Fever induced by macrophage inflammatory protein-1 (MIP-1) in rats: hypothalamic sites of action.

The purpose of this study was to clarify the central site of action as well as functional characteristics of the febrile response of the cytokine, macrophage inflammatory protein-1 (MIP-1). Guide cannulae for microinjection were implanted stereotaxically in the rat just above the pyrogen and thermosensitive area of the anterior hypothalamic, preoptic area (AH/POA). Following postoperative recovery, the body temperature of each rat (Tbo) was monitored during an experiment by a colonic thermistor probe at 0.5-1.0-h intervals. When MIP-1 was microinjected in a 0.5-microliter volume into the AH/POA in one of eight concentrations ranging from 0.0028 nanograms (ng) to 9.0 ng, an intense monophasic or biphasic fever was evoked. The MIP-1-induced increase in the Tbo of the rat was characterized by its short latency of 15 to 30 min and an inverse dose-response curve. Measures of mean latency and maximal rise in Tbo following MIP-1 confirmed the potency of this dose. Although the dose of 0.028 ng produced a fever of over 2.0 degrees C with a latency of only 15 min or less, the hyperthermic response became less intense as the dose of MIP-1 was increased. An anatomical mapping of sites of microinjection which reacted to MIP-1 in mediating fever revealed that the medial portion of the POA of the rat just rostral to the border of the AH was the region of maximum sensitivity to the cytokine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fever and feeding in the rat: Actions of intrahypothalamic interleukin-6 compared to macrophage inflammatory protein-1β (MIP-1β)

The chemokines, macrophage inflammatory protein-1 (MIP-1) and its subunit MIP-1β, induce an intense fever in the rat when they are injected directly into the anterior hypothalamic, pre-optic area (AH/POA), a region containing thermosensitive neurons. The purpose of this study was to compare the central action on body temperature (T_b) of MIP-1β with that of interleukin-6 (IL-6), which also has been implicated in the cerebral mechanism underlying the pathogenesis of fever. Following the stereotaxic implantation in the AH/POA of guide cannulae for repeated micro-injections, radio transmitters which monitor T_b continuously were inserted intraperitoneally in each of 15 male Sprague-Dawley rats. Each micro-injection was made in a site in the AH/POA in a volume of 1.0 μl of pyrogen-free artificial CSF, recombinant murine MIP-1β, or recombinant human IL-6. MIP-1β in a dose of 25 pg evoked an intense fever characterized by a short latency, a mean maximum rise in T_b of 2.4 ± 0.21°C reached by 3.7 ± 0.42 hr, and a duration exceeding 6.5 hr. Injected into homologous sites in the AH/POA, IL-6 induced a dose dependent fever of similar latency and a mean maximal increase in T_b of 1.2 ± 0.25°C, 1.8 ± 0.15°C, and 2.1 ± 0.22°C and duration of 6.2 ± 1.28 hr, 6.7 ± 0.49 hr, and 6.8 ± 0.65 hr when given in doses of 25, 50, and 100 ng, respectively. These results show that MIP-1β and the highest dose of IL-6 induce a fever of comparable intensity, but MIP-1β exerts its action in a much lower concentration. Thus, the de novo synthesis and subsequent action of the MIP-1 family of cytokines on neurons of the AH/POA in response to a pyrogen challenge apparently play a functional role in the pathogenesis of fever. Further, the endogenous activity of IL-6 in the hypothalamus which is enhanced in response to a lipopolysaccharide also may reflect its essential part in the acute phase response to a bacterial challenge. Copyright © 1994 Wiley-Liss, Inc.     

Anorexia and adipsia: dissociation from fever after MIP-1 injection in ventromedial hypothalamus and preoptic area of rats.

