The role of protein synthesis in the hypothalamic mechanism mediating pyrogen fever

The effects of inhibition of protein synthesis by anisomycin on the pathogenesis of fever and normal thermoregulatory processes were investigated in the conscious and unrestrained cat. Subcutaneous administraton of 5.0–25.0 mg/kg of anisomycin prevented the fever normally evoked by an intravenous infusion of either 1.0 ml (10⁸ organisms) of a 1:10 dilution of S. typhosa or 1.0–5.0 ml (3.5×10⁵−2.1×10⁷ cells/ml) of endogenous pyrogen. In addition, systemic pre-treatment with anisomycin delayed and/or blocked the fever typically elicited by a direct micro-injection into the anterior hypothalamic, preoptic area (AH/POA) at AP 12.5–16.0 of 1.0 μl of the endotoxin. Anisomycin did not alter the hyperthermic response to an anterior hypothalamic injection of either 1.0–7.0 μg/1.0 μl of serotonin (5-HT) or 100.0 ng/1.0 μl of prostaglandin (PGE). Inhibition of protein synthesis, furthermore, did not prevent the fall in body temperature usually produced by an intrahypothalamic micro-injection of 2.33–14.0 μg/1.0 μl of either norepinephrine (NE) or dopamine (DA). The thermoregulatory capacity of the cat was unaffected by the administration of comparable doses of anisomycin, i.e., the animal was able to maintain normal body temperature (±0.5°C) when exposed to an ambient temperature of either 10°C or 34°C. These results strongly suggest that the synthesis of new protein within the region of the AH/POA is a functional requisite for the development of a pyrogen-induced fever.     

Site-specific inhibition of receptivity by intracranial anisomycin in hamsters

The ventromedial nucleus of the hypothalamus (VMH) has been implicated in the mediation of the hormonal control of female rodent sexual behavior. However, in hamsters, progesterone (P) has been found to have effects on sexual receptivity in other diencephalic and mesencephalic sites as well. Progesterone is thought to exert its behavioral effects by altering protein synthesis in CNS target neurons. We tested the effects of 30 gauge implants of the protein synthesis inhibitor anisomycin in the preoptic area (POA), VMH, and ventral mesencephalon (VMES) 30 minutes before 500 micrograms P SC, on the facilitation of lordosis in ovariectomized estrogen-primed female hamsters. The same animals were tested one week later with estrogen and progesterone treatment but without anisomycin. Anisomycin reduced sexual receptivity (lordosis) when placed in the VMH or VMES, but not when delivered to the POA. The results confirm the importance of the VMH in the mediation of progesterone facilitation of female sexual behavior, but also provide evidence that ventral midbrain structures may play a role in female sexual receptivity in hamsters. These two structures may be important for different aspects of lordosis. Progesterone effects in both sites appear to be protein synthesis dependent.     

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.     

Hypothalamic Na and Ca ions and temperature set-point: New mechanisms of action of a central or peripheral thermal challenge and intrahypothalamic 5-HT, NE, PGE1, and pyrogen

