2.5. Histology After completion of the experiments, the animals received a microinjection of pontamine sky blue acetate (0.2 ml) through the cannulas. Then, the animals, under deep anesthesia with a large dose of pentobarbital sodium (i.p.), were perfused transcardially with 3.7% neutral formaldehyde and the brain was removed. The brain was stored in 3.7% formaldehyde for at least 24 h. Each brain was then sectioned coronally into 120 mm serial sections on a freezing microtome and the sections were stained with neutral red. The distribution of the microinjected dye in the brain was veri®ed microscopically. The maximal diameter of the spread of injected solution was ,0.6 mm. 2.6. Statistical analyses The data were presented as the mean ^ SEM. Analysis of variance (ANOVA) followed by Duncan's new multiple range test or Student's t-test for unmatched data was used to determine any statistical differences between the values
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the saline group (P , 0:01) and the PGE2 (500 pg) group and the saline group (P , 0:05). 3.2. Inhibitory effect of microinjection of SC19220 into the VMH on PGE2-induced prolongation of paw-withdrawal latency
Fig. 1. The effects of microinjection of PGE2 into the VMH on paw-withdrawal latency on a hot-plate. The paw-withdrawal latency was assessed 5, 10, 15 and 30 min after injection. Six groups of rats were microinjected with PGE2 at 50 fg (n 12), 500 fg (n 7), 5 pg (n 8), 500 pg (n 7), 50 ng (n 10) or physiological saline (n 11). Each column represents the mean ^ SEM. The symbols adjacent to columns represent the level of signi®cance (one-way ANOVA followed by Duncan's new multiple range test) when compared with the saline-injected controls. **P , 0:01, *P , 0:05.
of the vehicle control group and those of the drug-treated groups. P , 0:05 was regarded as statistically signi®cant.
3. Results 3.1. Effects of microinjections of PGE2 into the VMH on nociception Since our previous study (Oka et al., 1995) demonstrated that, among several nuclei in the hypothalamus, the VMH was the most sensitive site to microinjected IL-1b to elicit hypoalgesia, we investigated the effects of microinjection of PGE2 (50 fg±50 ng/0.2 ml) into the VMH on the paw-withdrawal latency on a hot plate. Saline-treated control rats responded to the hot plate with fairly constant latencies between 20:5^1:2 s and 22:2^1:6 s during the observation period of 30 min, which did not differ signi®cantly from the corresponding baseline latency (20.4^1.1 s). In comparison with the saline-injected controls, intraVMH injection of PGE2 at 5 and 500 pg showed statistically signi®cant prolongation of the paw-withdrawal latency 5 min after injection, producing a maximal response at 5 pg (Fig. 1). When the amount of PGE2 was increased to 50 ng, the paw-withdrawal latency was prolonged signi®cantly 10 min, but not 5 min, after injection. Microinjection of PGE2 at doses of 50 and 500 fg did not change the paw-withdrawal latency. Fig. 2 shows individual data of the maximal % changes in the paw-withdrawal latency from the corresponding baseline latency, i.e. (the most prolonged latency 2 baseline latency) £ 100/baseline latency. The maximal % changes after intraVMH injection of saline and PGE2 at 5 pg and 500 pg were 19:9 ^ 3:6%(n 11), 87:3 ^ 15:8% (n 8) and 67.7 ^ 19.2% (n 7), respectively. There are statistically signi®cant differences between the PGE2 (5 pg) group and
