Effects of endogenous pyrogen and prostaglandin E2 on hypothalamic neurons in guinea pig brain slices

To investigate the direct effects of endogenous pyrogen (EP) and prostaglandin E2 (PGE2) on the activity of neurons in the preoptic and anterior hypothalamic (PO-AH) region, single-unit activity was recorded from brain tissue slices prepared from the PO-AH region of guinea pigs. When EP was applied into the perfusate 18% of warm-responsive neurons decreased their activity, and 23% of warm-responsive neurons increased their activity. Most of the thermally insensitive neurons did not respond to EP. PGE2 inhibited 29% of warm-responsive neurons and facilitated 15% of them. Moreover, when EP and PGE2 were applied to the same neurons at different times, the same directions of changes in neuronal activity were observed in 72% of total neurons examined. These results suggest that EP and PGE2 change the neuronal activity of the thermoresponsive neurons in the PO-AH region involved in fever induction. However, by these results, the direction of neuronal response induced by these substances could not be generally categorized based on the thermoresponsiveness of the individual neuron.     

Effect of PGE2 on preoptic and anterior hypothalamic neurons using brain slice preparation

In the present study, effects of prostaglandin (PG) E2 on the hypothalamic neurons were investigated using slice preparations of rats. Warm- (57%) and cold-responsive (33.3%) and thermally insensitive (42.3%) neurons were facilitated by PGE applied in a culture chamber. Some neurons (5.1%) showed inhibitory response to PGE2. The remaining neurons did not show any responsiveness to PGE2. Moreover, we also examined the effects of PGE2 on the hypothalamic neurons both in normal Krebs-Ringer solution and synaptic blocking medium (low Ca2+, high Mg2+). Most of the neurons (12/15) retained their responsiveness to PGE2 in the synaptic blocking medium, indicating that PGE2 has a direct action on the hypothalamic neuron. Therefore, it is conceivable that neurons in the preoptic and anterior hypothalamic region that respond to PGE2 might play an important role in the development of fever. However, characteristics of neurons responding to PGE2 were not determined based on their thermoresponsiveness.     

葛根素、生石膏配伍对致热原作用下猫POAH温敏神经元放电的影响

应用微电极细胞外记录技术,在３４只猫ＰＯＡＨ区记录了温敏神经元单位放电,研究中药葛根素和生石膏解热可能的中枢机制。致热原使１４例热敏神经元放电频率减少;使１１例冷敏神经元放电频率增加。注射等量葛根素和生石膏能反转上述作用。致热原及二药对５例温度不敏感神经元放电无影响。结果显示,葛根素和生石膏是影响致热原作用下ＰＯＡＨ区温敏神经元的电活动而解热的。二者配伍使用对冷敏神经元放电频率的影响比单独使用作用强,提示在中枢水平二者有协同作用。     

Multimodal responses of preoptic and anterior hypothalamic neurons to thermal and nonthermal homeostatic parameters

The hypothesis that thermosensitive neurons in the preoptic anterior hypothalamic nuclei (POAH) have a principal role in central thermoregulation is based on numerous findings, suggesting correlations between the activity of thermosensitive neurons and thermoregulatory responses. Such relationships have been observed during thermal (local and peripheral) and pharmacological stimulation, during modulation of neural inputs from extra-POAH brain regions, and during actual thermoregulatory responses. Recent studies using in vitro slice preparations and conscious animals have revealed that 40-70% of POAH thermosensitive neurons respond to nonthermal homeostatic parameters such as local osmolality, blood pressure, and nonthermal emotional stimuli. About two-thirds of the POAH thermosensitive neurons, which responded in monkeys during bar press thermoregulatory tasks, changed their activity during bar press feeding behavior. A high degree of convergence of thermal and nonthermal homeostatic signals on the POAH neurons, together with abundant neural connections between the POAH and divergent areas of the brain, suggests that POAH thermosensitive neurons may be involved in the coordination of thermoregulation and nonthermal autonomic and behavioral responses controlled from the hypothalamus.     

