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Prostaglandin E2 may induce hyperthermia through EP1 receptor in the anterior wall of the third ventricle and neighboring preoptic regions
Brain Research 767 Ž1997. 92–99
Research report
Prostaglandin E 2 may induce hyperthermia through EP1 receptor in the anterior wall of the third ventricle and neighboring preoptic regions Kae Oka ) , Takakazu Oka, Tetsuro Hori Department of Physiology, Faculty of Medicine, Kyushu UniÕersity, Fukuoka 812-82, Japan Accepted 22 April 1997
Abstract We have previously reported that intracerebroventricular injection of prostaglandin E 2 ŽPGE 2 . induces hyperthermia possibly through EP1 receptors in the rat. In the present study, to determine the sites in the brain where PGE 2 induces hyperthermia through EP1 receptors, we microinjected an EP1 receptor agonist, 17-phenyl-v-trinor PGE 2 Ž17-Ph-PGE 2 , 100 ng. into different sites in the rat brain and observed the colonic temperature ŽTco . for 2 h in a 23 " 18C environment. Responsive sites where 17-Ph-PGE 2 Ž100 ng. produced a rise in the Tco of more than 1.18C within 60 min after injection were found in the medial preoptic area, the subchiasmatic portion of the median preoptic nucleus, the anterior wall of the third ventricle ŽA3V. and the ventral portion of the diagonal band of Broca. Among these sites, the A3V was the most responsive. In contrast, microinjection of neither butaprost Žan EP2 agonist, 100 ng. nor M & B28767 Žan EP3 agonist, 100 ng. into these four sites had any effect on the Tco . Intracerebroventricular pretreatment with SC-19220 Žan EP1 antagonist, 100 m g. inhibited the rise in the Tco which was induced by microinjection of PGE 2 Ž50 ng. into the A3V. These results thus suggest that PGE 2 induces hyperthermia by stimulating EP1 receptors in the A3V and the neighboring preoptic region. q 1997 Elsevier Science B.V. Keywords: Prostaglandin E 2 ; Hyperthermia; EP1 receptor; 17-Phenyl-v-trinor prostaglandin E 2 ; Diagonal band of Broca; Anterior wall of the third ventricle; Fever
1. Introduction The E type of prostaglandins ŽPGs. in the brain has long been assumed to be a principal mediator of fever w2,6,7,10,20x. The major findings to support this are Ž1. the ability to increase deep body temperature by PGE 2 or PGE 1 microinjected into the brain wfor review see w9xx, Ž2. the increased level of PGE 2 or PGE 1 in the cerebroventricle and the hypothalamus during pyrogen-induced fever w2,6,19x and Ž3. the antipyresis by cyclooxygenase inhibitors which is accompanied by a decreased level of PGE 2 w6x. Extensive microinjection studies have also revealed that the most sensitive site to PGE 1 w18,25x and PGE 2 w11x regarding the production of hyperthermia is located in the preoptic area and the anterior hypothalamus ŽPOArAH. in rats, rabbits and monkeys. However, a recent study has also demonstrated that the increase in deep body temperature by PGE 1 is greater when given into the organum vasculosum laminae terminalis ŽOVLT. re-
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Corresponding author. Fax: q81 Ž92. 633-6719.
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 5 6 2 - 3
gion which is located in the anterior wall of the third ventricle ŽA3V. than when injected into the POArAH w21x. Recent pharmacological and molecular biological studies have revealed that E-type prostaglandin ŽEP. receptors can be classified into four subtypes, i.e., EP1 , EP2 , EP3 and EP4 receptors w4,23x. Our intracerebroventricular Ži.c.v.. injection study using EP receptor agonists and an antagonist has shown that PGE 2-induced hyperthermia in the rat is mediated predominantly through EP1 receptors in the brain w13x. The present study was therefore undertaken to determine the sites in the hypothalamus and neighboring basal forebrain where the stimulation of EP1 receptors may increase deep body temperature in the rat. Some of these findings have already been reported in abstract form w14x. 2. Materials and methods 2.1. Subjects Male Wistar rats ŽKyudo, Tosu, Japan., weighing between 300 and 350 g, were used. They were housed two or
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During a recovery period of at least 7 days, the rats were transported to the experimental room and loosely restrained in individual cylindrical wire cages 4–5 h daily to familiarize them with the experimental procedures. 2.3. Drugs
Fig. 1. Effects of microinjection of 17-Ph-PGE 2 into the MPO Ža frontal section at y0.80 from bregma., the A3V Ž0.0., the subMnPO Žy0.26. and the VDB Ž0.70. on Tco . 17-Ph-PGE 2 at 100 ng Žv . or the vehicle Ž`. was microinjected at time zero into these sites and the changes in Tco Ž DTco . were observed. Each point represents the mean DTco "S.E.M. ns 4–6, respectively.
three per cage. The room was maintained at an ambient temperature of 23 " 18C on a 12:12 h light-dark cycle with lights on at 08.00 h. Food and water were given ad libitum. 2.2. Cannula implantation Rats were anesthetized with pentobarbital sodium Ž50 mgrkg, i.p.. and a 23-gauge stainless steel cannula containing 30-gauge stainless steel wire as a stylet was implanted stereotaxically into various sites of the hypothalamus and neighboring basal forebrain. The tip of the guide cannula was unilaterally placed 1.0 mm above the target nucleus Žleft side. according to the brain atlas w16x. Some animals additionally received an implantation of a guide cannula into the left lateral cerebroventricle ŽLCV.. The correct placement of the cannula in the LCV was confirmed by a rise of cerebrospinal fluid in the cannula. Then, the cannulas were fixed to the skull with acrylic dental cement. After surgery, the rats were administered sulfamethoxide Ž100 mg, i.p.. as postoperative prophylaxis against infection, returned to the colony, and housed individually.