Certain cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1) act centrally to affect eating behavior and thermoregulation and may be involved in the physiological mechanisms leading to anorexia, adipsia and loss in body weight. The newly discovered macrophage inflammatory protein-1 (MIP-1) infused into the anterior hypothalamic, preoptic area (AH/POA) evokes an intense hyperthermia. The present experiments were designed to determine whether MIP-1 affects the feeding mechanism in the ventromedial hypothalamus (VMH) independently of the thermoregulatory mechanism in the AH/POA. For the microinjection of MIP-1, guide cannulae were implanted stereotaxically in the rat just above the VMH or AH/POA. Following postoperative recovery, each unrestrained rat was adapted to procedures whereby body temperature and intakes of food and water available ad lib were monitored at predetermined intervals. When an efficacious dose of 5.6 picograms (pg) MIP-1 was microinjected in a volume of 0.5 microliters into the VMH, the intake of food in the rat was reduced significantly in the short term and throughout the following 22 h. Within intervals of 30 min and 4.0 h following MIP-1, the amount of food consumed was 4.0 and 10 g, respectively, below that eaten by control rats given the saline solvent vehicle injected at the same site in the VMH. Over the entire test period, the intake of water was similarly significantly below that of the control rats. Whereas MIP-1 injected into the AH/POA evoked fever accompanied by a transient decline in feeding, the body temperature of the rats was unaffected by the cytokine injected in the VMH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fever evoked by macrophage inflammatory protein-1 (MIP-1) injected into preoptic or ventral septal area of rats depends on intermediary protein synthesis

Macrophage inflammatory protein-1 (MIP-1), a novel cytokine composed of α/β subunits, is released from macrophages during infection, MIP-1 injected intravenously in the rabbit or into the anterior hypothalamic, preoptic area (AH/POA) of the rat causes an intense fever, which is not blocked by prostaglandin synthesis inhibitors, ibuprofin or indomethacin, respectively. The purpose of this study was to determine the role of de novo protein synthesis on the fever evoked by MIP-1 applied to thermosensitive cells of the AH/POA. Guide cannulae were implanted bilaterally above the AH/POA or ventral septal area (VSA) and medially above the third cerebral ventricle in each of 11 male Sprague-Dawley rats. Following postoperative recovery, body temperature (T_b) was monitored by a colonic thermistor probe. The bilateral microinjection of MIP-1 in a dose of 14 pg per 0.5 μ1 into the AH/POA caused a biphasic elevation in T_b to 0.9 ± 0.2 °C within 3.0 h, and persisted for over 6.0 h. An identical injection of MIP-1 into the VSA increased T_b biphasically to 0.1 ± 0.1 °C within 1.0 h and to 0.8 ± 0.3 °C within 3.0 h. The infusion into the third ventricle of 80 μg/10 μ1 of the inhibitor of protein synthesis, anisomycin, either 10 or 30 min before the microinjection of MIP-1 into the AH/POA, attenuated significantly the rise in T_b for 1.0 to 3.0 h or 2.5 to 3.0 h, respectively. These results coincide with the earlier finding that anisomycin inhibits both endotoxin- and IL-1β-induced fevers. Further, the synthesis of a new protein factor may be required functionally for the initiation and maintenance of a fever whose mechanism of induction apparently is metabolically independent of the cyclooxygenase pathway.     

Differential sensitivities of pyrogenic chemokine fevers to cyclooxygenase isozymes antibodies

It has been proposed that prostaglandin (PG)E(2) production via a process catalyzed by the inducible isoform of cyclooxygenase (COX)-2 and activation of specific PGE(2) receptor subtypes within the preoptic/anterior hypothalamus (AH/POA) is the last step and unique pathway in the induction of a fever. However, many data support the existence of a PG-independent pathway. That is, other more rapid mechanisms, which involve the constitutive COX-1 isozyme, may be more critical for a PG-dependent fever. Thus, we examined the role of both COX isoforms in the AH/POA in fevers induced by macrophage inflammatory protein (MIP)-1beta, a PG-independent pyrogen, and RANTES (regulated on activation, normal T-cells expressed and secreted), a PG-dependent pyrogen. In freely moving rats, two independent polyclonal antibodies were used which neutralize COX-1 and COX-2. The microinjection of either MIP-1beta or RANTES into the pyrogen-sensitive region of the AH/POA induced an intense fever of rapid onset. Peripheral pretreatment with an antipyretic dose of dexamethasone which prevents COX-2 expression, or the microinjections into the AH/POA of either anti-COX-1 or anti-COX-2, blocked the febrile response induced by RANTES but not that induced by MIP-1beta. These results provide strong evidence for the existence of rapid mechanisms in the AH/POA which involve both COX isozymes during the fever induced by RANTES, and further support the existence of an alternative PG-independent pathway in the febrile response.     

