Excitatory amino acid receptor activation in the raphe pallidus area mediates prostaglandin-evoked thermogenesis

Neuroscience 122 (2003) 5–15

EXCITATORY AMINO ACID RECEPTOR ACTIVATION IN THE RAPHE PALLIDUS AREA MEDIATES PROSTAGLANDIN-EVOKED THERMOGENESIS C. J. MADDEN* AND S. F. MORRISON

sympathetic, glutamate, medial preoptic area.

Neurological Sciences Institute, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, OR 97006-3448, USA

Prostaglandin has been suggested to play an essential role in the generation of the acute phase component of the febrile response (Cranston and Rawlins, 1972; Morimoto et al., 1988; Scammell et al., 1998; Ushikubi et al., 1998). Although the specific area responsible for prostaglandinevoked fever has been controversial (Stitt, 1991), several findings support a key role for medial preoptic area (MPA) neurons in mediating the febrile response to prostaglandin. Several subtypes of prostaglandin receptors are located within the MPA (Nakamura et al., 1999; Ek et al., 2000; Oka et al., 2000) and cells recorded in vitro in MPA alter their firing rate in response to prostaglandin E2 (PGE2; Matsuda et al., 1992; Boulant, 2000). The expression of Fos protein has been observed in the MPA in response to exogenous pyrogens (Sagar et al., 1995; Elmquist et al., 1996; Ek et al., 2000; Oka et al., 2000) and fever can be elicited by injection of prostaglandin directly into the MPA (Feldberg and Saxena, 1971; Stitt, 1973, 1991; Williams et al., 1977; Oka et al., 1997). While at least some portion of the febrile response is mediated by prostaglandin acting within the MPA, the neural pathways through which MPA neurons effect changes in sympathetically mediated thermogenesis are poorly understood. Recent observations suggest that neurons in the rostral raphe pallidus area (RPa) are an essential site of synaptic integration in the central pathways mediating the febrile response in rodents. Neurons in the RPa have been implicated in the control of sympathetic outflow to brown adipose tissue (BAT) and the activation of BAT contributes significantly to the thermogenic component of fever (Fyda et al., 1991; Rothwell, 1992). Retrograde tracing studies in which pseudorabies virus was injected into BAT indicate that the RPa contains sympathetic premotor neurons involved in the control of BAT function (Bamshad et al., 1999; Oldfield et al., 2002; Cano et al., 2003). Blockade of GABAergic inhibitory inputs to the RPa increases BAT SNA and thermogenesis (Morrison et al., 1999). In addition, microinjection of the GABAA receptor agonist, muscimol into the RPa reverses or prevents the sympathetically mediated increase in BAT thermogenesis evoked by central administration of PGE2 (Nakamura et al., 2002; Morrison, 2003). Together, these observations have led to the hypothesis that the RPa contains sympathetic premotor neurons controlling BAT thermogenesis which are tonically inhibited by GABAergic inputs (Morrison et al., 1999) and that during the febrile response, a PGE2-triggered mech-

Abstract—To investigate the role of excitatory amino acid neurotransmission within the rostral raphe pallidus area (RPa) in thermogenic and cardiovascular responses, changes in sympathetic nerve activity to brown adipose tissue (BAT), BAT temperature, expired CO2, arterial pressure, and heart rate were recorded after microinjection of excitatory amino acid (EAA) receptor agonists into the RPa in urethan-chloralose-anesthetized, ventilated rats. To determine whether EAA neurotransmission within the RPa is necessary for the responses evoked by disinhibition of the RPa or by prostaglandin E2 acting within the medial preoptic area, BAT sympathetic nerve activity, BAT temperature, expired CO2, arterial pressure, and heart rate were measured during these treatments both before and after blockade of EAA receptors within the RPa. Microinjection of EAA receptor agonists into the RPa resulted in significant increases in all measured variables; these increases were attenuated by prior microinjection of the respective EAA receptor antagonists into the RPa. Microinjection of prostaglandin E2 into the medial preoptic area or microinjection of bicuculline into the RPa resulted in respective significant increases in BAT sympathetic nerve activity (ⴙapproximately 190% and ⴙapproximately 235% of resting levels), in BAT temperature (approximately 1.8 °C and approximately 2 °C), in expired CO2 (approximately 1.1% and approximately 1.1%), and in heart rate (approximately 97 beats per minute (bpm) and approximately 100 bpm). Blockade of ionotropic EAA receptors within the RPa by microinjection of kynurenate completely reversed the prostaglandin E2 or bicuculline-evoked increases in all of the measured variables. Blockade of either N-methyl-D-aspartate (NMDA) receptors or non-NMDA receptors alone resulted in marked attenuations of the prostaglandin E2-evoked effects on all of the measured variables. These data demonstrate that activation of an EAA input to the RPa is necessary for the BAT thermogenic and the cardiovascular effects resulting from the actions of prostaglandin E2 within the medial preoptic area or from the disinhibition of local neurons in the RPa. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: fever, thermoregulation, brown adipose tissue, *Corresponding author. Tel: ⫹1-503-418-2671; fax: ⫹1-503-4182501. E-mail address: [email protected] (C. J. Madden). Abbreviations: AP, arterial pressure; AP5, aminophosphonopentanoic acid; BAT, brown adipose tissue; Bic, bicuculline; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DMH, dorsomedial hypothalamic nucleus; EAA, excitatory amino acid; EP, prostaglandin type E; KA, kainic acid; Kyn, kynurenic acid; MPA, medial preoptic area; NMDA, N-methyl-D-aspartate; PAG, periaqueductal gray; PGE2, prostaglandin E2; RPa, rostral raphe pallidus area; SNA, sympathetic nerve activity.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00527-X

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anism in MPA effectively relieves the tonic inhibition of BAT sympathetic premotor neurons to produce activation of BAT thermogenesis (Nakamura et al., 2002; Morrison, 2003). However, disinhibition of a neuronal population will not result in an increase in neuronal activity without a concomitant or preexisting source of excitation (be it synaptic or intrinsic). The present study was designed to determine whether activation of either of the two main classes of ionotropic excitatory amino acid (EAA) receptors in the RPa can elicit sympathetically mediated BAT thermogenesis and if so whether the sympathetically mediated BAT thermogenesis evoked by disinhibition of the RPa or by injection of PGE2 into the MPA is dependent upon activation of these receptors.