The effects of changes in ambient and central temperature, amines, PGE₁ and pyrogen were investigated with respect to the mechanism of Na⁺−Ca⁺⁺ ratio in the posterior hypothalamus of the unrestrained cat. Guide tubes were implanted bilaterally above the posterior hypothalamic area of 23 cats so as to accommodate push-pull cannulae. After a Na⁺ or Ca⁺⁺ sensitive site was identified by perfusion at 50 μ1/min of an artificial CSF containing 10.4 mM excess Ca⁺⁺ ions or 13.6 mM excess Na⁺ ions, several types of experiments were undertaken with the results summarized as follows: if the cat was exposed to a cold or warm environmental temperature as the posterior hypothalamus was perfused with excess cation, the typical hypothermia was produced by Ca⁺⁺ and hyperthermia by Na⁺ ions. However, if the cat was exposed to peripheral cooling or warming 30 min prior to the perfusion, the fall or rise produced by Ca⁺⁺ or Na⁺ was attenuated or prevented. In other experiments, 1.0 μCi ^{4 5}Ca⁺⁺ was injected in the ion sensitive site in the posterior hypothalamus to label stores of the cation. Raising of ambient temperature caused a retention of ^{4 5}Ca⁺⁺ in this hypothalamic area, whereas a cold environmental temperature enhanced the efflux of ^{4 5}Ca⁺⁺ at the same perfusion site. The magnitude of change in ^{4 5}Ca⁺⁺ efflux depended upon the intensity of the thermal challenge. Similarly, warming of the anterior hypothalamic, preoptic area by means of implanted thermodes caused an immediate diminution in ^{4 5}Ca⁺⁺ efflux in the posterior hypothalamus, whereas cooling of this anterior region augmented the extrusion of ^{4 5}Ca⁺⁺ ions from the posterior area. When substances which produce a temperature change were applied to the same thermosensitive zone, the direction of shift in ^{4 5}Ca⁺⁺ flux in the posterior area corresponded to the signal for heat production or heat loss. That is, the microinjection of 5-HT, PGE₁, or Salmonella typhosa into the anterior hypothalamus enhanced the efflux of ^{4 5}Ca⁺⁺ in the posterior hypothalamus as hyperthermia developed, whereas a similar microinjection of norepinephrine reduced the ^{4 5}Ca⁺⁺ output from the same sites. Finally, locally anesthetizing the cells of the anterior hypothalamus by the nerve blocker, procaine, prevented the cold and heat-induced ^{4 5}Ca⁺⁺ efflux and retention, respectively. These results suggest that if the Na⁴−Ca⁺⁺ ratio in the posterior hypothalamus establishes and maintains the set-point for body temperature of 37°–38°C, the mechanism of lability of Ca⁺⁺ through changes in binding characteristics, transport, or metabolism of the cation serves two purposes: (1) the active defense of the set-point temperature through gradations in ion shifts; and (2) the upward or downward change in set-point value, pathological or normal, triggered by virtue of impulses relayed from the anterior hypothalamus.     

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.     

Blockade of lipopolysaccharide-induced fever by subdiaphragmatic vagotomy in guinea pigs

It is generally believed that fever is mediated by certain cytokines produced by immune cells activated by exogenous pyrogens, e.g., lipopolysaccharides (LPS), released into the circulation and transported to the brain. There, the cytokines are thought to stimulate prostaglandin (PG) E₂ production within the organum vasculosum laminae terminalis region. PGE, then may act as a febrigenic mediator locally or in the surrounding preoptic area (POA). However, whereas the increases in preoptic PGE₂ and body (core) temperature (T_c) following the intravenous (i.0 administration of LPS correlate temporally, cytokine levels in blood lag both these increases. From recent data in the literature, we have conjectured that a possible, alternative communication pathway between the i.v. LPS-activated immune system and brain PGE₂ may be provided by the vagi. To test this possibility, we measured the levels of PGE₂ in the extracellular fluid of the POA (collected by microdialysis) of conscious, subdiaphragmatically vagotomized or sham-operated guinea pigs following LPS administration (2 μg/kg; i.v.); controls received pyrogen-free saline (PFS). The effluents from the microdialysis probes were collected over 30-min periods throughout the experiments and the samples analyzed by radioimmunoassay; (T_c) was monitored continuously using thermocouples inserted 5 cm into the colon. LPS induced a biphasic fall in T_c and failed to increase preoptic PGE₂ levels in the vagotomized guinea pigs (n = 10), whereas in their sham-operated controls (n = 10) it induced increases in both preoptic PGE₂ and (T_c) within 15 min after its injection; PFS (n = 13) had no effect on either variable. We postulate that peripheral immune cell-derived signals may be transmitted via the vagi to the medulla. From other data, we suggest further that they may be conveyed from here via the ventral noradrenergic bundle to the POA region, where the released norepinephrine induces the local synthesis of PGE₂ and, hence, fever onset.