Macrophage Inflammatory Protein-1β (MIP-1β) Produced Endogenously in Brain During E. coli Fever in Rats

Macrophage inflammatory protein-1 (MIP-1) evokes an intense fever, independent of a prostaglandin mechanism, and is now thought to play an important role in the defence response to bacterial pyrogens. The purpose of this study was 2-fold: (i) to determine whether the potent doublet of this cytokine, MIP-1β, is actually produced in the brain in response to a pyrogenic dose of a lipopolysaccharide of Escherichia coli and (ii) to determine the anatomical site of synthesis of this cytokine in the brain. Following the intense fever produced by intraperitoneal administration of lipopolysaccharide in the unrestrained rat, MIP-1β immunoreactivity was identified post mortem in two regions of the brain implicated in fever: the organum vasculosum laminae terminalis (OVLT) and the anterior hypothalamic, preoptic area (AH/POA). Microinjection of goat anti-mouse MIP-1β antibody (anti-MIP-1β) directly into the AH/POA markedly suppressed fever in rats in response to lipopolysaccharide. Further, anti-MIP-1β administered 180 min after the injection of lipopolysaccharide acted as an antipyretic and reversed the fever induced by the endotoxin. Anti-MIP-1β or control immunoglobulin G antibody microinjected into the hypothalamus immediately before the intraperitoneal injection of the control saline did not alter the temperature of the rats. Taken together, the present results demonstrate that MIP-1β is produced in the brain in response to a bacterial endotoxin. These observations, in the light of earlier data on fever induced by MIP-1β, further support the hypothesis that endogenously synthesized MIP-1β acts as an intermediary factor in the evocation of fever by acting on the thermosensitive cells of the brain.     

Prostaglandin E2 exerts an awaking effect in the posterior hypothalamus at a site distinct from that mediating its febrile action in the anterior hypothalamus.

The precise sites for prostaglandin E2 (PGE2)-related activity responsible for the promotion of wakefulness and elevation of brain temperature were determined in several regions of the monkey brain. PGE2 was administered through a microdialysis probe into 11 brain loci mainly in the preoptic area/anterior hypothalamic region (POA/AH) and the tuberomammillary region in the posterior hypothalamus (TuM-PH). Administration of PGE2 into the POA/AH resulted in a marked and dose-dependent febrile response. When a low dose (15 pmol/min) of PGE2 was administered into the POA/AH, brain temperature increased significantly up to a 0.6 degrees C rise, but sleep behavior and amounts of time in wakefulness, slow wave sleep (SWS), and REM sleep during the administration period were not significantly different from those of the control monkeys. Dose of PGE2 above 150 pmol/min elevated the brain temperature and heart rate more markedly and suppressed sleep. The degree of inhibition of sleep by PGE2 was closely correlated with the changes in these autonomic responses. On the other hand, when a low dose of PGE2 was administered into the TuM-PH, the time spent awake during the administration period increased up to 3.5-fold and the amount of time spent in SWS decreased to 50% of that of the control level, with negligible changes in brain temperature and heart rate. The awaking response of PGE2 in the TuM-PH was also dose dependent, but was not correlated with the change in brain temperature. Among 11 brain regions tested, the hyperthermic effect of PGE2 was most potent in the POA, while its awaking effect was most pronounced in the TuM-PH close to the mammillary complex. These findings demonstrate that the site of action of PGE2 for the regulation of sleep-wakefulness is clearly distinct from that for the temperature regulation. PGE2 may be involved in the neurochemical mechanism of wakefulness mediated in a specific site in the PH.     