EXPERIMENTAL PROCEDURES Male Sprague-Dawley rats (Charles River, Indianapolis, IN, USA; n⫽23) weighing 250 – 450 g were given ad libitum access to standard rat chow and water in a colony room maintained at 22–23 °C and kept on a 12-h light/dark cycle. Rats were anesthetized with isoflurane (2–3% in oxygen) and implanted with femoral arterial and venous catheters and transitioned to urethan and chloralose anesthesia (500 mg/kg and 80 mg/kg i.v., respectively) over a 10-min period. In order to record arterial pressure (AP) and HR, the arterial catheter was attached to a pressure transducer (Cobe). The trachea was cannulated, and the animals were paralyzed with D-tubocurarine (0.5 mg i.v., supplemented with 0.1 mg every hour) and ventilated (tidal volume: 1 ml/100 g body weight, 60 cycles per min) with 100% oxygen. Mixed expired CO2 was monitored using a capnometer (model 2200; Traverse Medical Monitors). Colonic temperature was monitored using a copperconstantan thermocouple (Physitemp, Clifton, NJ, USA) inserted 6 cm into the rectum and core body temperature was maintained between 37 °C and 38 °C with a heating plate and a heat lamp. Animals were placed in a stereotaxic instrument with the incisor bar positioned 4 mm below the interaural line. For microinjections into the MPA, small portions of the parietal and frontal bones were removed. Based on the coordinates at which microinjection of prostaglandin elicited hyperthermia (Williams et al., 1977), our coordinates for injections into the MPA were 0.0 – 0.3 mm caudal and 0.8 mm lateral to bregma and 8.0 mm ventral to dura. For microinjections into the RPa, a small portion of the interparietal bone was removed. The coordinates for injections into the RPa were 2.8 –3.2 mm caudal to lambda, 0.0 mm lateral to lambda and 9.2–9.5 mm ventral to dura. These coordinates correspond to the area from which postganglionic sympathetic nerves innervating BAT can be activated by electrical stimulation at the lowest intensity. Glass micropipettes (outer tip diameter, 20 –30 ␮m) were used for all microinjections which were given over a 10 –20 s period using a pressure injection system (model IIe; Toohey Co). All drugs were obtained from Sigma (St. Louis, MO, USA) except isofluorane which was obtained from Abbott Laboratories (Chicago, IL, USA). Postganglionic BAT sympathetic nerve activity (SNA) was recorded from the central cut end of a nerve bundle innervating the interscapular fat pad. A nerve bundle was isolated from the ventral surface of the right interscapular fat pad after dividing it along the midline and reflecting it laterally. The nerve bundle was placed on bipolar hook electrodes, and covered in mineral oil. Nerve activity was filtered (1–300 Hz) and amplified (50,000) with a Cyberamp 380 (Axon Instruments, Union City, CA, USA). The BAT temperature was monitored with a copper-constantan thermocouple (Physitemp) inserted beneath the intact, left interscapular fat pad. Physiological variables were digitized (Digidata