To investigate whether EP1 receptors are involved in the PGE2-induced prolongation of paw-withdrawal latency, we microinjected PGE2 (500 pg) or physiological saline with either SC19220 (150 ng), an EP1 receptor antagonist, or its vehicle (DMSO) into the VMH (total volume, 0.2 ml) and then observed the changes in nociception. Both the vehicle (saline/DMSO)-treated control rats and saline/SC19220treated rats responded to the hot-plate with fairly constant latencies, which were not signi®cantly different from each other and from the corresponding baseline latencies (Fig. 3). An intraVMH injection of PGE2/DMSO signi®cantly prolonged the paw-withdrawal latency, whereas that of PGE2/SC19220 did not. 3.3. Effects of microinjection of 17-phenyl-v -trinor PGE2 into the VMH on nociception To further con®rm the involvement of EP1 receptors in the PGE2-induced antinociception, we examined the effects of microinjection of 17-phenyl-v -trinor PGE2, an EP1 receptor agonist, at 500 pg on nociceptive behavior. In our previous studies, the potency of i.c.v. injected 17-phenyl-v trinor PGE2 to produce hypoalgesia is 10±50 times less than that of PGE2 to produce the equivalent hypoalgesic effect (Oka et al., 1994, 1997a). Therefore, we investigated the
Fig. 2. The maximal % increases in the paw-withdrawal latency from the baseline latency after intraVMH microinjection of drugs and their vehicles. Each dot represents the maximal % increase in the paw-withdrawal latency from each baseline latency ((maximal latency 2 baseline latency) £ 100/ baseline latency) after intraVMH injection of saline (n 11), PGE2 at 5 pg (n 8) and 500 pg (n 7), vehicle of 17-phenyl-v -trinor PGE2 (n 6) and 17-phenyl-v -trinor PGE2 at 500 pg (n 4). P represents the level of signi®cance of difference (PGE2 vs. saline, one-way ANOVA followed by Duncan's new multiple range test; 17-phenyl-v -trinor PGE2 vs. vehicle, Students's t-test for unmatched data).
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effect of 17-phenyl-v -trinor PGE2 at 500 pg. The paw-withdrawal latency was prolonged signi®cantly 5 min after the intraVMH injection of 17-phenyl-v -trinor PGE2, when compared with those at any time after injection of its vehicle (Fig. 4). The maximal % changes in the paw-withdrawal latency from the corresponding baseline latency after intraVMH injection of 17-phenyl-v -trinor PGE2 at 500 pg (53:5 ^ 8:4%, n 4) is also greater than that after injection of its vehicle (16:7 ^ 4:5%, n 6) (P , 0:01). The individual data are plotted in Fig. 2. We have observed no changes in the skin temperatures of the pad and tail as well as the colonic temperature after intraVMH injection of 17-phenyl-v -trinor PGE2 at 500 pg and even at 100 ng in the present and our previous studies (Oka et al., 1997). 3.4. The location of brain sites where the microinjection of PGE2 produced antinociceptive effect Fig. 5 is a schematic representation of the localization of responsive and non-responsive sites to microinjected saline (Fig. 5A left), PGE2 at 5 pg (Fig. 5B, left) and at 500 pg (Fig. 5B, right), vehicle of 17-phenyl-v -trinor PGE2 (Fig. 5C left), and 17-phenyl-v -trinor PGE2 at 500 pg (Fig. 5C right). The sites were plotted on the coronal sections according to the magnitude of the responses, expressed as the percentage of the maximal increase in the paw-withdrawal latency to the baseline latency. The microinjection of PGE2 at 5 pg and 500 pg and 17-phenyl-v -trinor PGE2 at 500 pg into the VMH, in all but one case, prolonged the paw-withdrawal latency (see individual data in Fig. 2). On the other hand, the maximal change did not exceed 37% after intraVMH injection of saline (19:9 ^ 3:6%) and the vehicle of 17-phenyl-v -trinor PGE2 (16:7 ^ 4:6%). Within the VMH, the microinjection of PGE2 at 5 pg into the dorsomedial part of the VMH tended to produce larger increase in the paw-withdrawal latency than that into the ventrolateral
Fig. 3. The effects of microinjection of SC19220 (150 ng) into the VMH on PGE2 (500 pg)-induced prolongation of the paw-withdrawal latency on a hot-plate. Four groups of rats were microinjected with physiological saline/ DMSO (n 8), physiological saline/SC19220 (n 10), PGE2/DMSO (n 10), and PGE2/SC19220 (n 5). The symbol adjacent to a column represents the level of signi®cance (one-way ANOVA followed by Duncan's new multiple range test) when compared with the physiological saline/DMSO-injected controls. *P , 0:05.