Cytokines and Thermoregulation: Interleukin-9 Injected in preoptic area fails to evoke fever in rats

A number of the members of the family of cytokines including IL-1, IL-2, IL-6, and IL-11 act directly in the brain to induce a febrile response in the rat and other species. The purpose of this study was to examine the effect of interleukin-9 (IL9) when this cytokine is applied directly to the thermosensitive and pyrogen reactive region of the anterior hypothalamic, preoptic area (AH/POA). In male Sprague-Dawley rats, guide cannulae for microinjection into the AH/POA were implanted stereotaxically, and radio transmitters for monitoring body temperature (T_b) were placed intraperitoneally. Following postoperative recovery, recombinant murine macrophage inflammatory protein (MIP)-1β was microinjected in the AH/POA of each rat in a dose of 28 pg/1μl to identify pyrogen reactive sites in the AH/POA. Then recombinant human IL-9 was suspended in pyrogen-free CSF vehicle and microinjected in the same sites in concentrations of 2.4, 24, and 240 U/μl. In contrast to the pyrexic action of MIP-1β, IL-9 failed to elicit a significant alteration in the T_b of the rats at any of the doses tested. IL-9 was also without effect on the intakes of either water or food. These results demonstrate that IL-9 applied to the region of the diencephalon in which other cytokines act to evoke fever may not play a direct role in the thermogenic component underlying the acute phase response. However, as demonstrated in several different cell systems, IL-9 may require a cofactor related to pyrogen for a febrile response to develop.     

Comparative pyrogenic potency of endogenous prostanoids and of prostanoid-mimetics injected into the anterior hypothalamic/preoptic region of the cat

In both pyrogen-induced fever and fever subsequent to acute hypothalamic trauma, pyrexia is believed to be mediated by cyclooxygenase products acting within the anterior hypothalamic/preoptic (AH/PO) region of the brain. The goal of the present study was to assess, through a potency analysis, the likely contributions of various prostanoids to pyrexia production. Prostanoids and prostanoid-mimetics were injected bilaterally into the AH/PO region of conscious, indomethacin pretreated cats, and partial dose-response curves for pyrexic activity were obtained. ED1 degrees doses (doses producing a 1 degree C fever) for PGE2, PGE1 and 6-keto-PGE1 (a metabolite of PGI2 and/or of the PGI2 hydrolysis product, 6-keto-PGF1 alpha) ranged between 2 and 15 pmol. PGF2 alpha and the stable PGI2-mimetics, iloprost and 6-beta-PGI1, required doses of 900-1100 pmol. PGD2 and 6-keto-PGF1 alpha had ED1 degrees doses of 2200-2400 pmol. PGI2, thromboxane (TX) B2 and the TXA2/PGH2-mimetics, SQ26655, 9,11-azo-PGH2 and U46619, were incapable of producing a 1 degrees C rise at the maximum dose of 30,000 pmol. The results offer no support for an involvement in fever of PGF2 alpha, PGD2, TXA2, TXB2, PGH2, PGI2 or 6-keto-PGF1 alpha. Only the 3 E-series prostaglandins were sufficiently potent to merit serious consideration as mediators of pyrexia. Of these, only PGE2 is known to be produced in abundance by cat brain; no information is available regarding PGE1 production, and our results with PGI2 and 6-keto-PGF1 alpha indicate that cat brain may not synthesize 6-keto-PGE1. The results thus suggest an important role for PGE2 in fever production in the cat and are compatible with an involvement of PGE1.     

Dissociation of locomotor impairment from mydriasis evoked by clonidine injected into cat''s rostral hypothalamus

