Anatomic patterns of Fos immunostaining in rat brain following systemic endotoxin administration.

To identify brain neurons that participate in the acute phase response, rat brains were examined immunocytochemically for Fos protein following the intravenous administration of bacterial endotoxin (lipopolysaccharide, LPS). Two to three hours after the injection of LPS, 150 micrograms/kg body weight, to adult male Long-Evans rats, a consistent anatomic pattern of Fos immunostained cell nuclei is seen. In the brain stem, prominant Fos immunostaining is induced in tyrosine hydroxylase immunoreactive neurons of the caudal ventral-lateral medulla (the A1 cell group), in both tyrosine hydroxylase positive and negative neurons of nu. tractus solitarius, in the parabrachial nu., and in a few neurons of the locus ceruleus. In the hypothalamus, endotoxin induces Fos expression in magnocellular neurons of the paraventricular and supraoptic nuclei and internuclear cell groups. A higher percentage of oxytocin-immunoreactive cells is double labeled for Fos nuclear immunostaining than vasopressin-immunoreactive cells. A minority of somatostatin immunoreactive periventricular hypothalamic neurons are Fos positive. Other hypothalamic nuclei that contain endotoxin-induced Fos nuclear immunostaining include the parvocellular neurons of the paraventricular nu., the dorsomedial and arcuate nuclei, the lateral hypothalamus, the dorsal hypothalamic area (zona incerta), and the median nucleus of the preoptic area. LPS induces numerous Fos-positive neurons in regions known to respond to a variety of stressful stimuli; these regions include the preoptic area, bed nucleus of the stria terminalis, lateral septum, and the central and medial nuclei of the amygdala. Moreover, Fos nuclear immunostaining is seen in neurons of circumventricular organs: the organum vasculosum of the lamina terminalis, the subfornical organ, and the area postrema. The maximum intensity of Fos nuclear immunostaining occurs 2-3 h after endotoxin administration and declines thereafter. It is attenuated by pretreatment with indomethacin, 25 mg/kg body weight Sc, or dexamethasone, 1 mg/kg IP. These observations are consistent with the participation of a variety of brain neuronal systems in the acute phase response and elucidate the functional neuroanatomy of that response at the cellular level.

Central interleukin-1 beta stimulation of vasopressin release into the rat brain: activation of an antipyretic pathway.

1. Arg8-vasopressin (AVP)-containing neurones of the bed nucleus of the stria terminalis (BST), which terminate in the ventral septal area (VSA) of the rat brain, provide a pathway which controls body temperature during fever. The present study was conducted to test the hypothesis that interleukin-1 beta (IL-1 beta) may trigger the antipyretic response by evoking AVP release from BST neurones projecting into the VSA. 2. The push-pull perfusion technique and radioimmunoassay were utilized to determine the AVP concentrations of retrieved VSA perfusion fluid in urethane-anaesthetized rats following BST infusion of vehicle or IL-1 beta (125 or 500 pg (2 microliters)-1). 3. Ventral septal AVP levels significantly increased from basal levels, in a dose-related manner, in response to IL-1 beta (0-500 pg). Electrical stimulation of the same areas of the BST also evoked AVP release into the VSA. 4. IL-1 beta infusions and electrical stimulation of the BST resulted in significant increases in rectal temperature. In IL-1 beta-treated animals (500 pg), the change in body temperature and VSA AVP release were negatively correlated (P < 0.001). However, external heating of the animals to approximately the same levels as electrically stimulated or IL-1 beta treated rats did not affect basal AVP release. 5. These data show that IL-1 beta is a potent stimulus for AVP release from BST neurones and supports BST involvement in neuro-immune interactions. We propose, that in addition to febrogenesis, IL-1 beta is also a key component in the process of endogenous antipyresis by activating vasopressinergic BST neurones to release AVP during fever.

Cardiovascular consequences of microinjection of vasopressin and angiotensin II in the area postrema.

