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Microinjection of prostaglandin E2 and muscimol into the preoptic area in conscious rats: Comparison of effects on plasma adrenocorticotrophic hormone (ACTH), body temperature, locomotor activity, and cardiovascular function
Neuroscience Letters 397 (2006) 291–296
Microinjection of prostaglandin E2 and muscimol into the preoptic area in conscious rats: Comparison of effects on plasma adrenocorticotrophic hormone (ACTH), body temperature, locomotor activity, and cardiovascular function Dmitry V. Zaretsky, Joseph L. Hunt, Maria V. Zaretskaia, Joseph A. DiMicco ∗ Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA Received 18 October 2005; received in revised form 30 November 2005; accepted 12 December 2005
Abstract The preoptic area (POA) is thought to play an important role in thermoregulation and fever. Local application of prostaglandin E2 (PGE2) to this region elicits increases in core body temperature, heart rate, and plasma levels of adrenocorticotrophic hormone (ACTH). Similar effects on body temperature and heart rate have also been reported after local application of the GABAA receptor agonist muscimol to the preoptic area. The purpose of this study was to assess and compare the effects of microinjection of PGE2 and muscimol into the preoptic area in the same chronically instrumented conscious rats on plasma levels of ACTH. Injection of either PGE2 (150 pmol/100 nL) or muscimol (20 or 80 pmol/100 nL) into the same sites in the preoptic area evoked increases in body temperature, heart rate, blood pressure, and plasma levels of ACTH, while significant increases in locomotor activity were apparent only after muscimol. These data confirm and extend previous findings and support the notion that neurons in the region of the preoptic area exert tonic inhibition on downstream mechanisms capable of increasing the activity of the hypothalamic–pituitary–adrenal (HPA) axis as well as sympathetic thermogenic and cardiac activity. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Body temperature; Heart rate; Blood pressure; Hypothalamus; Fever
The preoptic area (POA) of the mammalian hypothalamus plays a key role in a variety of hypothalamic functions, including thermoregulation. Among these is the integrated central nervous response to bacterial infection in mammals. This response includes a variety of physiological changes that are mediated through neural circuitry in the brain that has not been fully elucidated. Salient among these changes is fever, which appears to involve the generation of prostaglandins locally in the POA [2,18]. Accordingly, microinjection of prostaglandin E2 (PGE2) into this region elicits marked increases in body temperature in conscious and anesthetized animals, and these are accompanied by other physiologic components of the acute phase response seen in humans and other mammals, including activation of the hypothalamic–pituitary–adrenal (HPA) axis and sympathetically mediated tachycardia [13,17,19,21,34]. The increase in body temperature evoked by PGE2 under these conditions has
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Corresponding author. Tel.: +1 317 274 1571; fax: +1 317 274 7714. E-mail address: [email protected] (J.A. DiMicco).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.12.032
been employed as a model for the fever associated with bacterial infection [17,21,19,22,34]. PGE2-induced stimulation of the HPA axis manifest as an increase in plasma adrenocorticotrophic hormone (ACTH) is thought to reflect a negative feedback loop that serves to limit the intensity of the inflammatory/immune response to the invading microorganism [31,36]. Like PGE2, local microinjection or microdialysis of the POA with muscimol, a GABAA receptor agonist and thus a powerful inhibitor of most adult mammalian neurons, or with GABA itself has been reported to increase both body temperature and heart rate [23,26,24,12]. However, stimulation of GABAA receptors in the POA also increases locomotor activity [12,25], an effect not reported for PGE2. The effect of microinjection of muscimol into the POA on plasma ACTH, an important feature of the response to microinjection of PGE2 in this region, has not been studied previously. Therefore, our purpose was to characterize and compare the effect of similar microinjection of muscimol and PGE2 into the same sites in the POA on plasma ACTH as well as heart rate, blood pressure, body temperature, and locomotor activity in the same conscious freely moving rats.