The anterior hypothalamic preoptic area (AH/POA) was examined as a possible site of action of clonidine and other alpha noradrenergic receptor agonists which evoke motor and autonomic changes. Chronically indwelling guide cannulae were implanted stereotaxically in the diencephalon of the cat. Following post-operative recovery, a micro-injection into AH/POA was made in a volume of 1.0 microliter of one of the following compounds: 5.0-50.0 micrograms clonidine, 5.0-50.0 micrograms norepinephrine, 5.0-50.0 micrograms phenylephrine and 5.0-50.0 micrograms methoxamine. The smallest dose of 5.0 micrograms clonidine produced a brief period of restlessness, licking, retching and emesis but a much longer-lasting mydriasis. When the dose of clonidine was raised to 20 micrograms, the cat became behaviorally sedated, after a latency of about 15 min, for a period of up to 1.0-2.0 hr. This was accompanied by a prolonged period of mydriasis and preceded by a short interval of restlessness, licking, retching and emesis. After the highest dose of 50.0 micrograms clonidine was micro-injected in AH/POA, a profound impairment of motor activity, adynomia and restlessness developed within 15-20 min, persisted for 30 to 60 min and was accompanied also by mydriasis with maximal pupillary dilation lasting for up to six hr. When 5.0-50.0 micrograms phenylephrine or 5.0-50.0 micrograms norepinephrine were micro-injected at clonidine-reactive sites in AH/POA, only rarely were brief instances of restlessness, licking, retching and emesis observed; however, methoxamine at all doses tested failed to produce any visible signs of autonomic or motor disturbance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fever produced by intrahypothalamic pyrogen: effect of protein synthesis inhibition by anisomycin

In the unrestrained cat, the inhibition of protein synthesis by anisomycin, given either subcutaneously (5.0--25.0 mg/kg) or directly into the anterior hypothalamic, preoptic area (1.0--25.0 micrograms) impaired the development of a bacterial fever. S. typhosa infused intravenously (1:10 dilution in 1 ml) or into AH/POA (1.0 microliter) evoked an intense fever which was either significantly delayed or prevented by anisomycin. Conversely, anisomycin failed to affect the typical hyperthermia evoked by 100 ng PGE2 or 1.0--7.0 micrograms 5-HT similarly infused into AH/POA. These data demonstrate that an intermediary humoral factor of unknown nature is required in the hyperthermic effector pathway underlying the febrile response.     

Indomethacin-induced antipyresis in the rat: role of vasopressin receptors

Infusion of 15 micrograms/microliters (approximately 120 micrograms/kg/h) of indomethacin within the ventral septal area of the rat brain significantly reduced a centrally induced prostaglandin E1 (PGE1) hyperthermia when compared with infusions of artificial cerebrospinal fluid. A bolus injection of a V1 receptor antagonist, d(CH2)5Try(Me)AVP, (200, 2000, or 20,000 pmol) within the ventral septal area had no effect of body temperature alone but did suppress the PGE1-induced fever. Similar bolus injections of the V1 receptor antagonist within the ventral septal area failed to alter the antipyretic action of indomethacin on the hyperthermia resulting from centrally administered PGE1. Central injections of a V2 receptor antagonist failed to alter either the PGE1-induced fever or the indomethacin-evoked antipyresis. The results suggest that the V1 receptor antagonist may exert non-specific neurodepressant effects which may interfere with the expression or production of PGE1 hyperthermia and may further mask any contribution of arginine vasopressin to the antipyretic effects of indomethacin.     

Inhibition of interleukin-1beta and prostaglandin E(2) thermogenesis by glycyl-glutamine, a pro-opiomelanocortin-derived peptide

Interleukin-1beta (IL-1beta) and other cytokines produce fever by stimulating prostaglandin E(2) (PGE(2)) synthesis in thermoregulatory regions of the preoptic area and anterior hypothalamus (POA/AH). Prostaglandin E(2) is thought to raise body temperature, at least in part, by stimulating beta-endorphin release from pro-opiomelanocortin neurons that innervate the POA/AH. In this study, we investigated whether glycyl-glutamine (beta-endorphin(30-31)), an inhibitory dipeptide synthesized from beta-endorphin post-translationally, inhibits IL-1beta and PGE(2)-induced hyperthermia. Hyperthermic sites were identified by microinjecting PGE(2) (3 fmol/1 microl) into the medial preoptic area (mPOA) of conscious, unrestrained rats. Interleukin-1beta (1 U) injection into the same PGE(2) responsive thermogenic sites in the mPOA elicited a prolonged rise in colonic temperature (T(c)) (+1.02+/-0.06 degrees C) that persisted for at least 2 h. Glycyl-glutamine (3 nmol) co-injection into the mPOA inhibited IL-1beta thermogenesis completely (T(c)=-0.18+/-0.22 degrees C). Glycyl-glutamine had no effect on body temperature when given alone to normothermic rats. Co-injection of individual amino acids, glycine and glutamine (3 nmol each amino acid), failed to influence IL-1beta-induced thermogenesis, which indicates that Gly-Gln hydrolysis does not explain its inhibitory activity. Glycyl-glutamine (3 nmol) also prevented the rise in body temperature produced by PGE(2) (PGE(2)=0.89+/-0.05 degrees C; PGE(2) plus Gly-Gln=-0.16+/-0.14 degrees C), consistent with evidence that PGE(2) mediates IL-1beta-induced fever. These findings demonstrate that Gly-Gln inhibits the thermogenic response to endogenous pyrogens.     