PGE 2 and 17-phenyl-v-trinor PGE 2 Ž17-Ph-PGE 2 . were purchased from Sigma and Cayman Chemicals, respectively. The following compounds were generous gifts that we gratefully acknowledge: butaprost from Dr. P.J. Gardiner ŽBayer, UK., M & B28767 from Dr. L. Webb ŽRhone-Poulenc Rorer, UK. and SC-19220 from Dr. G. Fleet ŽSearle, USA.. PGE 2 was dissolved in saline. M & B28767 was dissolved in 1% NaHCO 3 in saline. Butaprost and 17-PhPGE 2 were dissolved in 2% ethanol in saline. They were all stored at y808C and diluted with saline before use. SC-19220 was dissolved in dimethyl sulfoxide ŽDMSO. making 3 m l of solution. The same saline dilution of ethanol or NaHCO 3 as the maximal dose of the test solutions was used as the control solution of each drug. The control solution of SC-19220 was 3 m l DMSO. All solutions were passed through a 0.22 m m Millipore filter ŽMillipore Lab, Tokyo, Japan. before injection. In addition, all glassware, syringes, and injection needles were autoclaved before use. 2.4. Experimental procedures On the experimental day, each rat was placed in a cylindrical wire cage at 09.00 h. A copper-constantan thermocouple probe was inserted into the colon 4 cm beyond the anus, and the colonic temperature ŽTco . was thus recorded on a strip chart recorder at 1 min intervals. The data were also stored in a computer. After the baseline Tco was observed for about 2 h, a stylet was removed and the guide cannula was opened. Then a 30-gauge injection needle which was connected to a 1.0 m l Hamilton microsyringe was inserted into the guide cannula. The tip of the injection needle was inserted 1.0 mm beyond the tip of the guide cannula. No animals were used for more than one measurement in each experiment. Microinjections were
Fig. 2. Effects of microinjection of 17-Ph-PGE 2 into the PVN, the VMH and the LH on Tco . 17-Ph-PGE 2 at 100 ng Žv . or the vehicle Ž`. was microinjected at time zero into the PVN, the VMH and the LH. Each point represents the mean DTco " S.E.M. n s 4–5, respectively.
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performed at a volume of 0.1 or 0.2 m l with a rate of 0.1 m lrmin. 2.4.1. Experiment 1 A fixed dose Ž100 ngr0.2 m l. of 17-Ph-PGE 2 or its vehicle was microinjected into various sites in the brain and the Tco was observed. 2.4.2. Experiment 2 17-Ph-PGE 2 Ž0.5, 5 or 50 ng. or its vehicle was microinjected into the A3V, the subchiasmatic portion of the median preoptic nucleus ŽsubMnPO., the medial preoptic area ŽMPO. or the ventral portion of the diagonal band of Broca ŽVDB. and the changes in Tco were observed. In this particular experiment, all injections were performed in a fluid volume of 0.1 m l to obtain a more precise localization. 2.4.3. Experiment 3 The effect of SC-19220, an EP1 antagonist, on the PGE 2-induced hyperthermia was examined. SC-19220 or its vehicle in a volume of 3 m l was injected into the LCV over 3 min. Fifteen minutes after the i.c.v. injection, PGE 2 Ž50 ngr0.1 m l. or its vehicle was microinjected into the A3V and the changes in Tco were then observed during the subsequent 2 h.
2.4.4. Experiment 4 Microinjections of butaprost Žan EP2 agonist. or M & B28767 Žan EP3 agonist. at a dose of 100 ngr0.2 m l were made into the brain sites similar to those examined in experiment 1 and the changes in Tco were observed. All experiments were performed between 09.00 and 15.00 h and the room temperature was maintained at 23 " 18C.
2.5. Histology After completing the experiments, the animals were administered a large dose of pentobarbital sodium Ži.p.. and then microinjected with 0.2 m l or 0.1 m l of Pontamine Sky Blue acetate into the target sites, using the same injection rate as that described in the experiment. Then, the animals were perfused transcardially with 3.7% neutral formaldehyde. The brain was removed and stored in 3.7% formaldehyde for at least 24 h. Each brain was then cut into 50 m m serial sections on a freezing microtome. They were dried and stained with Neutral Red. The distribution of the microinjected dye in the brain was verified microscopically. The maximal diameter of the spread of the injected solution in a volume of 0.1 and 0.2 m l was - 0.4 and - 0.6 mm, respectively.
Fig. 3. Frontal sections of the rat brain showing the distribution of the sites microinjected with 17-Ph-PGE 2 at 100 ngr0.2 m l, which were illustrated according to the magnitude of the changes in Tco . The magnitude of the changes in Tco was expressed as the maximal changes in Tco within 60 min after injection from the Tco at time zero. ^, - 0.68C; 0.68C - ' - 1.28C; v, ) 1.28C. LS, lateral septal nucleus; MS, medial septal nucleus; DBB, diagonal band of Broca; AC, anterior commissure; MnPO, median preoptic nucleus; A3V, anterior wall of the third ventricle; MPO, medial preoptic area; LPO, lateral preoptic area; SON, supraoptic nucleus; OX, optic chiasma; PVN, paraventricular nucleus; LH, lateral hypothalamic area; AH, anterior hypothalamus; VMH, ventromedial hypothalamus. Bar, 1 mm. The numbers on the right of the individual frontal sections indicate the distances in mm from bregma w19x.
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Fig. 4. Histological sections show the tip of the injection needle within the medial preoptic area ŽMPO; A. and the anterior wall of the third ventricle ŽA3V; B.. AC, anterior commissure.
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2.6. Statistical analyses The values are presented as the means " S.E.M. The results from studies of the dose dependency of 17-Ph-PGE 2 in the brain regions were tested by two-way analysis of variance ŽANOVA. and by one-way ANOVA followed by Scheffe’s test. Student’s t-test for unmatched data was also used when appropriate. Differences were considered to be significant if P - 0.05.