Fig. 4. The effects of microinjection of 17-phenyl-v -trinor PGE2 (17-ptPGE2; 500 pg) into the VMH on paw-withdrawal latency on a hot-plate. Two groups of rats were microinjected with 17-phenyl-v -trinor PGE2 (n 4) and vehicle (n 6). Each columns represents the mean ^ SEM. The symbol adjacent to a column represents the level of signi®cance (Student's t-test for unmatched data) when compared with the vehicleinjected control. **P , 0:01.
part. Furthermore, PGE2 at 5 pg (n 3) and 500 pg (n 3) microinjected into the dorsomedial hypothalamus (DMH) also showed an increase in the paw-withdrawal latency in all cases by 62:4 ^ 35:3% and 49:7 ^ 14:7%, respectively. In contrast, intraDMH injection of saline (n 2) did not increase the latency.
4. Discussion 4.1. Hypoalgesia induced by an intraVMH microinjection of PGE2 The present study demonstrated that microinjection of PGE2 and 17-phenyl-v -trinor PGE2 into the VMH prolonged paw-withdrawal latency on a hot plate. It has been known that PGE2 and 17-phenyl-v -trinor PGE2 induces hyperthermia when they are administered into the cerebroventricle (Oka and Hori, 1994) and the preoptic area and the neighboring basal forebrain (Oka et al., 1997). However, the prolongation of the paw-withdrawal latency was not caused secondarily by changes in body temperature, because an intraVMH microinjection of 17-phenyl-v -trinor PGE2 at 500 pg in the present study and even at 100 ng in the previous study (Oka et al., 1997) did not change the colonic temperature. The awaking action of PGE2 is also unlikely to explain the prolonged paw-withdrawal latency. It was shown that the decrease of sleep during intracerebral infusion of PGE2 was accompanied by a rise in body temperature in the rat (Matsumura et al., 1989), whereas a nonpyrogenic dose of the EP1 agonist prolonged the paw-withdrawal latency in the present study. Furthermore, our electrophysiological studies (Oka et al., 1997a,c) have demonstrated that i.c.v. injections of PGE2 at 0.1±1 nmol (35.3±353 ng) and 17-phenyl-v -trinor PGE2 at 1±10 nmol suppressed the nociceptive responses of wide dynamic range neurons in the trigeminal nucleus caudalis in the rat,
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Fig. 5. The location of the brain sites microinjected with saline (A, left), PGE2 at 5 pg (B, left) and 500 pg (B, right), vehicle of 17-phenyl-v -trinor PGE2 (C, left) and 17-phenyl-v -trinor PGE2 at 500 pg (C, right) according to the magnitude of the produced nociceptive responses. The magnitude of hypoalgesic responses was expressed as the percentage of maximal change in the paw-withdrawal latency to the baseline latency. DMH, dorsomedial hypothalamus; f, fornix; LH, lateral hypothalamic area; OX, optic tract; VMH, ventromedial hypothalamus; 3V, third ventricle. Bar 1 mm. The numbers above individual frontal sections indicate the distances from bregma (adapted from Paxinos and Watson, 1986).
with a similar time course (5±15 min after injection) as that of the changes in behavioral nociceptive responses in the present study. We have previously demonstrated that IL-1b produces the behavioral analgesia in a prostanoids-dependent way only when IL-1b was administered into the VMH (Oka et al., 1995). These ®ndings suggest that the prolonged paw-withdrawal latency observed in the present study re¯ects hypoalgesia. 4.2. The EP1 receptors mediation of the PGE2-induced hypoalgesia in the VMH The present study revealed that intraVMH microinjection of 17-phenyl-v -trinor PGE2 (an EP1 agonist) produced hypoalgesia. This is in agreement with our previous study which demonstrated that i.c.v. injection of 17-phenyl-v trinor PGE2 induces hypoalgesic effect both behaviorally (Oka et al., 1994) and electrophysiologically (Oka et al., 1997a). Furthermore, intraVMH microinjection of SC19220 (an EP1 receptor antagonist) blocked the PGE2induced hypoalgesia in the present study. These ®ndings suggest that the brain PGE2-induced hypoalgesia is mediated by EP1 receptors in the VMH. Although PGE2 binding is found in the VMH (Matsumura et al., 1992), an in situ hybridization showed that EP1 recep-