Differential sensitivity of estrogen target tissues: Implications for estrogen regulation of serum luteinizing hormone

In the rat, it is generally accepted that the primary site of estrogen's stimulatory (positive) effects on serum LH is the preoptic area-anterior hypothalamus (POA/AH). In contrast, the primary site of estrogen's inhibitory (negative) effects on serum LH levels has not been conclusively identified. There is evidence to suggest both a medial basal hypothalamic (MBH) and an anterior pituitary site of action. The present studies utilized a unique characteristic of these estrogen effects to investigate their putative loci. Extensive dose-response curves of estrogen's induction of positive and negative feedback indicated that the negative feedback response was activated at a lower concentration of serum estradiol than the positive feedback response. The differential sensitivities of these two responses suggested that the tissues mediating them might also be differentially sensitive to estradiol. In a previous paper, we showed that receptor translocation is an index of estrogen sensitivity. We measured receptor translocation in response to a series of estradiol doses in the POA/AH, the MBH, and the pituitary. Dose-response curves for estrogen's effect on receptor translocation showed that the pituitary receptor translocation mechanism is activated at significantly lower levels of serum estradiol than that of either the POA/AH or the MBH. These results are consistent with the POA/AH as a site of estrogen's positive feedback effects. In addition, they suggest that negative feedback in the rat may be mediated via estrogen's action at the anterior pituitary. Estrogen's negative feedback effect on serum LH occurs at a serum level of estrogen at which no receptor is translocated in the MBH. Therefore, the pituitary, which does possess nuclear receptors at these estradiol dose levels, is more likely to be the primary mediator of estrogen's negative feedback effects. In another experiment, pituitary, but not hypothalamic, receptor was translocated to the nuclear fraction with an injection of 100 μg clomiphere (Clomid). Under these conditions serum LH is depressed, thus strengthening the hypothesis that, in the rat, estrogen action on the pituitary can cause suppression of serum LH independently of the hormone's action in the hypothalamus.     

Organization of central hyperthermic mechanisms in helium-cold hypothermic hamsters

We have investigated the ability of three hyperthermic stimuli (PGE2, 5-HT and ACh) to elicit hyperthermia in the Helium-Cold (He-Cold) hypothermic hamster. Hamsters in these conditions are poikilothermic and will passively follow room temperature in a regulated cold room. Animals were injected centrally at AH/POA sites via an indwelling guide tube at body temperatures maintained between 9-12 degrees C. Active sites in the AH/POA were determined prior to the experiment by PGE2 injection. PGE2 injection at an effective AH/POA site immediately reversed the anesthetic induced hypothermia in warm air. Hamsters were induced into hypothermia by the He-Cold induction method and body temperatures were maintained in a 9 degrees C cold room. Colonic temperatures were monitored at 5 minute intervals by a YSI thermistor probe and telethermometer. Central injections of 5-HT (2 micrograms/microliter) and ACh (50 micrograms/microliter) at effective AH/POA sites evoked significant increases in colonic temperature in He-Cold hamsters. PGE2 injections were not different from saline control injections and did not elicit pronounced temperature changes in these animals. Specific blockade of the 5-HT and ACh temperature increases was demonstrated with specific antagonist injections. The results suggest that the central organization of heat-gain mechanisms in the AH/POA is the same as normothermic animals even at temperatures well below those previously investigated.     