3. Results 3.1. Brain sites where 17-phenyl-v-trinor PGE2 induces hyperthermia First, we microinjected 17-Ph-PGE 2 Ž100 ngr0.2 m l. or its vehicle into various sites in the hypothalamus and neighboring basal forebrain and thus examined the changes in the Tco for 2 h ŽFigs. 1–3.. The sites where microinjection of 17-Ph-PGE 2 produced a maximal rise in Tco of more than 1.18C were found in the MPO, the A3V, the subMnPO and the VDB ŽFig. 1.. Fig. 4 illustrates typical examples of the microinjected dye deposits within the MPO ŽFig. 4A. and the A3V ŽFig. 4B. in a volume of 0.2 m l. The regions surrounding these sites, such as the lateral preoptic area and the anterior hypothalamus ŽAH., were less responsive to microinjected 17-Ph-PGE 2 Ž100 ng. in producing hyperthermia ŽTco rise of 0.6–0.88C.. Microinjection into such remaining areas as the paraventricular nucleus ŽPVN., the ventromedial hypothalamus ŽVMH. and the lateral hypothalamic area ŽLH. did not induce hyperthermia Žless than 0.58C. ŽFig. 2.. The microinjected sites of 17-Ph-PGE 2 Ž100 ngr0.2 m l. in the brain were plotted according to the magnitude of the Tco changes in Fig. 3. The injection of the vehicle into all these sites had no effect on the Tco .
Fig. 5. Comparison of the effects of a microinjection of 17-Ph-PGE 2 into the MPO, the A3V, the subMnPO and the VDB on Tco . 17-Ph-PGE 2 at 0.5 ng ŽB., 5 ng Ž^., 50 ng Žv . or the vehicle Ž`. in a fluid volume of 0.1 m l was microinjected into the corresponding sites. Values are the mean DTco "S.E.M.
ing three areas Žtime to a peak, 40 min.. Furthermore, the intra-A3V injection of 17-Ph-PGE 2 at 5 and 0.5 ng elicited hyperthermia of 1.05 " 0.198C and 0.90 " 0.108C, respectively. On the other hand, the introduction of this compound at 5 and 0.5 ng into the other sites produced a significantly smaller rise in Tco Ž0.01 " 0.16–0.62 " 0.128C.. 3.3. Effects of SC-19220 on PGE2-induced hyperthermia To further investigate the possible involvement of EP1 receptors in PGE 2-induced hyperthermia, we microinjected SC-19220 Žan EP1 antagonist. or the vehicle ŽDMSO. into the LCV 15 min before the intra-A3V microinjection of either PGE 2 or saline and then observed changes in the Tco
3.2. Comparison of 17-Ph-PGE2-induced hyperthermic responses among the A3V, subMnPO, VDB and MPO Since different hyperthermic responsiveness to the microinjected PGE 1 within the rostral hypothalamus has been demonstrated w21x, we microinjected 17-Ph-PGE 2 at 0.5, 5 or 50 ng or the vehicle into the MPO, the A3V, the subMnPO and the VDB and compared the induced hyperthermic responses ŽFigs. 5 and 6.. In this experiment, all the injections were made in a fluid volume of 0.1 m l to localize the responsive sites more precisely. The mean Tco at time zero of each group ranged from 37.4 " 0.38C to 37.8 " 0.28C and did not differ significantly from each other. Although 17-Ph-PGE 2 at 50 ng, when administered into all these four areas, produced hyperthermia of a similar magnitude Ž0.84 " 0.07–1.26 " 0.068C., microinjection into the A3V induced a more rapid rise in Tco , which reached a peak at 30 min, than that into the remain-
Fig. 6. Dose-response curves for a maximal rise in Tco by the different amounts of 17-Ph-PGE 2 microinjected into the A3V Žv ., the subMnPO ŽB., the VDB Ž^. and the MPO Ž`.. The rats were injected with either vehicle or 17-Ph-PGE 2 at 0.5 ng, 5 ng or 50 ng Ž ns 5, respectively.. Each point represents the mean"S.E.M. The symbols represent the level of significance. ) P - 0.05, ) ) P - 0.01 Žone-way ANOVA followed by Scheffe’s test when compared with the value of the MPO.. q P - 0.05, qq P - 0.01 Žone-way ANOVA followed by Scheffe’s test when compared with the value of the vehicle..
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3.4. The effects of microinjection of butaprost and M & B28767 into the brain on Tc o We microinjected butaprost Žan EP2 agonist, 100 ngr0.2 m l. and M & B28767 Žan EP3 agonist, 100 ngr0.2 m l. into various brain sites which were similar to those of the 17-Ph-PGE 2 injection. However, microinjection of both substances into any sites, including the 17-Ph-PGE 2-responsive sites, demonstrated no effect on the Tco ŽFig. 8..
4. Discussion Fig. 7. Effects of SC-19220 on PGE 2 -induced fever. The rats were injected with SC-19220 at 100 m g or its vehicle ŽDMSO. into the LCV 15 min before microinjection of PGE 2 into the A3V at 50 ng or 0.9% saline. The time that the intra-A3V microinjection was started was determined as time zero. I, DMSOrPGE 2 ; B, SC-19220rPGE 2 ; `, DMSOrvehicle; v, SC-19220rvehicle; ns 5–6, respectively. Each point represents the mean"S.E.M. ) P - 0.05, ) ) P - 0.01 when compared with DMSOrvehicle-injected controls Žone-way ANOVA followed by Scheffe’s test..
over 120 min. The rats pretreated with DMSO Ž3 m l. followed by intra-A3V microinjection of PGE 2 Ž50 ngr0.1 m l. showed a rise in Tco of maximally 1.30 " 0.078C 30 min after injection. Pretreatment with SC-19220 Ž100 m gr3 m l, LCV. significantly attenuated the PGE 2 Ž50 ngr0.1 m l.-induced rise in the Tco observed 30–60 min after PGE 2 injection ŽFig. 7.. The maximal rise in the Tco of the rats pretreated with SC-19220 followed by PGE 2 microinjection was 0.48 " 0.148C Ž45 min after injection.. No changes in Tco occurred in the rats which received SC19220 followed by saline nor in the vehiclersaline-treated rats.