Ability of Human Leukocytic Pyrogen to Stimulate Brain Prostaglandin Synthesis In Vitro

Abstract: Fever is thought to be mediated by leukocytic pyrogen (LP), a polypeptide synthesized by phagocytic leukocytes and which is responsible for the upwards resetting of the hypothalamic thermostat. In an attempt to study the effects of LP directly on brain tissue, purified human LP was incubated with rabbit brain slices in vitro. Because of the well-documented role of prostaglandin (PG) synthesis in both the production of fever and antipyresis, PGE levels were measured on the supernates of brain slices incubated 30 min with LP. Levels of PGE increased 3- to 4-fold in rabbit anterior and posterior hypothalami. In addition, PGE levels were similarly increased in temporal cortex slices when exposed to LP. In another set of experiments, PGE levels increased 4- to 5-fold when brain tissue was incubated with a highly purified preparation of bacterial endotoxin (ET). The ability of ET to increase brain PGE levels was not affected by moderate heating (56°C, 30 min), whereas this temperature destroyed the PGE-inducing properties of LP. The antipyretic ibuprofen markedly reduced the amount of PGE measured in the brain slice supernates after stimulation with LP, suggesting that LP brings about synthesis of PGE and not the release of preformed PG. The results demonstrate that LP is a potent inducer of PGE synthesis in rabbit brain and that receptors for LP are not restricted to the thermoregulatory center, but rather may be distributed throughout the brain.     

葛根素,生石膏配伍对致热原作用下猫POAH温敏神经元放电…

应用微电极细胞外记录技术,在３４只猫ＰＯＡＨ区记录了温敏神经元单位放电,研究中药葛根素和生石膏解热可能的中机制。致热原使４例热敏神经元放电频率减少;使１１例冷敏神经元放电频率增加,注射等量葛根素和生石膏能反转上述作用。致热原及二药对５例温度不敏感神经元放电无影响。结果显示,葛根素和生石膏是影响致热原作用下ＰＯＡＨ区温敏神经元的电活动而解热的。二者配伍使用对冷敏神经元放电频率的影响比单独使用作用强,     

Effect of arecoline on neuronal activity in the posterior hypothalamus of rabbits with experimental fever

It has been found in experiments on unanesthetized rabbits that arecoline administered to the lateral ventricle of the brain produced an action which was opposite to that of leukocytic pyrogen. It inhibited the activity of individual neurons of the posterior hypothalamus and decreased the body temperature, with this decrease being attended by the signs of intensified heat emission. Arecoline injection coupled with the central action of PGE2 was followed by an increase in the neuronal activity in the posterior hypothalamus and reduction of hyperthermal response.     

Cyclic GMP alters the firing rate and thermosensitivity of hypothalamic neurons

The rostral hypothalamus, especially the preoptic-anterior hypothalamus (POAH), contains temperature-sensitive and -insensitive neurons that form synaptic networks to control thermoregulatory responses. Previous studies suggest that the cyclic nucleotide cGMP is an important mediator in this neuronal network, since hypothalamic microinjections of cGMP analogs produce hypothermia in several species. In the present study, immunohistochemisty showed that rostral hypothalamic neurons contain cGMP, guanylate cyclase (necessary for cGMP synthesis), and CNG A2 (an important cyclic nucleotide-gated channel). Extracellular electrophysiological activity was recorded from different types of neurons in rat hypothalamic tissue slices. Each recorded neuron was classified according to its thermosensitivity as well as its firing rate response to 2-100 microM 8-bromo-cGMP (a membrane-permeable cGMP analog). cGMP has specific effects on different neurons in the rostral hypothalamus. In the POAH, the cGMP analog decreased the spontaneous firing rate in 45% of temperature-sensitive and -insensitive neurons, an effect that is likely due to cGMP-enhanced hyperpolarizing K(+) currents. This decreased POAH activity could attenuate thermoregulatory responses and produce hypothermia during exposures to cool or neutral ambient temperatures. Although 8-bromo-cGMP did not affect the thermosensitivity of most POAH neurons, it did increase the warm sensitivity of neurons in other hypothalamic regions located dorsal, lateral, and posterior to the POAH. This increased thermosensitivity may be due to pacemaker currents that are facilitated by cyclic nucleotides. If some of these non-POAH thermosensitive neurons promote heat loss or inhibit heat production, then their increased thermosensitivity could contribute to cGMP-induced decreases in body temperature.     

Effects of electrical stimulation of ventral septal area on firing rates of pyrogen-treated thermosensitive neurons in preoptic anterior hypothalamus from rabbits