Chemokine ligand (CCL)-3 promotes an integrated febrile response when injected within pre-optic area (POA) of rats and induces calcium signaling in cells of POA microcultures but not TNF-α or IL-6 synthesis

Although studies have shown that chemokines are pyrogenic when injected into the brain, there are no data indicating which cell types and receptors in the CNS are employed by chemokines such as CCL3 (synonym: MIP-1α) to induce fever in rats. We aimed to study, whether CCL3 induces fever when injected directly into the thermoregulatory center within the pre-optic area (POA). Moreover, we investigated whether CCL3 activates cells from POA microcultures resulting in intracellular Ca⁺⁺ mobilization and synthesis/release of TNF-α and IL-6.Microinjections of CCL3 into the POA induced a dose-dependent fever, which was accompanied by a decrease in tail skin temperature. The primary microcultures of the POA (from topographically excised rat pup brain tissue) were stimulated by bolus administrations of 100 μl CCL3 (0.1 or 0.01 μg) or sterile PBS as control. We evaluated the responses of 261 (30.89%) neurons, 346 (40.94%) astrocytes and 238 microglia cells (29.17%). Stimulation of rat POA microcultures with CCL3 was capable of inducing Ca⁺⁺ signaling in 15.31% of all astrocytes and 5.75% of all neurons investigated. No cellular Ca⁺⁺-signals were observed after overnight incubation of the cultures with antiCCR1 or antiCCR5 antibodies. CCL3 did not alter the release of the pyrogenic cytokines IL-6 or TNF-α into the supernatant of the cultures.In conclusion the present study shows for the first time that CCL-3 injected directly into the rat POA, evoked an integrated febrile response. In parallel this chemokine induces Ca⁺⁺ signaling in astrocytes and neurons via both CCR1 and CCR5 receptors when administered to POA microcultures without stimulating the synthesis of TNF-α and IL-6. It is a possibility that CCL3-induced fever may occur via CCR1 and CCR5 receptors stimulation of astrocytes and neurons from POA.     

Activation of acute-phase responses by intrapreoptic injections of endogenous pyrogen in guinea pigs

The acute-phase reaction (APR) is the concatenation of events that develops in response to infectious or other acute inflammatory stimuli. It includes fever and changes in plasma trace metal and glycoprotein levels. Endogenous pyrogen (EP) is believed to be the mediator of the APR. It acts within the preoptic-anterior hypothalamus (PO) to initiate fever; prostaglandins E (PGE) may modulate this action. To determine whether the nonfebrile responses to EP also are mediated by the PO and through PGE, guinea pigs were injected bilaterally intra-PO (iPO) with homologous EP (1 microliter) or PGE2 (0.1 microgram), and their colonic temperatures (Tco) and plasma iron (Fe), zinc (Zn), copper (Cu), and N-acetylneuraminic acid (NANA) levels were measured. For comparison, EP (2 ml) also was injected intraperitoneally (IP). Heat-denatured EP (delta EP) or pyrogen-free saline (PFS) was the corresponding control. Fevers were induced by IP EP (1.0 +/- 0.1 degrees C [mean +/- SD]), iPO EP (1.1 +/- 0.2 degrees C), and iPO PGE2 (1.4 +/- 0.2 degrees C); neither delta EP nor PFS was pyrogenic. Plasma Fe and Zn levels were decreased significantly after IP EP, but unchanged after iPO EP and PGE2. Plasma Cu and NANA levels were elevated significantly following both IP and iPO EP, but not after iPO PGE2. delta EP or PFS did not cause any changes, by either route. It appears, therefore, that EP-induced fever and rises in plasma Cu and NANA are mediated by the PO, while the decreases of plasma Fe and Zn are direct, peripheral effects. On the other hand, PGE2 appears to be involved only in the central febrile response. Indeed, guinea pigs, pretreated with indomethacin (5 mg/kg, IP), and injected iPO with EP or IP with S. enteritidis endotoxin (2 micrograms/kg), did not develop fever, but exhibited the rise in plasma Cu and NANA.     