Fig. 8. Effects of microinjection of butaprost Ž100 ngr0.2 m l. and M&B28767 Ž100 ngr0.2 m l. into the MPO, the A3V, the subMnPO and the VDB on Tco . Butaprost ŽI. or M&B28767 Ž'. was microinjected at time zero into these sites. The injection of the vehicle, like those of butaprost and M&B28767, had no effect on the Tco and, therefore, the data on the vehicle study were omitted from the figure. Each point represents the mean DTco "S.E.M. ns 4–5, respectively.
The present study suggests that PGE 2 induces hyperthermia possibly through EP1 receptors in the MPO, the A3V, the subMnPO and the VDB in the rat. First, microinjection of 17-Ph-PGE 2 Ž50–100 ng. into these regions raised the Tco by more than 0.848C in the rat, whereas that of butaprost Ž100 ng. or M & B28767 Ž100 ng. into the same regions had no effect on the Tco . Second, SC-19220, an EP1 receptor antagonist, inhibited the rise in Tco after an intra-A3V injection of PGE 2 Ž50 ng. without affecting the Tco by itself. Many studies have demonstrated that the sites of actions for PGE 1 and PGE 2 to produce hyperthermia are confined to the POArAH which includes the MPO and the MnPO w11,15,17,18,25x. However, some researchers have revealed that the POArAH is not the sole site which can mediate PGE-induced hyperthermia. PGE-responsive sites in the extra-POArAH region include the ventral septum w17x, the VDB w11x, the VMH w11x and the OVLT region which corresponds to the A3V w15,21x. The present study demonstrated that the MPO, the subMnPO, the VDB and the A3V, but not the ventral septum and the VMH, showed pyrogenic sensitivity to microinjected 17-Ph-PGE 2 , and the A3V exhibited the greatest responsiveness. This finding is consistent with the results of previous studies which showed that the OVLT region was more responsive to PGE 1 in producing hyperthermia than the medial POArAH w21x. Furthermore, the neurons in the OVLT region were also found to respond to PGE 2 at an about 10 times lower dose than those in the MPO w8x. The maximal diameter of the spread of the dye solution in a volume of 0.1 m l was less than 0.4 mm and the dye did not spread to any other nuclei in the present study. Therefore, it seems unlikely that 17-Ph-PGE 2 which was injected into the MPO, the subMnPO and the VDB diffuses into the A3V including the OVLT region and thereby causes the rise in Tco . The stimulation of EP1 receptors results in the activation of different intracellular transduction pathways. The stimulation of EP1 receptors is suggested to increase the intracellular Ca2q probably by regulating the Ca2q channel gating via an unidentified G-protein w24x. However, it remains unknown as to how the increase in the intracellular Ca2q leads to the development of hyperthermia,
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although the decrease and increase in the extracellular concentration of Ca2q in the posterior hypothalamus in the cat have been demonstrated to raise and lower the body temperature, respectively w12x. We did not study the effects of the stimulation of EP4 receptors in the present study because no EP4 receptor agonist was available w4x. But EP2 and EP4 receptors are coupled to the Gs-protein and thus mediate increases in cyclic AMP ŽcAMP. concentration w23x. Since an EP2 agonist, butaprost, had no effect on the Tco after injection into the LCV w13x and the A3V and the neighboring basal forebrain in the present study, it is unlikely that EP4 receptors as well as EP2 receptors are involved in PGE 2-induced hyperthermia. Similarly, an EP3 receptor agonist, M & B28767, which is known to decrease the forskolin-stimulated cAMP level in cells expressing EP3 receptors w4x, failed to affect Tco . These findings do not support the possibility of the crucial involvement of the adenylate cyclase-cAMP system in PGE 2-induced hyperthermia. Previous studies on the microinjection of dibutyryl cAMP into the anterior hypothalamus revealed controversial findings, i.e., one study showed hyperthermia to be associated with hyperactivity w3x while the other showed hypothermia to be followed by hyperthermia which was probably due to convulsions w5x. An in situ hybridization study showed that EP1 receptor transcripts were found in nerve cells only in the PVN and the supraoptic nucleus ŽSON. in the hypothalamus of mice w1x. In contrast, the EP3 receptor mRNAs were widely distributed in the mouse brain and hybridization signals were found in various sites in the hypothalamus including the A3V and the MPO w22x. However, in the present study using the rat, no changes in Tco occurred after injection of the EP1 receptor agonist into the PVN and the SON, or after injection of the EP3 receptor agonist into the A3V and adjacent areas. Hyperthermia was obtained only when the EP1 agonist was injected into the A3V and the adjacent preoptic area, where no EP1 receptor transcripts were found in the mouse w1x. This might simply be due to a species difference in the distribution of EP1 receptors in the brain. Alternatively, it may be the result of technical problems in demonstrating the tissue distribution of EP1 receptors, since it has been pointed out that the expression of EP1 receptors is generally low in all tissues in comparison with other subtypes of EP receptors. Further study is called for using more sensitive methods to determine whether or not the A3V and its adjacent areas actually have EP1 receptors in the rat.
Acknowledgements This study was supported in part by Grants-in-Aid for Scientific Research Nos. 06454153 and 06557006 Žto T.H.. and grants from the Naito Foundation 93-183 Žto T.H.. and 95-185 Žto T.O... This experiment was reviewed by the Committee of Ethics on Animal Experiments in the Fac-
ulty of Medicine, Kyushu University and was carried out under the control of the Guidelines for Animal Experiments of the Faculty of Medicine, Kyushu University and the Law ŽNo. 105. and Notification ŽNo. 6. of the Government.