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All procedures conformed to guidelines set forth by NIH and were approved by the Institutional Animal Care and Use Committee. Male Sprague–Dawley rats (300 ± 10 g; Harlan, Indianapolis, IN, USA) maintained in 12-h light:12-h dark cycle and fed ad libitum were anesthetized (either 50 mg/kg pentobarbital, i.p. or 80 mg/kg ketamine and 11.5 mg/kg xylazine, i.p. supplemented as needed) and a telemetric transmitter was implanted intraperitoneally (Model TA11PA-PXT50; Data Sciences, MN, USA) as described previously [35]. Five days later, rats were again anesthetized and placed in a stereotaxic apparatus with the incisor bar set 3.3 mm below the interaural line for placement of a microinjection guide cannula (26 gauge, Plastics One, Roanoke, VA, USA) into the medial POA (coordinates: AP 0.0 mm; LR +0.7 mm; HD −8.2 mm; bregma as reference point) and secured by three stainless steel screws, Vetbond glue, and cranioplastic cement. Dummy wire cannulae were inserted in the guides, and rats were returned to their home cages for recovery. Three to four days later, rats were re-anesthetized and a femoral arterial catheter (1 cm of a 6 cm length of Teflon leader tubing inserted into a 20 cm length of Tygon tubing; Small Parts Inc., FL, USA) was implanted, routed subcutaneously, and exteriorized in the area between the shoulder blades, fixed to a harness (Rattus et al., USA), and flushed with saline. All microinjections were performed between 10.00 a.m. and 2.00 p.m. during the light phase of the 12-h light:12-h dark cycle in a laboratory where ambient temperature was maintained at 24–25 ◦ C. On the day of the experiment, rats were placed in their home cage on the telemetry receiver plate. A microinjector (33 gauge, Plastics One Inc., Roanoke, VA, USA) was connected to a 10 ul Hamilton syringe with Teflon FEP tubing (i.d. = 0.12 mm; o.d. = 0.65 mm; BAS, USA) and the entire system was loaded with vehicle or with a solution of the drug to be injected. The dummy cannula was then removed, the microinjector was inserted into the guide cannula, and the rat was left undisturbed for an additional 40–60 min. The Hamilton syringe was mounted in an infusion pump (KD-200) that was used to deliver a volume of 100 nL of injectate over 30 s. After the microinjection, the injector was left in place until the end of experiment while the animal remained undisturbed in its cage. The microinjection was considered successful if, immediately after removal of the microinjector, flow appeared within 5 s after the pump was reactivated, indicating that the injector was not clogged and that injectate had been successfully delivered. Blood samples (0.35 mL) were taken 5 min before and 15, 30, and 60 min after microinjection using a tuberculin syringe containing 0.06 ml of a mixture (1:1) of 20 mg/ml EDTA and
aprotinin (Sigma, USA). After withdrawal, the blood sample was immediately transferred into chilled Eppendorf tubes and centrifuged to obtain plasma that was stored at −80 ◦ C until analysis. ACTH was measured by radioimmunoassay according to the method of Li et al. [16] as we have described previously [35]. Five rats were subjected to trials in which the effect of microinjection of muscimol 80 pmol, muscimol 20 pmol, or saline vehicle (100 nL) or PGE2 150 pmol or its vehicle (0.15% DMSO in PBS) on body temperature, heart rate, and blood pressure were assessed. Thus, every rat received each of the five treatments at 2–3-day intervals in staggered order. After the last session, rats were deeply anesthetized with pentobarbital (100 mg/kg), and microinjection sites were marked with 100 nl of 2.5% alcian blue dye, brains were perfused, fixed in situ, removed, and processed for visualization of the injection site, determined according to the atlas of Paxinos and Watson [27]. A Dataquest telemetry system (Data Sciences, MN, USA) was used for the measurement of core body temperature (sensor placed intraperitoneally), arterial pressure, heart rate, and locomotor activity. Locomotor activity was assessed telemetrically by the degree of movement of the subject rat over telemetry receiver plate and expressed as arbitrary units (for details, see Ref. [3]). Results are expressed as mean ± S.E.M. Comparisons were made between groups with one-way or repeated measures ANOVA where appropriate. Fisher’s LSD test was employed for post hoc analysis. Limits of probability considered significant were 5%. Baseline data for all physiological parameters just prior to microinjections are given in Table 1. Although heart rate, body temperature, and plasma ACTH tended to be higher just prior to microinjection when injection cannulae loaded with PGE2 and muscimol were seated in the POA, no significant differences were found among the baselines for any parameter. As has been previously reported, microinjection of either PGE2 150 pmol or muscimol 80 pmol into the POA evoked marked increases in body temperature and heart rate, as well as moderate elevations in arterial pressure in conscious rats (Fig. 1). The increases in body temperature elicited by microinjection of PGE2 and muscimol 80 pmol into the POA were similar in magnitude, but the increase in heart rate evoked by muscimol was significantly greater than that evoked by PGE2. In fact, the increase in heart rate produced by PGE2 was nearly identical to that caused by microinjection of the lower dose of muscimol, a dose that failed to cause a significant effect on body temperature or blood pressure.
Table 1 Baselines prior to all microinjections Saline Heart rate (beats/min) Blood pressure (mmHg) Body temperature (◦ C) Plasma ACTH (pg/mL)
384 115 37.93 46
Muscimol 20 pmol ± ± ± ±
6 6 0.24 6
385 124 38.13 84
± ± ± ±
8 12 0.20 28
Muscimol 80 pmol 400 118 38.47 63
± ± ± ±
16 4 0.35 13
Means ± standard errors for given parameter just prior to microinjection into the POA as indicated (n = 5).