Microinjection of angiotensin II (ANG II) into the area postrema (AP) of urethan-anesthetized male Sprague-Dawley rats elicited statistically significant increases in mean arterial blood pressure at doses ranging from 10 pg to 500 ng (10 pg, mean +/- SE, 10.8 +/- 1.1 mmHg, P < 0.001; 250 ng, 15.2 +/- 2.6 mmHg, P < 0.001). Heart rate was also significantly increased at doses > 10 pg, although these increases were not dose dependent. Systemic administration of losartan (Dup-753), an AT1 antagonist, was able to significantly reduce the pressor response to 250 ng ANG (post-losartan: 81.9 +/- 9.5% reduction in blood pressure response, P < 0.0001), whereas PD123319, an AT2 antagonist, was without significant effect (P > 0.1). Microinjection of vasopressin (VP) (10 pg-500 ng) into the AP also resulted in statistically significant increases in blood pressure at doses ranging from 10 to 100 pg (10 pg, 7.0 +/- 1.5 mmHg, P < 0.05) and 100-500 ng (250 ng, 12.2 +/- 1.8 mmHg, P < 0.0001). Small but significant changes in heart rate were observed only at 100 pg and 100 ng. Systemic administration of a V1 antagonist significantly attenuated the increases in blood pressure in response to 50, 100, and 250 ng VP (250 ng, post-V1 antagonist: 66.4 +/- 8.6% reduction in blood pressure response, P < 0.001), whereas [desamino,D-Arg8]vasopressin (DDAVP), a V2 agonist, had a depressor effect when microinjected directly into the AP (250 ng, -9.9 +/- 1.6 mmHg, P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Vasopressin release within the ventral septal area of the rat brain during drug-induced antipyresis.

The techniques of push-pull perfusion and radioimmunoassay were used to determine concentrations of arginine vasopressin (AVP) in extracellular fluid derived from the ventral septal area (VSA) of the rat brain following antipyresis elicited by acetaminophen or indomethacin in conscious and unrestrained rats. Reduction of bacterial lipopolysaccharide (LPS)-induced fever by intraperitoneal indomethacin resulted in significant increases in AVP levels in VSA perfusion fluid (P < 0.05). In contrast, antipyresis after acetaminophen treatment was without significant effect on AVP output from VSA nerve terminals. In control animals (non-pyrogen treated), body temperature rose in apparent response to the perfusion procedure. Despite this elevation in core temperature, subsequent treatment with acetaminophen or indomethacin did not result in significant changes in AVP release from VSA perfusates. We conclude that AVP release into VSA extracellular fluids following intraperitoneal indomethacin is dependent upon the neuronal sequelae inherent to pyrogen-evoked fever and not nonspecific rises in body temperature. These results support the hypothesis that endogenous AVP, acting within the VSA, participates in the neuronal mechanisms mediating indomethacin-induced antipyresis.

Fever alters osmosensitivity of hypothalamic-vasopressin system in the rat.

The osmosensitivity of peripheral vasopressin release was studied during healthy thermoregulation and endotoxin-induced fever. There was an increase in osmosensitivity following bolus injection of saline in febrile rats. These animals displayed a steeper slope in the linear relationship between plasma osmolality and plasma vasopressin levels compared with afebrile animals. The change in regression slope was due to a significantly lower plasma osmolality in febrile rats. The plasma osmolality of animals infused with hypertonic saline was similarly decreased, but data analysis failed to show a significant change in the regression slope. Osmotic thresholds were not altered in either group. There was an increased urine output in febrile rats, and consequently these animals excreted greater amounts of salts than afebrile rats. This could account for the lower plasma osmolality observed in the febrile rat.

A simple cannula assembly for chronic bilateral brain infusion in freely moving rats.