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Research report
Prostaglandin E 2 may induce hyperthermia through EP1 receptor in the anterior wall of the third ventricle and neighboring preoptic regions Kae Oka ) , Takakazu Oka, Tetsuro Hori Department of Physiology, Faculty of Medicine, Kyushu UniÕersity, Fukuoka 812-82, Japan Accepted 22 April 1997
Abstract We have previously reported that intracerebroventricular injection of prostaglandin E 2 ŽPGE 2 . induces hyperthermia possibly through EP1 receptors in the rat. In the present study, to determine the sites in the brain where PGE 2 induces hyperthermia through EP1 receptors, we microinjected an EP1 receptor agonist, 17-phenyl-v-trinor PGE 2 Ž17-Ph-PGE 2 , 100 ng. into different sites in the rat brain and observed the colonic temperature ŽTco . for 2 h in a 23 " 18C environment. Responsive sites where 17-Ph-PGE 2 Ž100 ng. produced a rise in the Tco of more than 1.18C within 60 min after injection were found in the medial preoptic area, the subchiasmatic portion of the median preoptic nucleus, the anterior wall of the third ventricle ŽA3V. and the ventral portion of the diagonal band of Broca. Among these sites, the A3V was the most responsive. In contrast, microinjection of neither butaprost Žan EP2 agonist, 100 ng. nor M & B28767 Žan EP3 agonist, 100 ng. into these four sites had any effect on the Tco . Intracerebroventricular pretreatment with SC-19220 Žan EP1 antagonist, 100 m g. inhibited the rise in the Tco which was induced by microinjection of PGE 2 Ž50 ng. into the A3V. These results thus suggest that PGE 2 induces hyperthermia by stimulating EP1 receptors in the A3V and the neighboring preoptic region. q 1997 Elsevier Science B.V. Keywords: Prostaglandin E 2 ; Hyperthermia; EP1 receptor; 17-Phenyl-v-trinor prostaglandin E 2 ; Diagonal band of Broca; Anterior wall of the third ventricle; Fever
1. Introduction The E type of prostaglandins ŽPGs. in the brain has long been assumed to be a principal mediator of fever w2,6,7,10,20x. The major findings to support this are Ž1. the ability to increase deep body temperature by PGE 2 or PGE 1 microinjected into the brain wfor review see w9xx, Ž2. the increased level of PGE 2 or PGE 1 in the cerebroventricle and the hypothalamus during pyrogen-induced fever w2,6,19x and Ž3. the antipyresis by cyclooxygenase inhibitors which is accompanied by a decreased level of PGE 2 w6x. Extensive microinjection studies have also revealed that the most sensitive site to PGE 1 w18,25x and PGE 2 w11x regarding the production of hyperthermia is located in the preoptic area and the anterior hypothalamus ŽPOArAH. in rats, rabbits and monkeys. However, a recent study has also demonstrated that the increase in deep body temperature by PGE 1 is greater when given into the organum vasculosum laminae terminalis ŽOVLT. re-
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Corresponding author. Fax: q81 Ž92. 633-6719.
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 5 6 2 - 3
gion which is located in the anterior wall of the third ventricle ŽA3V. than when injected into the POArAH w21x. Recent pharmacological and molecular biological studies have revealed that E-type prostaglandin ŽEP. receptors can be classified into four subtypes, i.e., EP1 , EP2 , EP3 and EP4 receptors w4,23x. Our intracerebroventricular Ži.c.v.. injection study using EP receptor agonists and an antagonist has shown that PGE 2-induced hyperthermia in the rat is mediated predominantly through EP1 receptors in the brain w13x. The present study was therefore undertaken to determine the sites in the hypothalamus and neighboring basal forebrain where the stimulation of EP1 receptors may increase deep body temperature in the rat. Some of these findings have already been reported in abstract form w14x. 2. Materials and methods 2.1. Subjects Male Wistar rats ŽKyudo, Tosu, Japan., weighing between 300 and 350 g, were used. They were housed two or
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During a recovery period of at least 7 days, the rats were transported to the experimental room and loosely restrained in individual cylindrical wire cages 4–5 h daily to familiarize them with the experimental procedures. 2.3. Drugs
Fig. 1. Effects of microinjection of 17-Ph-PGE 2 into the MPO Ža frontal section at y0.80 from bregma., the A3V Ž0.0., the subMnPO Žy0.26. and the VDB Ž0.70. on Tco . 17-Ph-PGE 2 at 100 ng Žv . or the vehicle Ž`. was microinjected at time zero into these sites and the changes in Tco Ž DTco . were observed. Each point represents the mean DTco "S.E.M. ns 4–6, respectively.
three per cage. The room was maintained at an ambient temperature of 23 " 18C on a 12:12 h light-dark cycle with lights on at 08.00 h. Food and water were given ad libitum. 2.2. Cannula implantation Rats were anesthetized with pentobarbital sodium Ž50 mgrkg, i.p.. and a 23-gauge stainless steel cannula containing 30-gauge stainless steel wire as a stylet was implanted stereotaxically into various sites of the hypothalamus and neighboring basal forebrain. The tip of the guide cannula was unilaterally placed 1.0 mm above the target nucleus Žleft side. according to the brain atlas w16x. Some animals additionally received an implantation of a guide cannula into the left lateral cerebroventricle ŽLCV.. The correct placement of the cannula in the LCV was confirmed by a rise of cerebrospinal fluid in the cannula. Then, the cannulas were fixed to the skull with acrylic dental cement. After surgery, the rats were administered sulfamethoxide Ž100 mg, i.p.. as postoperative prophylaxis against infection, returned to the colony, and housed individually.