1320A; Axon Instruments) and recorded onto a PC hard drive using AxoScope acquisition software (Axon Instruments). In the first experimental protocol, each animal (n⫽5) received the following four-part series of microinjections into the RPa to characterize the responses to agonist application and the antagonist selectivity in blocking these responses. (A) Individual microinjections of the selective glutamate receptor agonists, N-methylD-aspartate (NMDA; 12 pmol/60 nl) and kainic acid (KA; 1.5 pmol/ 60 nl) were always given first and were separated by at least 5 min. The subsequent three series of microinjections were delivered in random order. (B) Individual microinjections of NMDA and KA were given 5–10 min after a microinjection of the NMDA receptor-selective antagonist, aminophosphonopentanoic acid (AP5; 300 pmol/60 nl), into the RPa area. (C) Individual microinjections of NMDA and KA were given 5–10 min after a microinjection of the non-NMDA receptor-selective antagonist, 6-cyano7-nitroquinoxaline-2,3-dione (CNQX; 120 pmol/60 nl), into the RPa area. Due to technical difficulties one animal did not undergo the CNQX protocol. (D) Individual microinjections of NMDA and KA were given 5–10 min after a microinjection of the non-selective, ionotropic EAA receptor antagonist, kynurenic acid (Kyn; 6 nmol/60 nl), into the RPa. Due to technical difficulties one animal did not receive a microinjection of Kyn followed by NMDA and KA. In addition to the test microinjection following each antagonist, individual microinjections of NMDA or KA were often given to characterize the duration of action of the antagonist and subsequent microinjections of other antagonists were not performed until both the NMDA and KA responses had recovered. In the second experimental protocol, a separate set of rats (n⫽6) received two microinjections of bicuculline (Bic; 30 pmol/ 60 nl) into the RPa. These two Bic injections were separated by approximately 2 h and were followed after 5 min by either (A) an experimental trial consisting of an 80 nl microinjection of the non-selective ionotropic EAA receptor antagonist, Kyn (100 mM), or (B) a control trial consisting of either placement of a microinjection pipette with no injection or with an injection of saline vehicle (150 mM, 60 nl). The four microinjections in each animal were made at the same site within the RPa and the order of experimental and control trials was varied among rats. No differences were observed between the responses to saline microinjections and the responses to no injection; therefore, these data were combined as a single control group in all analyses. In the third experimental protocol, rats (n⫽12) received a unilateral microinjection of PGE2 (60 ng, 60 nl) into the MPA. Twenty minutes after microinjection of PGE2 into the MPA, a 60 nl microinjection of one of the following was made into the RPa: saline vehicle (150 mM), Kyn (100 mM), the NMDA-selective EAA receptor antagonist, AP5 (5 mM), or the non-NMDA selective EAA receptor antagonist, CNQX (2 mM). The effects of the EAA receptor antagonists, AP5 and CNQX, were of a sufficiently short duration for the PGE2 response to fully recover following administration of these antagonists, allowing, in some cases, multiple tests of the effect of EAA receptor antagonism of RPa neurons on the BAT SNA response to a single microinjection of PGE2 into MPA. In these instances a period of at least 20 min was allowed between microinjections of EAA receptor antagonists, during which the BAT SNA response to PGE2 had recovered fully before an additional EAA receptor antagonist was administered. The individual effects to such administrations of more than one EAA receptor antagonist into RPa within a single PGE2 response did not differ from those in which only one EAA receptor antagonist was injected after microinjection of PGE2 into MPA. The doses of EAA receptor antagonists used in these experiments were selected on the basis of those shown to be both selective against their respective agonists and effective in previous studies involving microinjections into medullary regions controlling sympathetic functions (Kiely and Gordon, 1994; Morino et al., 1994; McManigle et al., 1995).

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Table 1. Effects of KA injected into the RPa alone or after injection of EAA antagonists into this areaa

BAT SNA (% BL) BAT temp (°C) Expired CO2 (%) HR (bpm)

Baseline

KA

Baseline after AP5

KA after AP5

Baseline after CNQX

KA after CNQX

Baseline after Kyn

KA after Kyn

100 35.6⫾0.4 5.2⫾0.4 392⫾28

⫹187⫾46* ⫹1.0⫾0.2* ⫹0.5⫾0.1* ⫹55⫾9*

100 35.6⫾0.2 5.0⫾0.2 409⫾25

⫹162⫾39* ⫹1.0⫾0.2* ⫹0.5⫾0.1* ⫹45⫾7*

100 35.4⫾0.2 4.8⫾0.2 391⫾25

⫹19⫾9# ⫺0.1⫾0.1# ⫹0.2⫾0.0* ⫹38⫾9*

100 35.3⫾0.1 5.0⫾0.3 395⫾26

⫹9⫾4# 0.0⫾0.1# ⫹0.3⫾0.0* ⫹27⫾4*

a

Values are mean⫾S.E.M; control, n⫽5; AP5, n⫽5; CNQX, n⫽4; Kyn, n⫽4. Baseline (BL) values do not differ among any of the trials. HR, heart rate. * Indicates P⬍0.05, compared to the respective baseline condition. # Indicates P⬍0.05 compared to the peak response evoked by an EAA agonist without prior injection of an EAA antagonist.

At the conclusion of each experiment, the final RPa microinjection site and that in MPA were marked by retracting the microinjection pipettes vertically, filling them with 2% Fast Green dye, repositioning them to the vertical coordinates of the microinjection sites, and electrophoretically depositing (20 ␮A anodal direct current for 10 min) the dye. Rats were perfused transcardially with a 10% paraformaldehyde solution. The brains were removed, post-fixed for 12–24 h and sectioned at 60 ␮m. Brain sections containing Fast Green dye deposits were mounted on slides and photographed using a digital microscope camera (Kodak DC 220). Photomicrographs were downloaded to a PC computer and assembled using Adobe Photoshop software; only brightness, sharpness, and contrast were adjusted. The locations of microinjection sites were plotted on atlas drawings (Paxinos and Watson, 1986). BAT SNA amplitude was derived from autospectral analysis using Datapac 2000 software (Run Technologies Co). For each experimental condition, the average autospectrum of a 101.6-s BAT SNA data segment was computed by dividing the data into 20 equal segments, computing the autospectrum for each of these 5.08 s segments and then averaging these individual autospectra (i.e. the average power at each frequency value is the mean of the powers at that frequency value in the 20 individual autospectra). The root mean square amplitude of the BAT SNA for each experimental condition was taken as the square root of the total power in the 0.19 –20 Hz band of the averaged autospectrum. Resting (baseline) values were obtained from the data during the 2-min period immediately prior to microinjection of chemical agents into RPa or of PGE2 into MPA. Response values of BAT SNA were obtained from the data during the 2-min period of peak change in BAT SNA following microinjection of chemical agents into RPa or of PGE2 into MPA. All statistics were performed using Systat software (version 10; SPSS Inc.). Values are expressed as mean⫾S.E.M. Statistical significance was assessed with the Student’s paired t-test and Bonferroni correction or ANOVA with Bonferroni post-hoc testing where appropriate, significance level was set at P⬍0.05.