Vehicle 390 116 38.09 55
± ± ± ±
PGE2 150 pmol 8 9 0.18 4
402 123 38.82 53
± ± ± ±
10 6 0.26 9
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Fig. 1. (A) Mean changes from average baseline values for all parameters measured after intrapreoptic microinjection of prostaglandin E2 (PGE2) and vehicle (100 nL 0.15% DMSO in PBS) above, and muscimol 80 and 20 pmol, and saline (100 nL) below in the same five chronically instrumented rats. Each parameter was averaged over a time interval that encompassed the maximal response elicited by microinjection of the substances. Thus, data for 0 to +40 min were averaged for heart rate (HR), mean blood pressure (MBP), and locomotor activity (LA; units = counts/min), and data for temperature (T) were averaged over +20 to +60 min. (* ) Levels significantly greater than corresponding levels seen after saline or vehicle; (#) levels significantly greater than corresponding levels seen after muscimol 20 pmol (one-way ANOVA and Fisher’s LSD post hoc test, p < 0.05). (B) Schematic coronal sections at the level of the POA adapted from the atlas of Paxinos and Watson [27] indicating approximate locations of sites of injection in all five experiments (filled circles). Numbers indicate distance in millimeters from bregma.
Muscimol also evoked marked elevations in plasma levels of ACTH (Figs. 1 and 2). Basal levels of plasma ACTH prior to the different treatments were at or below levels previously reported for unstressed freely moving Sprague–Dawley rats [1,4,7] and maintained in this low range following injection of saline or vehicle for PGE2. Plasma ACTH was significantly elevated 7 min after the microinjection of either dose of muscimol and peaked at 15 min. Microinjection of PGE2 also evoked increases in plasma ACTH as has been reported. The response to microinjection of the two agents differed most with respect to locomotor activity (Fig. 1). Microinjection of muscimol increased locomotor activity significantly, with peak stimulation occurring from 15 to 20 min after microinjection. In contrast, microinjection of PGE2 at these same sites had no significant effect on activity. Because microinjection of muscimol into the POA evoked significant increases in locomotor activity, we examined our results to determine whether either the increases in body temperature or the increases in plasma ACTH might be causally related to this behavioral effect. Seven minutes after the injection of muscimol when the first blood
sample was taken for measurement of ACTH, mean total activity counts were nearly identical after the two doses of muscimol (80 pmol = 1096 ± 437; 20 pmol = 1048 ± 435), whereas plasma ACTH had increased by an average of 226 pg/mL after 80 pmol but only by 98 pg/mL after 20 pmol. Examination of data from individual animals at this time point revealed no correlation between changes in body temperature and cumulative locomotor activity. Thus, the mean increase in body temperature after injection of 80 pmol of muscimol was 0.15 ± 0.06 ◦ C, but one of the largest increases (0.25 ◦ C) occurred in a rat whose total activity counts (540 counts) were less than half the group average, while the animal displaying the most activity (i.e., 2300 counts) registered an increase of only 0.09 ◦ C. Post-mortem processing and histology revealed that injection sites were all located in the preoptic area from 200 m anterior to 400 m posterior to bregma and within 1.1 mm of the midline (Fig. 1). Like the microinjection of PGE2 at the same sites, the microinjection of muscimol into the POA evoked a marked increase in plasma ACTH, signaling activation of the
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Fig. 2. Average (±S.E.) plasma levels of ACTH from blood samples taken 5 min before and at various times after microinjection (at t = 0 min) of (LEFT) prostaglandin E2 (PGE2) 150 pmol and vehicle (100 nL 0.15% DMSO in PBS–VEH), and (RIGHT) muscimol 80 and 20 pmol, and vehicle (saline 100 nL: SAL) into the POA in the same five chronically instrumented conscious rats.(* ) Levels significantly greater than corresponding levels seen after vehicle or saline; (#) levels significantly greater than corresponding levels seen after muscimol 20 pmol (repeated measures ANOVA and Fisher’s LSD post hoc test, p < 0.05).
hypothalamic–pituitary–adrenal axis. Our results demonstrate for the first time that muscimol acts in the POA to increased plasma levels of ACTH in conscious rats as has been described for PGE2 [13,32]. Microinjection of either agent also increased body temperature, heart rate, and arterial pressure. Application of muscimol to the POA using microdialysis has been reported to increase body temperature and heart rate in conscious rats or anesthetized rats and locomotor activity in conscious animals [12,23–26]. These same effects of microinjected PGE2 have been well studied and are thought to represent an experimental model for the acute phase response [11,17,22]. To our knowledge, however, this study represents the first where all these parameters were measured in the same conscious rats. Also, by seating the injection cannula prior to the baseline period and thus well in advance of the microinjection itself, we avoided effects resulting from the manipulation associated with the microinjection. Therefore, the injection itself was not associated with the artifacts evident in a previous report [32]. The finding that muscimol, thought to be a powerful inhibitor of the vast majority of adult mammalian neurons by virtue if its GABAA receptor agonist properties, produces a pattern of effects so closely resembling those produced by PGE2 when both these agents are injected into the POA has significant implications. With regard to thermoregulation, current hypotheses point to the preoptic area as a source of tonic inhibition of downstream thermogenic mechanisms [5,20]. One such mechanism resides in neurons in the dorsomedial hypothalamus (DMH), where blockade of GABAA receptor-mediated inhibition in conscious rats has been shown to increase core body temperature [35]. In fact, disinhibition of the DMH results in a pattern of changes closely resembling that produced by presumptive inhibition of neuronal activity in the POA in the present study, including marked increases in plasma ACTH as well as in heart