tor transcripts are observed in nerve cells only in the paraventricular nucleus and the supraoptic nucleus in the hypothalamus of mice (Batshake et al., 1995). Further study is required to determine whether or not the VMH actually have EP1 receptors in the rat. The hypoalgesia after intraVMH injection of PGE2 in the present study is in good contrast with the hyperalgesia after its injection into the preoptic hypothalamus and the neighboring basal forebrain (for review see Hori et al., 1998; Oka and Hori, 1999). First, hypoalgesia was observed by larger amounts of PGE2 at 330 ng injected i.c.v. (Oka et al., 1994) and at 5 pg±50 ng injected into the VMH in the present study, whereas hyperalgesia was caused by only smaller doses of PGE2 at 3.3 pg±3.3 ng injected i.c.v. (Oka et al., 1994) and 5±50 fg injected into the preoptic hypothalamus (Hosoi et al., 1997). Second, the hypoalgesic response is rapid and short-lasting, i.e. peaking at 5 min after i.c.v. injection (Oka et al., 1994) and 5±10 min after intraVMH injection in the present study, whereas the hyperalgesic response is long-lasting, i.e. 5±60 min (peak time, 15 min) after i.c.v. injection of PGE2 (Oka et al., 1994) and 15 min after intrapreoptic injection (Hosoi et al., 1997). Finally, the hypoalgesia and hyperalgesia caused by PGE2 is mediated by its actions on EP1 and EP3 receptors in the brain, respectively (Oka et al., 1994; Hosoi et al., 1997). Thus, the oppo-
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site effects of PGE2 on nociception are caused by its concentration dependent actions on different types EP receptors in different sites in the hypothalamus. 4.3. Possible mechanisms that PGE2 in the VMH produces hypoalgesia There are several lines of evidence that the VMH is involved in nociception. First, electrical stimulation of the VMH induces hypoalgesia (Rhodes and Liebeskind, 1978; Culhane and Carstens, 1988; Duysens et al., 1989) and its lesioning induces hyperalgesia (Vidal and Jacob, 1980). Second, the VMH receives nociceptive inputs from the mesencephalic parabrachial area (mPB) via spino(trigemino)parabrachiohypothalamic pathway (Bester et al., 1995). A recent study using a sensitive and selective anterograde axonal tracer, Phaseolus vulgaris-leucoagglutinin, has shown that the mPB region projects much densely to almost the entire dorsomedial subdivision of the ipsilateral VMH nucleus. The DMH receives more diffuse but less dense projections from the parabrachial area compared with the VMH (Bester et al., 1997). It is interesting that PGE2 (5 pg)induced analgesic effect was pronounced in the dorsomedial subdivision of the VMH and it was also observed in the DMH in the present study. On the other hand, the VMH projects to the PAG which exerts a descending inhibitory control on the spinal and trigeminal nociceptive transmission (Mokha et al., 1987; Hori et al., 1998). Therefore, it is possible to suggest that PGE2-induced analgesic effect in the VMH is mediated by its actions on the spinoparabrachiohypothalamic pathway and/or by in¯uencing the activity of the descending inhibitory system of nociception. Our previous studies have suggested that IL-1, IL-6 and TNFa in the hypothalamus may modulate pain, at least in part, by local synthesis of PGE2, although the involvement of the other mediators such as the other arachidonate metabolites and nitric oxide remains to be elucidated. Our ®ndings, together with the present one, show that small amounts of IL-1b induce hyperalgesia via an action of PGE2 on EP3 receptors in the preoptic hypothalamic area and large amounts of IL-1b produce analgesia via an action of PGE2 on EP1 receptors in the VMH area (Oka et al., 1994, 1995, 1997a,c; Hosoi et al., 1997). The hyperalgesia produced by IL-1b and PGE2 at non-pyrogenic doses may explain systemic decreases in nociceptive threshold clinically observed in the early phase of infectious diseases and thereby alarming the infection before the development of sickness symptoms such as fever. When the disease becomes intense, the nociception is switched to analgesia with the development of typical sickness symptoms. This may re¯ect the changes in the strategy of the host for ®ghting microbial invasion (Oka and Hori, 1999). Acknowledgements We thank to Dr R.A. Marks (Searle) for kind supply of
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