RESULTS EAA agonists in RPa Resting values of BAT temperature, expired CO2, and HR for animals receiving microinjections of KA and NMDA into

the RPa are shown in Tables 1 and 2, respectively. Under resting conditions, BAT SNA exhibited only a few bursts, resulting in low amplitude values. There were no differences between trials in the mean resting values of any of the measured variables. BAT SNA increased immediately following microinjection of NMDA or KA into the RPa and reached peak levels within 2 min (Fig. 1A–C). Activation of EAA receptors in the RPa with NMDA or KA elicited increases in BAT temperature and expired CO2 which followed and paralleled the increase in BAT SNA (Fig. 1A–C). In addition, activation of EAA receptors (either NMDA or non-NMDA) within the RPa elicited an immediate and marked tachycardia (Fig. 1A–C). Although activation of EAA receptors within the RPa did not consistently change mean AP, when present, these consisted of small (⬍20 mm Hg) increases in mean AP (data not shown). Group mean data for the EAA receptor agonist-evoked responses are presented in Tables 1 and 2. Prior blockade of ionotropic EAA receptors by microinjection of Kyn into the RPa prevented the increases in BAT SNA and BAT temperature evoked by microinjection of either KA or NMDA into RPa (Fig. 1D; Tables 1 and 2). Kyn administered into the RPa also prevented the increases in expired CO2, and HR evoked by microinjection of NMDA into RPa, although Kyn did not block the increases in expired CO2 and HR evoked by microinjection of KA into RPa. Prior selective blockade of local NMDA receptors by microinjection of AP5 into the RPa significantly attenuated the NMDA-evoked increases in all measured variables but did not attenuate the KA-evoked increases in any of the measured variables (Fig. 1B; Tables 1 and 2). Conversely, prior selective blockade of nonNMDA receptors by microinjection of CNQX into the RPa significantly attenuated the KA-evoked increases in BAT SNA and BAT temperature but did not attenuate the NMDA-evoked increases in any of the measured variables

Table 2. Effects of NMDA injected into the RPa alone or after injection of EAA antagonists into this areaa

BAT SNA (% BL) BAT temp (°C) Expired CO2 (%) HR (bpm) a

Baseline

NMDA

Baseline after AP5

NMDA after AP5

Baseline after CNQX

NMDA after CNQX

Baseline after Kyn

NMDA after Kyn

100 35.3⫾0.3 5.1⫾0.3 392⫾34

⫹227⫾47* ⫹1.3⫾0.2* ⫹0.7⫾0.1* ⫹56⫾4*

100 35.5⫾0.2 5.0⫾0.2 411⫾23

⫹14⫾8# ⫹0.0⫾0.0# ⫹0.1⫾0.0# ⫹12⫾3*#

100 35.2⫾0.2 4.7⫾0.2 388⫾40

⫹239⫾71* ⫹1.1⫾0.1* ⫹0.7⫾0.1* ⫹56⫾13*

100 35.3⫾0.2 5.0⫾0.3 402⫾27

⫹4⫾2# ⫹0.2⫾0.1# ⫹0.1⫾0.1# ⫹19⫾8#

Values are mean⫾S.E.M.; control, n⫽5; AP5, n⫽5; CNQX, n⫽4; Kyn, n⫽4. HR, heart rate. * Indicates P⬍0.05, compared to the respective baseline (BL) condition. # Indicates P⬍0.05 compared to the peak response evoked by an EAA agonist without prior injection of an EAA antagonist.

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Fig. 1. Thermogenic, metabolic and cardiovascular responses evoked by injection of NMDA or KA into the RPa are differentially blocked by microinjection of several EAA receptor antagonists into RPa. A. microinjection of NMDA or KA into RPa (open arrows) markedly increased BAT SNA, BAT temperature, expired CO2, and heart rate (HR). B. Microinjection of AP5 into RPa (filled arrow) completely prevented the NMDA-evoked increases in BAT SNA, BAT temperature, and expired CO2, but did not prevent the KA-evoked increases in these variables. C. Microinjection of CNQX into RPa (filled arrow) prevented the KA-evoked increase in BAT SNA, BAT temperature, and expired CO2, but did not prevent the NMDA-evoked increases in these variables. However, microinjection of CNQX in RPa did not prevent the KA-evoked increase in HR. D. Microinjection of Kyn into RPa (filled arrow) prevented the NMDA-evoked and KA-evoked responses. Vertical scale bar for BAT SNA⫽400 ␮V peak-to-peak.

Abbreviations used in the figures 3V 4V 7 ac DC icp LPGi LPO MnPO MVe ox

third ventricle fourth ventricle facial nucleus anterior commissure dorsal cochlear nucleus inferior cerebellar peduncle lateral paragigantocellular nucleus lateral preoptic area median preoptic nucleus medial vestibular nucleus optic chiasm

PrH py RMg ROb RPa sol Sp5 Sp50 SpVe VCP

prepositus hypoglossal nucleus pyramidal tract raphe magnus raphe obscurus raphe pallidus nucleus of the solitary tract spinal trigeminal tract spinal trigenminal nucleus spinal vestibular nucleus ventral cochlear nucleus posterior

C. J. Madden and S. F. Morrison / Neuroscience 122 (2003) 5–15

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Table 3. Effects of Kyn or vehicle into RPa following disinhibition of RPa neuronsa Control

BAT SNA (% baseline) BAT temp (°C) expired C02 (%) HR (bpm)