PGE 2 and 17-phenyl-v-trinor PGE 2 Ž17-Ph-PGE 2 . were purchased from Sigma and Cayman Chemicals, respectively. The following compounds were generous gifts that we gratefully acknowledge: butaprost from Dr. P.J. Gardiner ŽBayer, UK., M & B28767 from Dr. L. Webb ŽRhone-Poulenc Rorer, UK. and SC-19220 from Dr. G. Fleet ŽSearle, USA.. PGE 2 was dissolved in saline. M & B28767 was dissolved in 1% NaHCO 3 in saline. Butaprost and 17-PhPGE 2 were dissolved in 2% ethanol in saline. They were all stored at y808C and diluted with saline before use. SC-19220 was dissolved in dimethyl sulfoxide ŽDMSO. making 3 m l of solution. The same saline dilution of ethanol or NaHCO 3 as the maximal dose of the test solutions was used as the control solution of each drug. The control solution of SC-19220 was 3 m l DMSO. All solutions were passed through a 0.22 m m Millipore filter ŽMillipore Lab, Tokyo, Japan. before injection. In addition, all glassware, syringes, and injection needles were autoclaved before use. 2.4. Experimental procedures On the experimental day, each rat was placed in a cylindrical wire cage at 09.00 h. A copper-constantan thermocouple probe was inserted into the colon 4 cm beyond the anus, and the colonic temperature ŽTco . was thus recorded on a strip chart recorder at 1 min intervals. The data were also stored in a computer. After the baseline Tco was observed for about 2 h, a stylet was removed and the guide cannula was opened. Then a 30-gauge injection needle which was connected to a 1.0 m l Hamilton microsyringe was inserted into the guide cannula. The tip of the injection needle was inserted 1.0 mm beyond the tip of the guide cannula. No animals were used for more than one measurement in each experiment. Microinjections were
Fig. 2. Effects of microinjection of 17-Ph-PGE 2 into the PVN, the VMH and the LH on Tco . 17-Ph-PGE 2 at 100 ng Žv . or the vehicle Ž`. was microinjected at time zero into the PVN, the VMH and the LH. Each point represents the mean DTco " S.E.M. n s 4–5, respectively.
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performed at a volume of 0.1 or 0.2 m l with a rate of 0.1 m lrmin. 2.4.1. Experiment 1 A fixed dose Ž100 ngr0.2 m l. of 17-Ph-PGE 2 or its vehicle was microinjected into various sites in the brain and the Tco was observed. 2.4.2. Experiment 2 17-Ph-PGE 2 Ž0.5, 5 or 50 ng. or its vehicle was microinjected into the A3V, the subchiasmatic portion of the median preoptic nucleus ŽsubMnPO., the medial preoptic area ŽMPO. or the ventral portion of the diagonal band of Broca ŽVDB. and the changes in Tco were observed. In this particular experiment, all injections were performed in a fluid volume of 0.1 m l to obtain a more precise localization. 2.4.3. Experiment 3 The effect of SC-19220, an EP1 antagonist, on the PGE 2-induced hyperthermia was examined. SC-19220 or its vehicle in a volume of 3 m l was injected into the LCV over 3 min. Fifteen minutes after the i.c.v. injection, PGE 2 Ž50 ngr0.1 m l. or its vehicle was microinjected into the A3V and the changes in Tco were then observed during the subsequent 2 h.
2.4.4. Experiment 4 Microinjections of butaprost Žan EP2 agonist. or M & B28767 Žan EP3 agonist. at a dose of 100 ngr0.2 m l were made into the brain sites similar to those examined in experiment 1 and the changes in Tco were observed. All experiments were performed between 09.00 and 15.00 h and the room temperature was maintained at 23 " 18C.
2.5. Histology After completing the experiments, the animals were administered a large dose of pentobarbital sodium Ži.p.. and then microinjected with 0.2 m l or 0.1 m l of Pontamine Sky Blue acetate into the target sites, using the same injection rate as that described in the experiment. Then, the animals were perfused transcardially with 3.7% neutral formaldehyde. The brain was removed and stored in 3.7% formaldehyde for at least 24 h. Each brain was then cut into 50 m m serial sections on a freezing microtome. They were dried and stained with Neutral Red. The distribution of the microinjected dye in the brain was verified microscopically. The maximal diameter of the spread of the injected solution in a volume of 0.1 and 0.2 m l was - 0.4 and - 0.6 mm, respectively.
Fig. 3. Frontal sections of the rat brain showing the distribution of the sites microinjected with 17-Ph-PGE 2 at 100 ngr0.2 m l, which were illustrated according to the magnitude of the changes in Tco . The magnitude of the changes in Tco was expressed as the maximal changes in Tco within 60 min after injection from the Tco at time zero. ^, - 0.68C; 0.68C - ' - 1.28C; v, ) 1.28C. LS, lateral septal nucleus; MS, medial septal nucleus; DBB, diagonal band of Broca; AC, anterior commissure; MnPO, median preoptic nucleus; A3V, anterior wall of the third ventricle; MPO, medial preoptic area; LPO, lateral preoptic area; SON, supraoptic nucleus; OX, optic chiasma; PVN, paraventricular nucleus; LH, lateral hypothalamic area; AH, anterior hypothalamus; VMH, ventromedial hypothalamus. Bar, 1 mm. The numbers on the right of the individual frontal sections indicate the distances in mm from bregma w19x.
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Fig. 4. Histological sections show the tip of the injection needle within the medial preoptic area ŽMPO; A. and the anterior wall of the third ventricle ŽA3V; B.. AC, anterior commissure.
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2.6. Statistical analyses The values are presented as the means " S.E.M. The results from studies of the dose dependency of 17-Ph-PGE 2 in the brain regions were tested by two-way analysis of variance ŽANOVA. and by one-way ANOVA followed by Scheffe’s test. Student’s t-test for unmatched data was also used when appropriate. Differences were considered to be significant if P - 0.05.