Osmotic minipumps provide a means for the chronic infusion of agents into the brain of the awake unrestrained animal. This paper describes the construction of a simple and inexpensive cannula assembly to be used with an osmotic minipump for the bilateral infusion of brain regions in the rat. The cannula/catheter assembly employs the use of a Y-joint, thereby allowing the investigator to use only one osmotic minipump per animal. This reduces the flow rate and decreases the cost of an experiment. Chronic infusion of the ventral septal area with saline using this system failed to alter the circadian rhythm of body temperature, except for a significant decrease in the amplitude of the rhythm.

A critical role for central vasopressin in regulation of fever during bacterial infection.

Previous investigations on the antipyretic properties of arginine vasopressin have used bacterial endotoxins or pyrogens to induce fever. Because these experimental models of fever fail to mimic all aspects of the responses to infection, we felt it was important to examine the role of endogenously released vasopressin as a neuromodulator in febrile thermoregulation during infection. Therefore the present study examines the effects of chronic infusion of a V1-receptor antagonist or saline (via osmotic minipumps into the ventral septal area of the brain) on a fever induced by injection of live bacteria. Telemetry was used for continuous measurement of body temperature in the awake unhandled rat. Animals infused with the V1-antagonist exhibited fevers that were greater in duration compared with those of saline-infused animals. These results support the hypothesis that vasopressin functions as an antipyretic agent or fever-reducing agent in brain. Importantly, they suggest that endogenously released vasopressin may play a role as a neuromodulator in natural fever.

Effects of interleukins on plasma arginine vasopressin and oxytocin levels in conscious, freely moving rats.

To elucidate whether interleukins are involved in vasopressin or oxytocin release during cytokine-related stressful conditions, we examined the effects of human interleukin-1 beta and interleukin-6 on plasma vasopressin and oxytocin levels in rats. Interleukin-1 beta administrated intravenously stimulated both the vasopressin and oxytocin secretion in dose-dependent manners. Neither hormone release was observed following interleukin-6 administration. Pretreatment with aspirin significantly attenuated the effects of interleukin-1 beta on both the vasopressin and oxytocin levels. SC-19220, a prostaglandin E2 receptor antagonist, did not affect the interleukin-1 beta-induced increase of plasma oxytocin levels, but almost completely abolished its effect on plasma vasopressin levels. These results suggest that under certain stressful conditions which accompany the stimulation of cytokine production, interleukin-1 is involved in the increase of plasma vasopressin and oxytocin levels and, moreover, different kinds of prostaglandins are suggested to participate in these interleukin-1-induced hormone release.

Central vasopressin V1-blockade prevents salicylate but not acetaminophen antipyresis.

Recent evidence has suggested that the endogenous antipyretic arginine vasopressin (AVP) may participate in drug-induced antipyresis. This study sought to further those investigations by comparing the effects of two other antipyretic drugs, sodium salicylate and acetaminophen, administered intraperitoneally, during AVP V1-receptor blockade within the ventral septal area (VSA) of the rat brain. During endotoxin-evoked fever, V1-receptor blockade within the VSA of the conscious unrestrained rat significantly antagonized the antipyretic effects of salicylate. The effects of the V1-antagonist on salicylate-induced antipyresis were dose related. In contrast, the antipyresis elicited by acetaminophen was unaffected by VSA V1-antagonist pretreatment. Neither saline nor the V1-antagonist microinjected into the VSA of febrile or nonfebrile rats had any significant effects on the normal progression of endotoxin fever or normal core temperature, respectively. These data suggest that the mechanism of action of salicylate-induced antipyresis includes activation of AVP V1-type receptors within the VSA, as has been shown for indomethacin. However, the lack of effect of the V1-antagonist on antipyresis induced by acetaminophen indicates that not all antipyretic drugs act through the same mechanism in the brain.

Centrally acting vasopressin contributes to endotoxin tolerance.