Kynurenate

Baseline

Bic RPa

Control RPa

Baseline

Bic RPa

Kyn RPa

100 34.1⫾0.3 5.5⫾0.2 381⫾21

⫹242⫾43 ⫹2.3⫾0.5 ⫹1.2⫾0.3 ⫹99⫾22

⫺98⫾37 ⫺0.3⫾0.2 ⫺0.4⫾0.2 ⫺35⫾14

100 34.4⫾0.3* 5.5⫾0.2 376⫾13

⫹226⫾46 ⫹1.9⫾0.4 ⫹1.0⫾0.2 ⫹97⫾20

⫺185⫾49* ⫺1.1⫾0.2* ⫺0.8⫾0.2* ⫺75⫾10*

a Values are given for BAT thermogenic, metabolic and cardiovascular variables. Columns labeled “Baseline” provide values under resting conditions. The peak increases in the thermogenic and cardiovascular variables within 5 min of the microinjection of Bic into the RPa are provided in the columns labeled “Bic RPa.” The maximal decrease (within 15 min of the Bic microinjection) from the peak Bic response evoked by a microinjection of saline (or no microinjection) into the RPa are presented in the column labeled “Control RPa.” The column labeled “Kyn RPa” provides the maximal decreases from the peak responses of the measured variables within 15 min of the microinjection of Bic into the RPa when a microinjection of the excitatory amino acid antagonist, Kyn was made into RPa at 5 min after the microinjection of Bic. Values are mean⫾S.E.M. (n⫽6); * indicates P⬍0.05, compared to the control trials (i.e. Control RPa). HR, heart rate.

(Fig. 1C; Tables 1 and 2). Microinjection of CNQX into RPa did not block the increases in expired CO2 and HR (Fig. 1C; Table 1) evoked by KA administration into RPa, although the rate of rise in the HR response to KA was markedly reduced (compare Fig. 1A, B and Fig. 1C). Blockade of EAA receptors within RPa following disinhibition of RPa Resting values of BAT temperature, mixed expired CO2, and HR for animals receiving a microinjection of Bic into the RPa are shown in Table 3. Under resting conditions, BAT SNA exhibited only a few bursts, resulting in low amplitude values. There was no difference in the mean resting BAT SNA values between the control trials and those trials in which Kyn was microinjected into the RPa. BAT SNA increased immediately following microinjection of Bic into the RPa, reaching peak levels within approximately 5 min (Fig. 2A, B; peak increases: ⫹212%, ⫹169%, respectively). Accompanying the increase in BAT SNA, Bic application to RPa elicited a rise in BAT temperature (Fig. 2A, B; peak increases: ⫹1.7 °C, ⫹1.2 °C, respectively) and increases in expired CO2 (Fig. 2A, B; peak increases: ⫹0.5% in each), and HR (Fig. 2A, B; peak increases: ⫹81 bpm, ⫹54 bpm, respectively). Group data for these evoked responses are presented in Table 3. Blockade of EAA receptors within the RPa produced an immediate reversal of the BAT thermogenic and HR responses evoked by disinhibition of RPa neurons with Bic (Fig. 2B). We characterized the effects of EAA receptor blockade both on response duration and on response amplitude. The BAT SNA response to Bic microinjection into RPa was characterized by a sustained, high amplitude bursting pattern (Fig. 2A) which terminated relatively abruptly as the effects of the Bic dissipated. Blockade of EAA receptors in RPa significantly reduced the duration of the BAT thermogenic response to prior disinhibition of RPa neurons. In control trials, the mean time after microinjection of Bic into the RPa for BAT SNA amplitude to decline to 50% of the peak response amplitude was 20⫾4 min (Fig. 2A; latency to 50% of peak: 29 min). The mean control duration of the increase in BAT SNA (i.e. time from microinjection of Bic to a return to control level of BAT SNA) following disinhibition of RPa neurons was 28⫾4 min

(Fig. 2A; response duration: 35 min). In trials in which Kyn was microinjected into RPa 5 min after disinhibition of RPa neurons with Bic, the BAT SNA response had declined to 50% of the peak response level by 10⫾3 min (Fig. 2B; latency to 50% of peak: 8 min) and BAT SNA had returned to control levels by 18⫾4 min (Fig. 2B; response duration: 20 min). Microinjection of Kyn into RPa significantly (P⬍0.05) reduced both measures of the duration of the evoked excitation of RPa thermogenic neurons following blockade of local GABAA receptors. Blockade of EAA receptors in RPa also significantly reduced the magnitude of the BAT thermogenic responses to prior disinhibition of RPa neurons. As illustrated in Fig. 2, at 15 min after the microinjection of Bic, the increase in BAT SNA was lower (⫹21% of pre-Bic control level; Fig. 2B) after Kyn microinjection into RPa than in the control trial (⫹199% of pre-Bic control level; Fig. 2A). Similarly, the declines at 15 min from the peaks in BAT temperature, HR and expired CO2 evoked by microinjection of Bic into the RPa were greater after microinjection of Kyn into the RPa (Fig. 2B, ⫺1.3 °C, ⫺79 bpm, ⫺0.8%, respectively) compared with control trials (Fig. 2A, ⫺0.1 °C, ⫺16 bpm, ⫺0.1%, respectively). The group means for these responses are presented in Table 3 and indicate that blockade of EAA receptors in RPa significantly (P⬍0.05) reduced the magnitudes of the BAT thermogenic responses elicited by disinhibition of neurons in RPa. Blockade of EAA receptors in RPa following PGE2 in MPA Resting values of HR, BAT temperature and mixed expired CO2 for animals receiving a microinjection of PGE2 into the MPA are shown in Table 4. Under resting conditions, BAT SNA was typically low with only a few, small-amplitude bursts being recorded every minute (Fig. 3) Unilateral microinjection of PGE2 into the MPA produced significant (P⬍0.01) increases in BAT thermogenesis, expired CO2 and HR (Fig. 3A; Table 4). These responses were rapid in onset, with initial increases beginning within minutes and typical peak responses occurring within 15 min (Fig. 3A) of the injection of PGE2. The PGE2-evoked responses were also long lasting: in rats receiving saline microinjections in the RPa, the response levels remained within 20% of peak