rate, locomotor activity, and arterial pressure [3,6,30,35]. If the POA represents a source of tonic GABA-mediated inhibition of neurons in the DMH whose activation results in increased heart rate, body temperature, and plasma ACTH, then it is tempting to speculate that these same effects of microinjected PGE2, like those of muscimol, are a consequence of inhibition of these critical neurons in the POA. In support of this hypothesis, warmsensitive neurons in the POA are inhibited by local application of PGE2 [28,29]. Since the hyperthermic and tachycardic effects of microinjection of PGE2 into the POA appear to be mediated through neuronal activity in the DMH [17,34], it seems likely that the same is true for PGE2-induced increases in plasma ACTH and for the same effects of microinjected muscimol in the present study. Thus, neuronal activity of these critical thermoregulatory neurons in the POA appears to exert tonic inhibition over central nervous circuits capable of generating tachycardia, activation of the hypothalamic–pituitary–adrenal axis, and hyperthermia. While the responses to microinjection of muscimol and PGE2 into the POA were similar, they were not identical. At doses that evoked equivalent increases in body temperature, muscimol appeared to elicit greater increases in heart rate and plasma ACTH. However, the two agents were most different with regard to their behavioral effects. Only muscimol produced increased locomotor activity. Osborne and colleagues [25,26] likewise reported that microdialysis of the lateral POA with either muscimol or PGE2 evoked increases in body temperature while only muscimol increased locomotor activity in conscious rats. This difference is paralleled by differences in the behavioral components of the different responses that microinjection of these agents has been used to model. Thus, while microinjection of PGE2 into the POA represents an established model for the acute phase response, microinjection of muscimol into the same
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region has been examined in the context of the role of endogenous GABAergic inhibition in thermoregulatory responses seen in a cold environment [12,23]. Interestingly, tachycardia, activation of thermogenic and/or heat conserving mechanisms, and excitation of the HPA axis are features common to both these contexts [9]. However, exposure to cold may be associated with locomotor stimulation [10], while the acute phase response or the response to centrally administered PGE is characterized by behavioral depression [8,14,15,33]. One possible explanation is that the shared features of the response to microinjected muscimol and PGE2 result from the inhibition of warm-sensitive neurons in the POA that is elicited by both agents, while the behavioral stimulation seen after muscimol is a consequence of inhibition of warm-insensitive neurons in the region, neurons that are instead excited by PGE2 [28,29]. In summary, our results demonstrate that microinjection of muscimol into the POA increases plasma ACTH, thus replicating the effect of similar microinjection of PGE2. This finding points to an important role for neurons in the POA in the tonic inhibition of central nervous circuits whose activation evokes diverse effects, including increased heart rate, body temperature, locomotor activity, and secretion of ACTH. Acknowledgements This work was supported by USPHS grants NS 19883 and MH 65697. References [1] V.C. Anseloni, F. He, S.I. Novikova, M. Turnbach-Robbins, I.A. Lidow, M. Ennis, M.S. Lidow, Alterations in stress-associated behaviors and neurochemical markers in adult rats after neonatal short-lasting local inflammatory insult, Neuroscience 131 (2005) 635–645. [2] D.M. Aronoff, E.G. Neilson, Antipyretics: mechanisms of action and clinical use in fever suppression, Am. J. Med. 111 (2001) 304–315. [3] T.W. Bailey, J.A. DiMicco, Chemical stimulation of the dorsomedial hypothalamus elevates plasma ACTH in conscious rats, Am. J. Physiol. 280 (2001) R8–R15. [4] O. Chan, K. Inouye, E. Akirav, E. Park, M.C. Riddell, M. Vranic, S.G. Matthews, Insulin alone increases hypothalamo-pituitary-adrenal activity, and diabetes lowers peak stress responses, Endocrinology 146 (2005) 1382–1390. [5] X.M. Chen, T. Hosono, T. Yoda, Y. Fukuda, K. Kanosue, Efferent projection from the preoptic area for the control of non-shivering thermogenesis in rats, J. Physiol. 512 (1998) 883–892. [6] V. DeNovellis, E. Stotz-Potter, S.M. Morin, F. Rossi, J.A. DiMicco, Hypothalamic sites mediating cardiovascular effects of microinjected bicuculline and excitatory amino acids in rats, Am. J. Physiol. 269 (1995) R131–R140. [7] M.M. Faraday, K.H. Blakeman, N.E. Grunberg, Strain and sex alter effects of stress and nicotine on feeding, body weight, and HPA axis hormones, Pharmacol. Biochem. Behav. 80 (2005) 577–589. [8] U. Forstermann, R. Heldt, G. Hertting, Effects of intracerebroventricular administration of prostaglandin D2 on behaviour, blood pressure and body temperature as compared to prostaglandins E2 and F2 alpha, Psychopharmacology 80 (1983) 365–370. [9] K. Fukuhara, R. Kvetnansky, G. Cizza, K. Pacak, H. Ohara, D.S. Goldstein, I.J. Kopin, Interrelations between sympathoadrenal system and hypothalamo-pituitary-adrenocortical/thyroid systems in rats exposed to cold stress, J. Neuroendocrinol. 8 (1996) 533–541.