3. Results 3.1. Brain sites where 17-phenyl-v-trinor PGE2 induces hyperthermia First, we microinjected 17-Ph-PGE 2 Ž100 ngr0.2 m l. or its vehicle into various sites in the hypothalamus and neighboring basal forebrain and thus examined the changes in the Tco for 2 h ŽFigs. 1–3.. The sites where microinjection of 17-Ph-PGE 2 produced a maximal rise in Tco of more than 1.18C were found in the MPO, the A3V, the subMnPO and the VDB ŽFig. 1.. Fig. 4 illustrates typical examples of the microinjected dye deposits within the MPO ŽFig. 4A. and the A3V ŽFig. 4B. in a volume of 0.2 m l. The regions surrounding these sites, such as the lateral preoptic area and the anterior hypothalamus ŽAH., were less responsive to microinjected 17-Ph-PGE 2 Ž100 ng. in producing hyperthermia ŽTco rise of 0.6–0.88C.. Microinjection into such remaining areas as the paraventricular nucleus ŽPVN., the ventromedial hypothalamus ŽVMH. and the lateral hypothalamic area ŽLH. did not induce hyperthermia Žless than 0.58C. ŽFig. 2.. The microinjected sites of 17-Ph-PGE 2 Ž100 ngr0.2 m l. in the brain were plotted according to the magnitude of the Tco changes in Fig. 3. The injection of the vehicle into all these sites had no effect on the Tco .
Fig. 5. Comparison of the effects of a microinjection of 17-Ph-PGE 2 into the MPO, the A3V, the subMnPO and the VDB on Tco . 17-Ph-PGE 2 at 0.5 ng ŽB., 5 ng Ž^., 50 ng Žv . or the vehicle Ž`. in a fluid volume of 0.1 m l was microinjected into the corresponding sites. Values are the mean DTco "S.E.M.
ing three areas Žtime to a peak, 40 min.. Furthermore, the intra-A3V injection of 17-Ph-PGE 2 at 5 and 0.5 ng elicited hyperthermia of 1.05 " 0.198C and 0.90 " 0.108C, respectively. On the other hand, the introduction of this compound at 5 and 0.5 ng into the other sites produced a significantly smaller rise in Tco Ž0.01 " 0.16–0.62 " 0.128C.. 3.3. Effects of SC-19220 on PGE2-induced hyperthermia To further investigate the possible involvement of EP1 receptors in PGE 2-induced hyperthermia, we microinjected SC-19220 Žan EP1 antagonist. or the vehicle ŽDMSO. into the LCV 15 min before the intra-A3V microinjection of either PGE 2 or saline and then observed changes in the Tco
3.2. Comparison of 17-Ph-PGE2-induced hyperthermic responses among the A3V, subMnPO, VDB and MPO Since different hyperthermic responsiveness to the microinjected PGE 1 within the rostral hypothalamus has been demonstrated w21x, we microinjected 17-Ph-PGE 2 at 0.5, 5 or 50 ng or the vehicle into the MPO, the A3V, the subMnPO and the VDB and compared the induced hyperthermic responses ŽFigs. 5 and 6.. In this experiment, all the injections were made in a fluid volume of 0.1 m l to localize the responsive sites more precisely. The mean Tco at time zero of each group ranged from 37.4 " 0.38C to 37.8 " 0.28C and did not differ significantly from each other. Although 17-Ph-PGE 2 at 50 ng, when administered into all these four areas, produced hyperthermia of a similar magnitude Ž0.84 " 0.07–1.26 " 0.068C., microinjection into the A3V induced a more rapid rise in Tco , which reached a peak at 30 min, than that into the remain-
Fig. 6. Dose-response curves for a maximal rise in Tco by the different amounts of 17-Ph-PGE 2 microinjected into the A3V Žv ., the subMnPO ŽB., the VDB Ž^. and the MPO Ž`.. The rats were injected with either vehicle or 17-Ph-PGE 2 at 0.5 ng, 5 ng or 50 ng Ž ns 5, respectively.. Each point represents the mean"S.E.M. The symbols represent the level of significance. ) P - 0.05, ) ) P - 0.01 Žone-way ANOVA followed by Scheffe’s test when compared with the value of the MPO.. q P - 0.05, qq P - 0.01 Žone-way ANOVA followed by Scheffe’s test when compared with the value of the vehicle..
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3.4. The effects of microinjection of butaprost and M & B28767 into the brain on Tc o We microinjected butaprost Žan EP2 agonist, 100 ngr0.2 m l. and M & B28767 Žan EP3 agonist, 100 ngr0.2 m l. into various brain sites which were similar to those of the 17-Ph-PGE 2 injection. However, microinjection of both substances into any sites, including the 17-Ph-PGE 2-responsive sites, demonstrated no effect on the Tco ŽFig. 8..
4. Discussion Fig. 7. Effects of SC-19220 on PGE 2 -induced fever. The rats were injected with SC-19220 at 100 m g or its vehicle ŽDMSO. into the LCV 15 min before microinjection of PGE 2 into the A3V at 50 ng or 0.9% saline. The time that the intra-A3V microinjection was started was determined as time zero. I, DMSOrPGE 2 ; B, SC-19220rPGE 2 ; `, DMSOrvehicle; v, SC-19220rvehicle; ns 5–6, respectively. Each point represents the mean"S.E.M. ) P - 0.05, ) ) P - 0.01 when compared with DMSOrvehicle-injected controls Žone-way ANOVA followed by Scheffe’s test..
over 120 min. The rats pretreated with DMSO Ž3 m l. followed by intra-A3V microinjection of PGE 2 Ž50 ngr0.1 m l. showed a rise in Tco of maximally 1.30 " 0.078C 30 min after injection. Pretreatment with SC-19220 Ž100 m gr3 m l, LCV. significantly attenuated the PGE 2 Ž50 ngr0.1 m l.-induced rise in the Tco observed 30–60 min after PGE 2 injection ŽFig. 7.. The maximal rise in the Tco of the rats pretreated with SC-19220 followed by PGE 2 microinjection was 0.48 " 0.148C Ž45 min after injection.. No changes in Tco occurred in the rats which received SC19220 followed by saline nor in the vehiclersaline-treated rats.