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Fig. 2. Thermogenic, metabolic and cardiovascular responses evoked by disinhibition of neurons within the RPa are reversed by microinjection of the EAA receptor antagonist, Kyn, into RPa. A. microinjection of Bic into RPa (filled arrows) markedly increased BAT SNA, BAT temperature, expired CO2, and heart rate (HR). Microinjection of Kyn into RPa (open arrow) completely reversed the Bic-evoked responses. Vertical scale bar for BAT SNA⫽80 ␮V peak-to-peak.

values for at least 1 h (Fig. 3A). In the responses illustrated in Fig. 3A and B, PGE2 produced peak respective increases in BAT SNA of ⫹222% and ⫹212%, in BAT temperature of ⫹0.9 °C and ⫹2.0 °C, in mixed expired CO2 of ⫹1.0% and ⫹0.9% and in HR of ⫹107 bpm and ⫹67 bpm. The increases in BAT SNA, BAT temperature, expired CO2 and HR produced by PGE2 administration into the MPA was significantly attenuated by microinjection of EAA receptor antagonists into RPa (Fig. 3B), but not by microinjection of saline vehicle into the RPa (Fig. 3A; Table 4). BAT SNA began to decrease immediately following microinjection of AP5, CNQX, or Kyn into RPa, with maximum

reductions in BAT SNA occurring at 1⫾0 min, 6⫾3 min and 3⫾1 min after their respective microinjections. Microinjection of AP5 into RPa elicited a maximum fall in BAT SNA that was ⫺57⫾12% of the PGE2-stimulated level of BAT SNA during the 2 min prior to microinjection of AP5. Microinjection of CNQX into RPa produced a maximum attenuation of ⫺65⫾10% of the PGE2-evoked level of BAT SNA (Fig. 3B; Table 4). Microinjection of Kyn into the RPa completely reversed the PGE2-evoked increase in BAT SNA, producing a maximum attenuation of 100% from the PGE2-evoked level (Fig. 3B; Table 4) in all cases. Similarly, the increase in BAT temperature evoked by PGE2 administration was attenuated by approximately ⫺40% by microinjection of AP5 or CNQX into the RPa (Fig. 3B; Table 4), whereas injection of Kyn into the RPa completely reversed the PGE2-evoked increase in BAT temperature (Fig. 3B; Table 4). The times to the nadirs in BAT temperature following microinjections of AP5 or CNQX were 8⫾4 min and 7⫾2 min, respectively. Following microinjection of Kyn into RPa, BAT temperature returned to pre-PGE2 levels within 15 min. Administration of AP5 or CNQX into the RPa attenuated the PGE2-evoked increase in expired CO2 by ⫺46⫾14% (at 3⫾1 min) and ⫺44⫾13% (at 6⫾1 min), respectively, whereas Kyn completely reversed the PGE2-evoked increase in expired CO2 within 10 min (Fig. 3B; Table 4). The tachycardic response to PGE2 was also attenuated by AP5 and by CNQX microinjected into the RPa (⫺50⫾18% and ⫺39⫾9%, respectively) and was eliminated by microinjection of Kyn into the RPa (Fig. 3B; Table 4). The maximum reduction in the HR response produced by the EAA receptor antagonists occurred 7⫾3 min, 5⫾1 min, and 19⫾4 min after microinjections of AP5, CNQX or Kyn, respectively. The locations of the microinjection sites within the RPa and MPA are shown in Fig. 4. The approximate centers of the injection sites targeting the RPa were either located within the RPa at the level of the caudal half of the facial nucleus, within the central portion of the caudal raphe magnus or along the dorsomedial border of the pyramidal tracts. The approximate centers of the injection sites targeting the MPA were located primarily dorsal and medial to the anteroventral preoptic nucleus within the boundaries of the MPA (Paxinos and Watson, 1986).

DISCUSSION The present study is the first to demonstrate that activation of either NMDA or non-NMDA ionotropic EAA receptors within the RPa stimulates BAT SNA and BAT thermogenesis. Our results also indicate that activation of both NMDA and non-NMDA ionotropic EAA receptors within the RPa is necessary for the thermogenic and HR responses evoked by disinhibition of RPa neurons or by PGE2 administration into the MPA of anesthetized rats. These results support a model for the central regulation of BAT thermogenesis in which BAT sympathetic premotor neurons within RPa receive both a tonically active, GABA-mediated inhibition as well as an EAA-mediated excitatory input, the latter being primarily responsible for the excitatory drive to BAT sym-

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Table 4. Effect of EAA receptor antagonists into RPa on the thermogenic responses following PGE2 into MPAa Saline

BAT SNA (% baseline) BAT temp (°C) Expired CO2 (%) HR (bpm)

AP5

Baseline PGE2 MPA

Saline RPa

100

381⫾65 100

347⫾64*

Baseline

CNQX PGE2 MPA

AP5 RPa

263⫾35* 167⫾17#

Kyn

Baseline PGE2 MPA

CNQX RPa

100

159⫾21# 100

269⫾37*

Baseline PGE2 MPA 281⫾23*

Kyn RPa 105⫾5#

33.6⫾0.6 34.9⫾0.7* 34.8⫾0.6 32.9⫾0.3 34.7⫾0.6* 34.2⫾0.5# 33.0⫾0.3 35.2⫾0.4* 34.4⫾0.5 33.0⫾0.2 34.9⫾0.4* 32.9⫾0.2# 4.9⫾0.5 5.8⫾0.4* 5.9⫾0.4 4.9⫾0.2 6.1⫾0.5* 5.6⫾0.4* 5.1⫾0.2 6.5⫾0.4 6.0⫾0.4# 5.1⫾0.1 6.2⫾0.3* 4.9⫾0.1# 364⫾9