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Microinjection of prostaglandin E2 and muscimol into the preoptic area in conscious rats: Comparison of effects on plasma adrenocorticotrophic hormone (ACTH), body temperature, locomotor activity, and cardiovascular function Dmitry V. Zaretsky, Joseph L. Hunt, Maria V. Zaretskaia, Joseph A. DiMicco ∗ Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA Received 18 October 2005; received in revised form 30 November 2005; accepted 12 December 2005
Abstract The preoptic area (POA) is thought to play an important role in thermoregulation and fever. Local application of prostaglandin E2 (PGE2) to this region elicits increases in core body temperature, heart rate, and plasma levels of adrenocorticotrophic hormone (ACTH). Similar effects on body temperature and heart rate have also been reported after local application of the GABAA receptor agonist muscimol to the preoptic area. The purpose of this study was to assess and compare the effects of microinjection of PGE2 and muscimol into the preoptic area in the same chronically instrumented conscious rats on plasma levels of ACTH. Injection of either PGE2 (150 pmol/100 nL) or muscimol (20 or 80 pmol/100 nL) into the same sites in the preoptic area evoked increases in body temperature, heart rate, blood pressure, and plasma levels of ACTH, while significant increases in locomotor activity were apparent only after muscimol. These data confirm and extend previous findings and support the notion that neurons in the region of the preoptic area exert tonic inhibition on downstream mechanisms capable of increasing the activity of the hypothalamic–pituitary–adrenal (HPA) axis as well as sympathetic thermogenic and cardiac activity. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Body temperature; Heart rate; Blood pressure; Hypothalamus; Fever
The preoptic area (POA) of the mammalian hypothalamus plays a key role in a variety of hypothalamic functions, including thermoregulation. Among these is the integrated central nervous response to bacterial infection in mammals. This response includes a variety of physiological changes that are mediated through neural circuitry in the brain that has not been fully elucidated. Salient among these changes is fever, which appears to involve the generation of prostaglandins locally in the POA [2,18]. Accordingly, microinjection of prostaglandin E2 (PGE2) into this region elicits marked increases in body temperature in conscious and anesthetized animals, and these are accompanied by other physiologic components of the acute phase response seen in humans and other mammals, including activation of the hypothalamic–pituitary–adrenal (HPA) axis and sympathetically mediated tachycardia [13,17,19,21,34]. The increase in body temperature evoked by PGE2 under these conditions has
∗
Corresponding author. Tel.: +1 317 274 1571; fax: +1 317 274 7714. E-mail address: [email protected] (J.A. DiMicco).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.12.032
been employed as a model for the fever associated with bacterial infection [17,21,19,22,34]. PGE2-induced stimulation of the HPA axis manifest as an increase in plasma adrenocorticotrophic hormone (ACTH) is thought to reflect a negative feedback loop that serves to limit the intensity of the inflammatory/immune response to the invading microorganism [31,36]. Like PGE2, local microinjection or microdialysis of the POA with muscimol, a GABAA receptor agonist and thus a powerful inhibitor of most adult mammalian neurons, or with GABA itself has been reported to increase both body temperature and heart rate [23,26,24,12]. However, stimulation of GABAA receptors in the POA also increases locomotor activity [12,25], an effect not reported for PGE2. The effect of microinjection of muscimol into the POA on plasma ACTH, an important feature of the response to microinjection of PGE2 in this region, has not been studied previously. Therefore, our purpose was to characterize and compare the effect of similar microinjection of muscimol and PGE2 into the same sites in the POA on plasma ACTH as well as heart rate, blood pressure, body temperature, and locomotor activity in the same conscious freely moving rats.