Repeated daily intravenous injections of bacterial endotoxin induce a refractory state to their usual pyrogenic effects. The neuropeptide arginine vasopressin (AVP) has been implicated in natural fever suppression and may be involved in the process of pyrogenic tolerance to intravenous endotoxin. This study was conducted to test this hypothesis. Tolerance was induced by two successive daily intravenous injections of Escherichia coli endotoxin (50 micrograms/kg) into conscious unrestrained rats. This tolerance was maintained, unaltered, after a third or fourth subsequent injection. However, bilateral administration of an AVP V1-receptor antagonist (0.43-4.3 nmol) into the ventral septal area (VSA) of the rat brain markedly enhanced the thermoregulatory response to a third or fourth endotoxin challenge compared with saline controls. The effect of the V1 antagonist was dose related. In contrast, an AVP V2 antagonist (0.43 nmol) bilaterally injected into the VSA did not affect the tolerant reaction to endotoxin. Furthermore, neither saline nor the V1 antagonist significantly affected core temperature when administered within the VSA without subsequent endotoxin. These results are consistent with the hypothesis that AVP acts as an endogenous antipyretic within the VSA during fever. Moreover, the data suggest a possible role for centrally acting vasopressin during pyrogenic tolerance to E. coli endotoxin.

Antipyretic doses of centrally administered vasopressin reach physiologically meaningful concentrations in the brain of the rat as evaluated by microdialysis.

It was important to determine whether vasopressin (AVP) injected intracerebroventricularly (i.c.v.) in the rat reached the site of action within the ventral septal area (VSA) in sufficient concentrations to account for its physiological effects. Microdialysis was used to evaluate this hypothesis. The exchange rate across the dialysis tubing was determined in vitro to be 0.40%. After placement of the microdialysis cannula in the VSA of the rat the recovery of i.c.v. injected labelled or cold AVP was 0.23 and 0.20%, respectively. Maximum concentrations of AVP in the extracellular fluid of the VSA was determined to be 10.7 nM after 10 ng i.c.v. and hence extrapolated to be 1.07 nM after 1 ng i.c.v. or 2.65 nM after 2.5 ng i.c.v. between which lies the threshold dose of AVP for its antipyretic effects. This can be compared with a reported Kd for these receptors of 1.06 nM as determined by receptor binding assay.

Afferent renal nerve effects on plasma vasopressin and oxytocin in conscious rats.

Experiments were done in conscious, unrestrained, baroreceptor-intact rats to investigate the effects of afferent renal nerve (ARN) stimulation on circulating levels of arginine vasopressin (AVP) and oxytocin (OXY). Electrical stimulation of ARN elicited a rise in arterial pressure (AP) of 12 +/- 2 mmHg and no significant change in heart rate (HR). Plasma concentrations of AVP and OXY measured by radioimmunoassay were significantly increased immediately after the stimulation period. AVP increased from control levels of 1.89 +/- 0.72 to 4.59 +/- 1.19 pg/ml after stimulation of ARN and OXY increased from 4.3 +/- 0.35 to 10.0 +/- 1.00 pg/ml. AVP remained significantly elevated at 1 h after stimulation and gradually returned to control levels by 3 h after stimulation. On the other hand, OXY values were at control levels at 1 h after stimulation. Stimulation of ARN, after cutting ARN proximal to the stimulating electrode, and sham ARN stimulation did not alter AP, HR, or plasma levels of AVP and OXY. Plasma osmolality was not altered during the experiments. These data demonstrate that, in the conscious rat, afferent information from the kidney alters the release of AVP and OXY from the neurohypophysis and suggests that ARNs are important components of a neuronal circuit that modulates differentially the release of these hormones, which function in the homeostatic regulation of AP and fluid balance.

Central vasopressin V1-receptors mediate indomethacin-induced antipyresis in rats.