458⫾18*

456⫾13

366⫾21

471⫾11* 414⫾28

369⫾16 471⫾8*

433⫾14#

375⫾18 462⫾12*

366⫾15#

a Values for physiological variables under resting conditions, peak responses within 30 min of the microinjection of PGE2 into the MPA and at the nadir after subsequent microinjection of an EAA receptor antagonist or saline into the RPa. Values are mean⫾S.E.M.; Saline, n⫽6; AP5, n⫽4; CNQX, n⫽5; Kyn, n⫽5. * Indicates P⬍0.05, increase compared to the baseline value. # Indicates P⬍0.05, reduction compared to the peak PGE2-evoked response. HR, heart rate.

pathetic premotor neurons when their tonic inhibition is relieved either by blockade of GABAA receptors in RPa or by the actions of PGE2 on thermoregulatory pathways emanating from the MPA. Our data provide no information on the possible location of the glutamatergic neurons that provide excitation to RPa thermogenic neurons since our microinjection experiments could not distinguish among the possible roles of (a) glutamate inputs to RPa from neurons outside of RPa, (b) glutamatergic interneurons within RPa or (c) glutamatergic inputs to local interneurons that may excite RPa projection neurons with a non-glutamatergic transmitter. By determining one of the principal excitatory neurotransmitters responsible for the discharge of BAT sympathetic premotor neurons in RPa following PGE2 application to MPA, the present data extend earlier studies in which inhibition of RPa neurons with the GABAA agonist muscimol (Nakamura et al., 2002; Morrison, 2003), indicated the critical role of RPa neurons in mediating the BAT thermogenic component of the PGE2-evoked febrile response. We interpret the PGE2-evoked increases in BAT temperature to be secondary to the increase in BAT SNA, since sympathetic activation has been shown to drive increases in BAT thermogenesis (Fyda et al., 1991) and in the present study the time course of the increase in BAT temperature always followed and paralleled that of the BAT SNA. It is noteworthy that the BAT temperature (approximately 34 –35 °C) was substantially lower than colonic temperature (maintained between 37 and 38 °C). We attribute this difference to the fact that BAT is superficially located and surgically exposed in our preparation. We also consider the augmented BAT metabolism necessary to increase BAT thermogenesis to be an important factor in the increase in expired CO2 following PGE2 administration. In this regard, central administration of PGE2 has been shown to result in an increase in oxygen consumption (indicative of an increased metabolic rate and resulting in an increase in CO2 production) and that this response is not blocked by prevention of the PGE2-evoked tachycardia or hypertensive response (Hoffman et al., 1986). These data do not preclude, however, a significant contribution to the increased expired CO2 from the augmented cardiac muscle metabolism accompanying the marked tachycardia

following PGE2. Indeed, the present study demonstrated that the increase in expired CO2 following stimulation of the RPa was not eliminated solely by preventing the BAT thermogenic response but also required blockade of the evoked tachycardia. Our finding that the Kyn-induced blockade of EAA receptors in RPa eliminated the tachycardia evoked either by disinhibition of neurons in RPa or by PGE2 administration in the MPA indicates that the RPa contains a population of neurons capable of exciting the sympathetic outflow to the cardiac sinoatrial node, that these neurons are under a tonically active GABAergic inhibition and that a significant fraction of their excitation during the febrile response is mediated by a glutamatergic excitatory input. Although our data do not directly address the function of the evoked tachycardia, we speculate that the evoked tachycardia provides a synergistic increase in cardiac output that accompanies activation of BAT thermogenesis to facilitate a rapid distribution of the heat generated in BAT to the rest of the body. It is noteworthy that tachycardia has also been shown to accompany the febrile response evoked by systemic administration of an endotoxin in both rat and human (Linthorst et al., 1995; Cabrera et al., 2000; Mullington et al., 2000). Additionally, activation of neurons in the dorsomedial hypothalamus (DMH), thought to mediate autonomic components of the stress response, also produces a simultaneous increase in BAT temperature (Zaretskaia et al., 2002) and heart rate (Zaretsky et al., 2003) and the latter has been attributed to activation of RPa neurons (Zaretsky et al., 2003). We cannot, however, eliminate the possibility that our microinjection approach evoked a nonspecific, simultaneous activation of pathways activating BAT thermogenesis and those increasing HR. In the current study, the tachycardia evoked by microinjection of KA into the RPa was unaffected by prior treatment with CNQX, whereas prior local treatment with CNQX did prevent the thermogenic response to microinjection of KA into RPa. This finding implies that two distinct populations of RPa neurons are responsible for the BAT thermogenic stimulation and for the tachycardia. A differential availability of CNQX to these two neuronal populations could account for the current results.

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Fig. 3. Reversal of the PGE2-evoked increases in BAT thermogenesis and heart rate by microinjection of EAA receptor antagonists into the RPa. A. microinjection of PGE2 into the MPA (filled arrow) markedly increased BAT SNA, BAT temperature, expired CO2, and heart rate (HR). Microinjection of saline vehicle into the RPa (open arrow) had no effect on any of the measured variables. Please note that due to the compressed time scale of this figure it is not possible to appreciate the decrease in the frequency of bursts of SNA in approximately the last 30 min of the response. B. in a different rat, microinjection of PGE2 into the MPA (filled arrow) increased BAT thermogenesis and HR. Microinjection of CNQX (first open arrow) or AP5 (second open arrow) into the RPa partially reversed the PGE2-evoked responses, whereas microinjection of Kyn into the RPa (third open arrow) completely eliminated the PGE2-evoked responses. Vertical scale bar⫽peak-to-peak BAT SNA of 100 ␮V in A and 80 ␮V in B.