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All procedures conformed to guidelines set forth by NIH and were approved by the Institutional Animal Care and Use Committee. Male Sprague–Dawley rats (300 ± 10 g; Harlan, Indianapolis, IN, USA) maintained in 12-h light:12-h dark cycle and fed ad libitum were anesthetized (either 50 mg/kg pentobarbital, i.p. or 80 mg/kg ketamine and 11.5 mg/kg xylazine, i.p. supplemented as needed) and a telemetric transmitter was implanted intraperitoneally (Model TA11PA-PXT50; Data Sciences, MN, USA) as described previously [35]. Five days later, rats were again anesthetized and placed in a stereotaxic apparatus with the incisor bar set 3.3 mm below the interaural line for placement of a microinjection guide cannula (26 gauge, Plastics One, Roanoke, VA, USA) into the medial POA (coordinates: AP 0.0 mm; LR +0.7 mm; HD −8.2 mm; bregma as reference point) and secured by three stainless steel screws, Vetbond glue, and cranioplastic cement. Dummy wire cannulae were inserted in the guides, and rats were returned to their home cages for recovery. Three to four days later, rats were re-anesthetized and a femoral arterial catheter (1 cm of a 6 cm length of Teflon leader tubing inserted into a 20 cm length of Tygon tubing; Small Parts Inc., FL, USA) was implanted, routed subcutaneously, and exteriorized in the area between the shoulder blades, fixed to a harness (Rattus et al., USA), and flushed with saline. All microinjections were performed between 10.00 a.m. and 2.00 p.m. during the light phase of the 12-h light:12-h dark cycle in a laboratory where ambient temperature was maintained at 24–25 ◦ C. On the day of the experiment, rats were placed in their home cage on the telemetry receiver plate. A microinjector (33 gauge, Plastics One Inc., Roanoke, VA, USA) was connected to a 10 ul Hamilton syringe with Teflon FEP tubing (i.d. = 0.12 mm; o.d. = 0.65 mm; BAS, USA) and the entire system was loaded with vehicle or with a solution of the drug to be injected. The dummy cannula was then removed, the microinjector was inserted into the guide cannula, and the rat was left undisturbed for an additional 40–60 min. The Hamilton syringe was mounted in an infusion pump (KD-200) that was used to deliver a volume of 100 nL of injectate over 30 s. After the microinjection, the injector was left in place until the end of experiment while the animal remained undisturbed in its cage. The microinjection was considered successful if, immediately after removal of the microinjector, flow appeared within 5 s after the pump was reactivated, indicating that the injector was not clogged and that injectate had been successfully delivered. Blood samples (0.35 mL) were taken 5 min before and 15, 30, and 60 min after microinjection using a tuberculin syringe containing 0.06 ml of a mixture (1:1) of 20 mg/ml EDTA and
aprotinin (Sigma, USA). After withdrawal, the blood sample was immediately transferred into chilled Eppendorf tubes and centrifuged to obtain plasma that was stored at −80 ◦ C until analysis. ACTH was measured by radioimmunoassay according to the method of Li et al. [16] as we have described previously [35]. Five rats were subjected to trials in which the effect of microinjection of muscimol 80 pmol, muscimol 20 pmol, or saline vehicle (100 nL) or PGE2 150 pmol or its vehicle (0.15% DMSO in PBS) on body temperature, heart rate, and blood pressure were assessed. Thus, every rat received each of the five treatments at 2–3-day intervals in staggered order. After the last session, rats were deeply anesthetized with pentobarbital (100 mg/kg), and microinjection sites were marked with 100 nl of 2.5% alcian blue dye, brains were perfused, fixed in situ, removed, and processed for visualization of the injection site, determined according to the atlas of Paxinos and Watson [27]. A Dataquest telemetry system (Data Sciences, MN, USA) was used for the measurement of core body temperature (sensor placed intraperitoneally), arterial pressure, heart rate, and locomotor activity. Locomotor activity was assessed telemetrically by the degree of movement of the subject rat over telemetry receiver plate and expressed as arbitrary units (for details, see Ref. [3]). Results are expressed as mean ± S.E.M. Comparisons were made between groups with one-way or repeated measures ANOVA where appropriate. Fisher’s LSD test was employed for post hoc analysis. Limits of probability considered significant were 5%. Baseline data for all physiological parameters just prior to microinjections are given in Table 1. Although heart rate, body temperature, and plasma ACTH tended to be higher just prior to microinjection when injection cannulae loaded with PGE2 and muscimol were seated in the POA, no significant differences were found among the baselines for any parameter. As has been previously reported, microinjection of either PGE2 150 pmol or muscimol 80 pmol into the POA evoked marked increases in body temperature and heart rate, as well as moderate elevations in arterial pressure in conscious rats (Fig. 1). The increases in body temperature elicited by microinjection of PGE2 and muscimol 80 pmol into the POA were similar in magnitude, but the increase in heart rate evoked by muscimol was significantly greater than that evoked by PGE2. In fact, the increase in heart rate produced by PGE2 was nearly identical to that caused by microinjection of the lower dose of muscimol, a dose that failed to cause a significant effect on body temperature or blood pressure.
Table 1 Baselines prior to all microinjections Saline Heart rate (beats/min) Blood pressure (mmHg) Body temperature (◦ C) Plasma ACTH (pg/mL)
384 115 37.93 46
Muscimol 20 pmol ± ± ± ±
6 6 0.24 6
385 124 38.13 84
± ± ± ±
8 12 0.20 28
Muscimol 80 pmol 400 118 38.47 63
± ± ± ±
16 4 0.35 13
Means ± standard errors for given parameter just prior to microinjection into the POA as indicated (n = 5).
Vehicle 390 116 38.09 55
± ± ± ±
PGE2 150 pmol 8 9 0.18 4
402 123 38.82 53
± ± ± ±
10 6 0.26 9
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Fig. 1. (A) Mean changes from average baseline values for all parameters measured after intrapreoptic microinjection of prostaglandin E2 (PGE2) and vehicle (100 nL 0.15% DMSO in PBS) above, and muscimol 80 and 20 pmol, and saline (100 nL) below in the same five chronically instrumented rats. Each parameter was averaged over a time interval that encompassed the maximal response elicited by microinjection of the substances. Thus, data for 0 to +40 min were averaged for heart rate (HR), mean blood pressure (MBP), and locomotor activity (LA; units = counts/min), and data for temperature (T) were averaged over +20 to +60 min. (* ) Levels significantly greater than corresponding levels seen after saline or vehicle; (#) levels significantly greater than corresponding levels seen after muscimol 20 pmol (one-way ANOVA and Fisher’s LSD post hoc test, p < 0.05). (B) Schematic coronal sections at the level of the POA adapted from the atlas of Paxinos and Watson [27] indicating approximate locations of sites of injection in all five experiments (filled circles). Numbers indicate distance in millimeters from bregma.