ACh and 5-HT stimulated thermogenesis at different core temperatures in the He-Cold hypothermic hamster

Hamsters in deep experimentally induced hypothermia, at body temperatures between 7 degrees C and 11.5 degrees C, were microinjected with 5-HT and ACh at brain sites in the anterior-preoptic area of the hypothalamus (AH/POA). ACh or 5-HT was injected into an AH/POA site at different starting core temperatures in different groups of hypothermic hamsters. Colonic temperatures (Tc) were maintained, following He-Cold induction, in a temperature controlled environmental chamber and measured with a YSI thermister probe and YSI telethermometer. Injections of either 5-HT or ACh at Tc's between 7.0 degrees C and 9.0 degrees C elicited only modest increases in Tc i.e., 0.3 degrees C--0.6 degrees C, respectively. As Tc increased, however, to ranges between 9.1 degrees C--10.0 degrees C and in different animals to greater than 10 degrees C both ACh and 5-HT at the same sites elicited significant increases in Tc, 1.5 degrees C for 5-HT and 2.2 degrees C for ACh compared to saline injections. These data suggest that at the lowest Tc's we are observing a "cold block" of temperature sensitive sites in the AH/POA. Increasing the starting Tc beyond 9.0 degrees C however, evokes significant increases in heat-gain following AH/POA injection of either ACh or 5-HT. These data are consistent with Myers' observations concerning the organization of heat-gain mechanisms at AH/POA sites. In addition, they suggest that both the afferent limb of the heat-gain circuit (5-HT) and the efferent limb of the circuit (ACh) are functionally impaired when Tc is close to the physiological limit in the He-Cold hypothermic hamster.     

Antibodies to macrophage inflammatory protein-1β in preoptic area of rats fail to suppress PGE2 hyperthermia

This study determined whether macrophage inflammatory protein-1β (MIP-1β) plays a role in the hyperthermia caused by prostaglandin E₂ (PGE₂) given intracerebroventricularly (i.c.v.) in the rat. In these experiments, anti-murine MIP-1β antibody (anti-MIP-1β) was micro-injected in the anterior hypothalamic, preoptic area (AH/POA) just before i.c.v. PGE₂. The results showed that anti-MIP-1β failed to alter the PGE₂ hyperthermia. However, immunocytochemical studies revealed MIP-1β immunoreactivity detectable in both the organum vasculosum laminae terminalis (OVLT) and AH/POA in the febrile rat. These data thus demonstrate that MIP-1β is sequestered in diencephalic structures underlying thermoregulation even though it is not involved in PGE₂ hyperthermia. This dissociation supports the viewpoint that at least two distinct systems exist in the brain which underlie a febrile response: MIP-1β underlies one component whereas PGE₂ comprises the other.     

Rostral hypothalamus: A new neuroanatomical site of neurochemically-induced emesis in the cat

The localized effect of noradrenergic agonists administered directly in the anterior hypothalamic preoptic area (AH/POA) in inducing emesis in the cat was investigated. Of the noradrenergic agonists tested, which included norepinephrine, clonidine, phenylephrine and methoxamine, only clonidine in doses of 5.0-50.0 micrograms was found to evoke emesis consistently when micro-injected in a volume of 1.0 microliter into AH/POA of the unrestrained cat. The emetic response to clonidine was short-lasting, generally dose-dependent in terms of latency and frequency, and occurred in bouts of one to three episodes. The sequence of the vomiting response, beginning with licking and retching, functionally resembled a normal pattern of an emetic response. The clonidine-induced emesis was not antagonized by the following antagonists micro-injected in AH/POA just prior to clonidine: alpha-adrenergic blocking agents, yohimbine, RX 781094 and phentolamine; the antimuscarinic drug, atropine; the serotonin antagonist, methysergide; the opioid antagonist, naloxone; and the dopamine antagonist, chlorpromazine. Therefore, it would appear that clonidine-induced emesis is not mediated by alpha noradrenergic, serotonergic, dopaminergic, muscarinic and opiate receptor systems within the AH/POA of the cat. Finally, the obtained results show that apart from the area postrema and a circumscribed zone of the brain-stem reticular formation, the hypothalamus is now implicated as a neuroanatomical site in the central nervous system mechanism underlying neurochemically-induced emesis.