Central microinjection or infusion of an arginine vasopressin (AVP) V1-receptor antagonist within the brain of the conscious, unrestrained, and febrile rat inhibited or abolished the antipyretic effects of peripherally administered indomethacin (Indo). The degree of Indo-induced antipyresis was determined by 2-h thermal indexes (degree C.h) calculated from the time of Indo injection. Microinjection of saline or V1-receptor antagonist within the ventral septal area (VSA) of the rat brain immediately followed by intraperitoneal Indo evoked antipyretic responses of -1.63 +/- 0.17 and -0.24 +/- 0.09 degrees C.h, respectively (P less than 0.01). Infusion of the VSA with saline or V1-receptor antagonist before and after Indo resulted in thermal indexes of -1.35 +/- 0.16 and 0.13 +/- 0.30 degree C.h, respectively (P less than 0.01). Central microinjection of a V2-receptor antagonist did not significantly effect Indo-induced antipyresis compared with paired saline controls. Neither saline nor the V1-receptor antagonist affected nonfebrile body temperature when microinjected into the VSA. These data indicate the importance of AVP V1-receptors within the VSA in mediating the potent fever-reducing properties of the antipyretic drug Indo. Furthermore, these data call into question whether prostaglandin synthesis inhibition is a sufficient explanation of drug-induced antipyresis.

Criteria for establishing a physiological role for brain peptides. A case in point: the role of vasopressin in thermoregulation during fever and antipyresis.

This paper has attempted to present and discuss the criteria necessary for the evaluation of a specific physiological role for a peptide in the CNS. These criteria are based on many experimental approaches to the problem and conclusions must be supported by the weight of the evidence. These criteria were illustrated by examining the hypothesis that AVP is an antipyretic neurotransmitter involved in regulating febrile increases in Tb by release and action in the VSA of the brain. The weight of the evidence in this case implies that this hypothesis is essentially correct. The only serious conflicting evidence comes from the work with Brattleboro rats. It is hoped that further research will resolve these discrepancies or result in a suitably modified hypothesis.

Angiotensin acts at the subfornical organ to increase plasma oxytocin concentrations in the rat.

We have examined the effects of systemic angiotensin II (AII) on plasma oxytocin (OXY) concentrations in freely moving male Sprague-Dawley rats. We have also examined the role of the subfornical organ (SFO) as a CNS site at which circulating AII acts to influence secretion of this neurohypophysial peptide. OXY concentrations were measured by radioimmunoassay in plasma samples obtained by drawing blood samples through indwelling atrial catheters. In SFO intact animals (n = 8) AII infusion (1.0 microgram/kg/min) resulted in increases in plasma OXY concentrations from baseline values of 6.8 +/- 2.5 pg/ml to postinfusion concentrations of 44.9 +/- 11.9 pg/ml. In a second series of experiments electrolytic lesions were placed in the region of the SFO prior to testing the effects of AII infusion on OXY concentrations. Two further experimental groups were thus established according to the histologically verified location of lesions in either the rostral or caudal SFO. In the caudal SFO lesioned group AII infusion resulted in increases in plasma OXY concentrations from control values of 6.9 +/- 1.4 pg/ml to postinfusion levels of 45.1 +/- 9.8 pg/ml. These changes were not significantly different from the SFO intact group. In contrast rostral SFO lesions resulted in significantly elevated basal concentrations of OXY (17.4 +/- 3.4 pg/ml, n = 6) while postinfusion concentrations were found to be 22.8 +/- 4.9 pg/ml indicating that AII infusion was without effect following such lesions. These data are in accordance with the hypothesis that circulating AII acts at the SFO to influence SFO efferents which in turn activate OXY secreting neurons in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. These neuroendocrine cells then release this peptide into the systemic circulation from the posterior pituitary.

Enhanced fever following castration: possible involvement of brain arginine vasopressin.

Arginine vasopressin (AVP) is thought to act as an antipyretic in the ventral-septal area (VSA) of the brain. As AVP content of this area has been shown to be virtually eliminated following long-term castration, we have tested the hypothesis that castrated rats would display enhanced fevers. Four months after castration (or sham castration), male Wistar rats were given prostaglandin E1 (200 ng), purified interleukin 1 (25 U), or saline (5 microliters) into a lateral cerebral ventricle. Castrated rats displayed fevers of longer duration, reflected as significantly enhanced thermal indexes, than did age-matched sham-operated controls. Castrated rats also were less able to defend their body temperatures to ambient heat stress but not to ambient cold. AVP content of VSA and lateral septum, but not of hippocampus, of castrated rats was significantly reduced; oxytocin content of the three areas was unchanged following castration. These data support earlier studies concerning effects of castration on septal AVP content and are consistent with the possibility that AVP is an antipyretic in the VSA of the rat.