Based on the observations that RPa neurons controlling BAT thermogenesis are tonically inhibited by GABAergic inputs (Morrison et al., 1999), that inhibition of RPa neurons prevents PGE2-evoked BAT thermogenesis, and that some MPA neurons containing GAD67, a marker for GABAergic neurons, project to the region of the RPa (Na-

kamura et al., 2002), it has been proposed that disinhibition of the RPa is responsible for PGE2-evoked BAT thermogenesis and fever (Nakamura et al., 2002). Our findings suggest that this model must be extended to include the addition of an EAA input to the RPa that provides the excitatory drive to BAT sympathetic premotor neurons that

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Fig. 4. Location of microinjection sites in the RPa and MPA. Upper panels: Representative photomicrographs illustrating the Fast Green dye deposits (arrows) at the sites of microinjections within the area of the RPa (left) and MPA (right). Lower panels: Locations of the microinjection sites for all 22 rats used in these studies plotted on atlas drawings of the rat medulla (bregma ⫺11.3 mm) and hypothalamus (bregma ⫺0.26 mm; Paxinos and Watson, 1986).

is essential for the PGE2-evoked increase in BAT thermogenesis. While our data do not allow us to distinguish among the following possibilities that could explain the relationship between the tonic GABAergic inhibition and the glutamatergic excitation that underlies the PGE2evoked thermogenic responses, their brief consideration provides the interpretation of our results. (A) Similar to the application of Bic to RPa, administration of PGE2 into the MPA could release RPa neurons from a tonic GABAergic inhibition, thereby revealing a tonically active, EAA-mediated excitation of RPa neurons that had been completely suppressed. (B) Administration of PGE2 into the MPA could produce an increased glutamate release onto RPa thermogenic neurons, thereby overcoming their tonic GABAergic inhibition. (C) PGE2-evoked thermogenic and cardiovascular responses could be mediated by the simultaneous withdrawal of a tonically active GABAergic input to RPa neurons and a concomitant release of glutamate from an input to the RPa that was not previously active.

An obvious issue arising from the present data is the source of the excitatory, glutamatergic input to the RPa. One possibility is that the PGE2-sensitive cells within the MPA send excitatory projections directly to the neurons of the RPa. Indeed, Nakamura et al. (2002) have demonstrated the existence of neurons within the MPA that project to the RPa and also contain prostaglandin type E (EP) receptors. In contrast, since the majority of EP3 receptor-immunoreactive cells within the preoptic area also exhibit GAD-67 mRNA, these authors proposed that administration of PGE2 to MPA activates BAT thermogenesis by inhibiting a tonically active, direct inhibitory input to RPa neurons from MPA (Nakamura et al., 2002). This hypothesis is weakened, however, by earlier demonstrations that transections of the neuraxis between the hypothalamus and the pons have no effect on BAT temperature (Rothwell, 1983; Shibata et al., 1987), whereas sectioning a tonically active inhibitory projection to RPa would be expected to increase BAT temperature (Rothwell et al., 1983;

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Shibata et al., 1987). Retrograde tracing of inputs to rostral RPa (Hermann et al., 1997; Nogueira et al., 2000) in conjunction with studies employing pseudorabies virus injections into BAT (Oldfield et al., 2002; Cano et al., 2003) suggests potential brain regions that could provide a thermogenic EAA input to the RPa: the preoptic area, the DMH, the paraventricular nucleus of the hypothalamus, the periaqueductal gray (PAG), the retrorubral fields, the lateral parabrachial nucleus, the rostroventrolateral medulla, and the nucleus of the solitary tract. Physiologically, activation of neurons in the lateral and ventral regions of the caudal PAG increases BAT temperature (Chen et al., 2002) and DMH neurons have been shown to play a significant role in the thermogenesis and tachycardia evoked by microinjection of PGE2 into the preoptic area (Zaretskaia et al., 2003). Whether neurons in either the caudal PAG or the DMH are a source of direct EAA input to BAT sympathetic premotor neurons in the RPa and how the release of EAA is stimulated by Bic application or PGE2 administration remain to be determined. Interestingly, the interaction between excitatory and inhibitory amino acid regulation of the discharge of the DMH neurons influencing heart rate (Soltis and DiMicco, 1991a, b) parallels that demonstrated here for RPa neurons regulating BAT SNA and thermogenesis. Together with the finding that disinhibition of DMH neurons also increases BAT thermogenesis (Zaretskaia et al., 2002), these results raise the possibility that an EAA-mediated excitation of neurons tonically inhibited by a variable GABAergic input may represent a common regulatory mechanism at multiple synaptic integration sites in the central pathway mediating fever. In summary, the present study demonstrates that activation of EAA receptors within the RPa is capable of driving sympathetically mediated thermogenic responses and that activation of these receptors is required for the thermogenic and accompanying cardiovascular effects resulting from microinjection of PGE2 into the MPA or from disinhibition of neurons within the RPa. Further studies will be required to determine the source(s) of the EAA inputs responsible for the excitation of the neurons in the RPa involved in thermogenic and tachycardic responses.

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(Accepted 3 July 2003)