Muscimol also evoked marked elevations in plasma levels of ACTH (Figs. 1 and 2). Basal levels of plasma ACTH prior to the different treatments were at or below levels previously reported for unstressed freely moving Sprague–Dawley rats [1,4,7] and maintained in this low range following injection of saline or vehicle for PGE2. Plasma ACTH was significantly elevated 7 min after the microinjection of either dose of muscimol and peaked at 15 min. Microinjection of PGE2 also evoked increases in plasma ACTH as has been reported. The response to microinjection of the two agents differed most with respect to locomotor activity (Fig. 1). Microinjection of muscimol increased locomotor activity significantly, with peak stimulation occurring from 15 to 20 min after microinjection. In contrast, microinjection of PGE2 at these same sites had no significant effect on activity. Because microinjection of muscimol into the POA evoked significant increases in locomotor activity, we examined our results to determine whether either the increases in body temperature or the increases in plasma ACTH might be causally related to this behavioral effect. Seven minutes after the injection of muscimol when the first blood
sample was taken for measurement of ACTH, mean total activity counts were nearly identical after the two doses of muscimol (80 pmol = 1096 ± 437; 20 pmol = 1048 ± 435), whereas plasma ACTH had increased by an average of 226 pg/mL after 80 pmol but only by 98 pg/mL after 20 pmol. Examination of data from individual animals at this time point revealed no correlation between changes in body temperature and cumulative locomotor activity. Thus, the mean increase in body temperature after injection of 80 pmol of muscimol was 0.15 ± 0.06 ◦ C, but one of the largest increases (0.25 ◦ C) occurred in a rat whose total activity counts (540 counts) were less than half the group average, while the animal displaying the most activity (i.e., 2300 counts) registered an increase of only 0.09 ◦ C. Post-mortem processing and histology revealed that injection sites were all located in the preoptic area from 200 m anterior to 400 m posterior to bregma and within 1.1 mm of the midline (Fig. 1). Like the microinjection of PGE2 at the same sites, the microinjection of muscimol into the POA evoked a marked increase in plasma ACTH, signaling activation of the
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Fig. 2. Average (±S.E.) plasma levels of ACTH from blood samples taken 5 min before and at various times after microinjection (at t = 0 min) of (LEFT) prostaglandin E2 (PGE2) 150 pmol and vehicle (100 nL 0.15% DMSO in PBS–VEH), and (RIGHT) muscimol 80 and 20 pmol, and vehicle (saline 100 nL: SAL) into the POA in the same five chronically instrumented conscious rats.(* ) Levels significantly greater than corresponding levels seen after vehicle or saline; (#) levels significantly greater than corresponding levels seen after muscimol 20 pmol (repeated measures ANOVA and Fisher’s LSD post hoc test, p < 0.05).
hypothalamic–pituitary–adrenal axis. Our results demonstrate for the first time that muscimol acts in the POA to increased plasma levels of ACTH in conscious rats as has been described for PGE2 [13,32]. Microinjection of either agent also increased body temperature, heart rate, and arterial pressure. Application of muscimol to the POA using microdialysis has been reported to increase body temperature and heart rate in conscious rats or anesthetized rats and locomotor activity in conscious animals [12,23–26]. These same effects of microinjected PGE2 have been well studied and are thought to represent an experimental model for the acute phase response [11,17,22]. To our knowledge, however, this study represents the first where all these parameters were measured in the same conscious rats. Also, by seating the injection cannula prior to the baseline period and thus well in advance of the microinjection itself, we avoided effects resulting from the manipulation associated with the microinjection. Therefore, the injection itself was not associated with the artifacts evident in a previous report [32]. The finding that muscimol, thought to be a powerful inhibitor of the vast majority of adult mammalian neurons by virtue if its GABAA receptor agonist properties, produces a pattern of effects so closely resembling those produced by PGE2 when both these agents are injected into the POA has significant implications. With regard to thermoregulation, current hypotheses point to the preoptic area as a source of tonic inhibition of downstream thermogenic mechanisms [5,20]. One such mechanism resides in neurons in the dorsomedial hypothalamus (DMH), where blockade of GABAA receptor-mediated inhibition in conscious rats has been shown to increase core body temperature [35]. In fact, disinhibition of the DMH results in a pattern of changes closely resembling that produced by presumptive inhibition of neuronal activity in the POA in the present study, including marked increases in plasma ACTH as well as in heart