Simultaneous and independent release of vasopressin and oxytocin in the rat.

The relative dependence or independence of the secretion of the neurohypophysial hormones, arginine vasopressin and oxytocin, was investigated using a wide variety of stimuli reported to cause the secretion of one or the other hormone. Differences in species, animal preparations, sampling techniques, assays, and other factors make comparison of many previous studies difficult. The aim of this study was to overcome these problems by using the same methodology, animal species, and assays to compare vasopressin and oxytocin release. To further strengthen the analysis, determinations of vasopressin and oxytocin were done in the same blood samples. The results demonstrated that during simultaneous release of both hormones, vasopressin is released in greater proportion following restraint stress, hemorrhage, isotonic hypovolemia, and nicotine, whereas oxytocin is released in greater proportion following endotoxin or hypertonic saline. Vasopressin was released without oxytocin following diethylstilbestrol. Oxytocin was released without concomitant vasopressin release following exercise, hypothermia, hyperthermia, labour, and lactation. Neither oxytocin nor vasopressin release was observed following thyroid-releasing hormone or insulin-induced hypoglycemia. These data illustrate the marked flexibility of the hypothalamo-neurohypophysial system that regulates secretion of vasopressin and oxytocin.

Antipyresis due to centrally administered vasopressin differentially alters thermoregulatory effectors depending on the ambient temperature.

The intracerebroventricular (i.c.v.) administration of arginine vasopressin (AVP), in the febrile rat elicits an antipyresis at cold, warm and neutral ambient temperatures. These experiments were conducted, therefore, to elucidate the thermoregulatory effector mechanisms responsible for this antipyretic effect. At 25 degrees C, AVP-induced antipyresis was mediated by tail skin vasodilation while metabolic rate was unaffected. At 4 degrees C, the antipyresis produced by AVP was approximately double that seen at 25 degrees C. This effect appeared to be mediated exclusively by inhibition of heat production since the metabolic rate decreased markedly following AVP. This antipyresis at 4 degrees C was accompanied by cutaneous vasoconstriction. At 32 degrees C, neither vasomotor tone, metabolic rate nor evaporative heat loss could be shown to contribute to the small antipyretic effect elicited by AVP. We conclude from these data that i.c.v. AVP is producing antipyresis by affecting the febrile body temperature set-point mechanism since the thermoregulatory strategy to lose heat varies at different ambient temperatures and the decrease in body temperature cannot be shown to be due to changes in a single effector mechanism.

Activation of subfornical organ efferents stimulates oxytocin secretion in the rat.

The effects of activation of subfornical organ (SFO) efferents on plasma oxytocin concentrations were examined in conscious freely moving male Sprague-Dawley rats. Blood samples were obtained through chronically implanted atrial catheters and SFO efferents were activated electrically using chronically implanted bipolar stimulating electrodes. Hormone concentrations were measured by radioimmunoassay, and experimental animals were assigned to one of 3 experimental groups according to histologically verified anatomical locations of stimulating electrodes in either the SFO, the hippocampal commissure (HC), or the medial septum (MS). Electrical stimulation in the SFO resulted in increased plasma concentrations of oxytocin from control values of 2.54 +/- 0.9 pg/ml, to a post-stimulation level of 65.6 +/- 27.0 pg/ml. In contrast, stimulation in immediately adjacent structures including HC and MS was found to be without effect on plasma concentrations of oxytocin. These studies provide the first definitive evidence that SFO efferents may play a significant role in controlling the secretion of oxytocin from the posterior pituitary.