Fig. 8. Effects of microinjection of butaprost Ž100 ngr0.2 m l. and M&B28767 Ž100 ngr0.2 m l. into the MPO, the A3V, the subMnPO and the VDB on Tco . Butaprost ŽI. or M&B28767 Ž'. was microinjected at time zero into these sites. The injection of the vehicle, like those of butaprost and M&B28767, had no effect on the Tco and, therefore, the data on the vehicle study were omitted from the figure. Each point represents the mean DTco "S.E.M. ns 4–5, respectively.
The present study suggests that PGE 2 induces hyperthermia possibly through EP1 receptors in the MPO, the A3V, the subMnPO and the VDB in the rat. First, microinjection of 17-Ph-PGE 2 Ž50–100 ng. into these regions raised the Tco by more than 0.848C in the rat, whereas that of butaprost Ž100 ng. or M & B28767 Ž100 ng. into the same regions had no effect on the Tco . Second, SC-19220, an EP1 receptor antagonist, inhibited the rise in Tco after an intra-A3V injection of PGE 2 Ž50 ng. without affecting the Tco by itself. Many studies have demonstrated that the sites of actions for PGE 1 and PGE 2 to produce hyperthermia are confined to the POArAH which includes the MPO and the MnPO w11,15,17,18,25x. However, some researchers have revealed that the POArAH is not the sole site which can mediate PGE-induced hyperthermia. PGE-responsive sites in the extra-POArAH region include the ventral septum w17x, the VDB w11x, the VMH w11x and the OVLT region which corresponds to the A3V w15,21x. The present study demonstrated that the MPO, the subMnPO, the VDB and the A3V, but not the ventral septum and the VMH, showed pyrogenic sensitivity to microinjected 17-Ph-PGE 2 , and the A3V exhibited the greatest responsiveness. This finding is consistent with the results of previous studies which showed that the OVLT region was more responsive to PGE 1 in producing hyperthermia than the medial POArAH w21x. Furthermore, the neurons in the OVLT region were also found to respond to PGE 2 at an about 10 times lower dose than those in the MPO w8x. The maximal diameter of the spread of the dye solution in a volume of 0.1 m l was less than 0.4 mm and the dye did not spread to any other nuclei in the present study. Therefore, it seems unlikely that 17-Ph-PGE 2 which was injected into the MPO, the subMnPO and the VDB diffuses into the A3V including the OVLT region and thereby causes the rise in Tco . The stimulation of EP1 receptors results in the activation of different intracellular transduction pathways. The stimulation of EP1 receptors is suggested to increase the intracellular Ca2q probably by regulating the Ca2q channel gating via an unidentified G-protein w24x. However, it remains unknown as to how the increase in the intracellular Ca2q leads to the development of hyperthermia,
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although the decrease and increase in the extracellular concentration of Ca2q in the posterior hypothalamus in the cat have been demonstrated to raise and lower the body temperature, respectively w12x. We did not study the effects of the stimulation of EP4 receptors in the present study because no EP4 receptor agonist was available w4x. But EP2 and EP4 receptors are coupled to the Gs-protein and thus mediate increases in cyclic AMP ŽcAMP. concentration w23x. Since an EP2 agonist, butaprost, had no effect on the Tco after injection into the LCV w13x and the A3V and the neighboring basal forebrain in the present study, it is unlikely that EP4 receptors as well as EP2 receptors are involved in PGE 2-induced hyperthermia. Similarly, an EP3 receptor agonist, M & B28767, which is known to decrease the forskolin-stimulated cAMP level in cells expressing EP3 receptors w4x, failed to affect Tco . These findings do not support the possibility of the crucial involvement of the adenylate cyclase-cAMP system in PGE 2-induced hyperthermia. Previous studies on the microinjection of dibutyryl cAMP into the anterior hypothalamus revealed controversial findings, i.e., one study showed hyperthermia to be associated with hyperactivity w3x while the other showed hypothermia to be followed by hyperthermia which was probably due to convulsions w5x. An in situ hybridization study showed that EP1 receptor transcripts were found in nerve cells only in the PVN and the supraoptic nucleus ŽSON. in the hypothalamus of mice w1x. In contrast, the EP3 receptor mRNAs were widely distributed in the mouse brain and hybridization signals were found in various sites in the hypothalamus including the A3V and the MPO w22x. However, in the present study using the rat, no changes in Tco occurred after injection of the EP1 receptor agonist into the PVN and the SON, or after injection of the EP3 receptor agonist into the A3V and adjacent areas. Hyperthermia was obtained only when the EP1 agonist was injected into the A3V and the adjacent preoptic area, where no EP1 receptor transcripts were found in the mouse w1x. This might simply be due to a species difference in the distribution of EP1 receptors in the brain. Alternatively, it may be the result of technical problems in demonstrating the tissue distribution of EP1 receptors, since it has been pointed out that the expression of EP1 receptors is generally low in all tissues in comparison with other subtypes of EP receptors. Further study is called for using more sensitive methods to determine whether or not the A3V and its adjacent areas actually have EP1 receptors in the rat.
Acknowledgements This study was supported in part by Grants-in-Aid for Scientific Research Nos. 06454153 and 06557006 Žto T.H.. and grants from the Naito Foundation 93-183 Žto T.H.. and 95-185 Žto T.O... This experiment was reviewed by the Committee of Ethics on Animal Experiments in the Fac-
ulty of Medicine, Kyushu University and was carried out under the control of the Guidelines for Animal Experiments of the Faculty of Medicine, Kyushu University and the Law ŽNo. 105. and Notification ŽNo. 6. of the Government.
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