rate, locomotor activity, and arterial pressure [3,6,30,35]. If the POA represents a source of tonic GABA-mediated inhibition of neurons in the DMH whose activation results in increased heart rate, body temperature, and plasma ACTH, then it is tempting to speculate that these same effects of microinjected PGE2, like those of muscimol, are a consequence of inhibition of these critical neurons in the POA. In support of this hypothesis, warmsensitive neurons in the POA are inhibited by local application of PGE2 [28,29]. Since the hyperthermic and tachycardic effects of microinjection of PGE2 into the POA appear to be mediated through neuronal activity in the DMH [17,34], it seems likely that the same is true for PGE2-induced increases in plasma ACTH and for the same effects of microinjected muscimol in the present study. Thus, neuronal activity of these critical thermoregulatory neurons in the POA appears to exert tonic inhibition over central nervous circuits capable of generating tachycardia, activation of the hypothalamic–pituitary–adrenal axis, and hyperthermia. While the responses to microinjection of muscimol and PGE2 into the POA were similar, they were not identical. At doses that evoked equivalent increases in body temperature, muscimol appeared to elicit greater increases in heart rate and plasma ACTH. However, the two agents were most different with regard to their behavioral effects. Only muscimol produced increased locomotor activity. Osborne and colleagues [25,26] likewise reported that microdialysis of the lateral POA with either muscimol or PGE2 evoked increases in body temperature while only muscimol increased locomotor activity in conscious rats. This difference is paralleled by differences in the behavioral components of the different responses that microinjection of these agents has been used to model. Thus, while microinjection of PGE2 into the POA represents an established model for the acute phase response, microinjection of muscimol into the same
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region has been examined in the context of the role of endogenous GABAergic inhibition in thermoregulatory responses seen in a cold environment [12,23]. Interestingly, tachycardia, activation of thermogenic and/or heat conserving mechanisms, and excitation of the HPA axis are features common to both these contexts [9]. However, exposure to cold may be associated with locomotor stimulation [10], while the acute phase response or the response to centrally administered PGE is characterized by behavioral depression [8,14,15,33]. One possible explanation is that the shared features of the response to microinjected muscimol and PGE2 result from the inhibition of warm-sensitive neurons in the POA that is elicited by both agents, while the behavioral stimulation seen after muscimol is a consequence of inhibition of warm-insensitive neurons in the region, neurons that are instead excited by PGE2 [28,29]. In summary, our results demonstrate that microinjection of muscimol into the POA increases plasma ACTH, thus replicating the effect of similar microinjection of PGE2. This finding points to an important role for neurons in the POA in the tonic inhibition of central nervous circuits whose activation evokes diverse effects, including increased heart rate, body temperature, locomotor activity, and secretion of ACTH. Acknowledgements This work was supported by USPHS grants NS 19883 and MH 65697. References [1] V.C. Anseloni, F. He, S.I. Novikova, M. Turnbach-Robbins, I.A. Lidow, M. Ennis, M.S. Lidow, Alterations in stress-associated behaviors and neurochemical markers in adult rats after neonatal short-lasting local inflammatory insult, Neuroscience 131 (2005) 635–645. [2] D.M. Aronoff, E.G. Neilson, Antipyretics: mechanisms of action and clinical use in fever suppression, Am. J. Med. 111 (2001) 304–315. [3] T.W. Bailey, J.A. DiMicco, Chemical stimulation of the dorsomedial hypothalamus elevates plasma ACTH in conscious rats, Am. J. Physiol. 280 (2001) R8–R15. [4] O. Chan, K. Inouye, E. Akirav, E. Park, M.C. Riddell, M. Vranic, S.G. Matthews, Insulin alone increases hypothalamo-pituitary-adrenal activity, and diabetes lowers peak stress responses, Endocrinology 146 (2005) 1382–1390. [5] X.M. Chen, T. Hosono, T. Yoda, Y. Fukuda, K. Kanosue, Efferent projection from the preoptic area for the control of non-shivering thermogenesis in rats, J. Physiol. 512 (1998) 883–892. [6] V. DeNovellis, E. Stotz-Potter, S.M. Morin, F. Rossi, J.A. DiMicco, Hypothalamic sites mediating cardiovascular effects of microinjected bicuculline and excitatory amino acids in rats, Am. J. Physiol. 269 (1995) R131–R140. [7] M.M. Faraday, K.H. Blakeman, N.E. Grunberg, Strain and sex alter effects of stress and nicotine on feeding, body weight, and HPA axis hormones, Pharmacol. Biochem. Behav. 80 (2005) 577–589. [8] U. Forstermann, R. Heldt, G. Hertting, Effects of intracerebroventricular administration of prostaglandin D2 on behaviour, blood pressure and body temperature as compared to prostaglandins E2 and F2 alpha, Psychopharmacology 80 (1983) 365–370. [9] K. Fukuhara, R. Kvetnansky, G. Cizza, K. Pacak, H. Ohara, D.S. Goldstein, I.J. Kopin, Interrelations between sympathoadrenal system and hypothalamo-pituitary-adrenocortical/thyroid systems in rats exposed to cold stress, J. Neuroendocrinol. 8 (1996) 533–541.
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