Although there is considerable evidence supporting that fever evolved as a host defense response, it is important that the rise in body temperature would not be too high. Many endogenous cryogens or antipyretics that limit the rise in body temperature have been identified. Endogenous antipyretics attenuate fever by influencing the thermoregulatory neurons in the preoptic anterior hypothalamus (POAH) and in adjacent septal areas including ventral septal area (VSA). Our previous study showed that intracerebroventricular (I.C.V.) injection of interleukin-1beta (IL-1beta) affected electrophysiological activities of thermosensitive neurons in VSA regions, and electrical stimulation of POAH reversed the effect of IL-1beta. To further investigate the functional electrophysiological connection between POAH and VSA and its mechanisms in thermoregulation, the firing rates of thermosensitive neurons in POAH of forty-seven unit discharge were recorded by using extracellular microelectrode technique in New Zealand white rabbits. Our results show that the firing rates of the warm-sensitive neurons decreased significantly and those of the cold-sensitive neurons increased in POAH when the pyrogen (IL-1beta) was injected I.C.V. The effects of IL-1beta on firing rates in thermosensitive neurons of POAH were reversed by electrical stimulation of VSA. An arginine vasopressin (AVP) V1 antagonist abolished the regulatory effects of VSA on the firing rates in thermosensitive neurons of POAH evoked by IL-1beta. However, an AVP V2 antagonist had no effects. These data indicated that VSA regulates the activities of the thermosensitive neurons of POAH through AVP V1 but not AVP V2 receptor.     

Effect of beta-endorphin on the thermal excitability of preoptic neurons in the unanesthetized rabbit

The opioid peptide, beta-endorphin (beta-E), will promote changes in body temperature when injected into the brain. It is possible that beta-E alters body temperature by affecting the activity of thermoregulatory neurons in the preoptic anterior hypothalamus (POAH). Single unit activity in the POAH was recorded in unanesthetized rabbits while radiant heat was applied to the dorsal skin. Beta-E was then microinjected into the POAH, and the peripheral heating was repeated. Seventy-seven percent of the POAH neurons were responsive to skin heating. Beta-E and equal excitatory and inhibitory effects on warm-excited and warm-inhibited neurons. Four of six warm-excited neurons were converted to warm-inhibited or unresponsive following beta-E injection. Six out of ten warm-inhibited neurons were converted to warm-excited or unresponsive by beta-E. Beta-E-induced shifts in thermal excitability of POAH neurons may be responsible for the ability of POAH injections of beta-E to elevate body temperature in the rabbit.     

Carbon dioxide and pH effects on temperature-sensitive and -insensitive hypothalamic neurons.

The preoptic-anterior hypothalamus (POAH) controls body temperature, and thermoregulatory responses are impaired during hypercapnia. If increased CO(2) or its accompanying acidosis inhibits warm-sensitive POAH neurons, this could provide an explanation for thermoregulatory impairment during hypercapnia. To test this possibility, extracellular electrophysiological recordings determined the effects of CO(2) and pH on the firing rates of both temperature-sensitive and -insensitive neurons in hypothalamic tissue slices from 89 male Sprague-Dawley rats. Firing rate activity was recorded in 121 hypothalamic neurons before, during, and after changing the CO(2) concentration aerating the tissue slice chamber or changing the pH of the solution bathing the tissue slices. Increasing the aeration CO(2) concentration from 5% (control) to 10% (hypercapnic) had no effect on most (i.e., 69%) POAH temperature-insensitive neurons; however, this hypercapnia inhibited the majority (i.e., 59%) of warm-sensitive neurons. CO(2) affected similar proportions of (non-POAH) neurons in other hypothalamic regions. These CO(2) effects appear to be due to changes in pH since the CO(2)-affected neurons responded similarly to isocapnic acidosis (i.e., normal CO(2) and decreased pH) but were not responsive to isohydric hypercapnia (i.e., increased CO(2) and normal pH). These findings may offer a neural explanation for some heat-related illnesses (e.g., exertional heat stroke) where impaired heat loss is associated with acidosis.     

Effect of protein synthesis inhibitors on leukocytic pyrogen-induced in vitro hypothalamic prostaglandin production

In order to study the antipyretic effect of inhibitors of protein synthesis, hypothalamic tissue was incubated in vitro under controlled conditions and the amount of prostaglandin E2 (PGE2) measured in the supernatant medium. Rabbit anterior hypothalamic tissue was incubated with purified human leukocytic pyrogen (LP) and after 60 minutes the supernatant fluid was assayed for PGE2 by radioimmunoassay. Control tissue incubated with Eagle's medium (MEM) released elevated levels of PGE2; however, the addition of polymyxin B (PmxB), a cationic antibiotic which blocks the activities of bacterial endotoxins, significantly reduced PGE2. In addition, endotoxin added to MEM induced from the brain tissue PGE2 production which could be reduced by the addition of PmxB. Thus, commercial culture media such as MEM may contain sufficient amounts of endotoxin to stimulate brain PGE2 production in vitro. Purified human LP incubated with hypothalamic tissue in the presence of PmxB induced PGE2 production in a dose-dependent fashion. This release could be reduced (p less than 0.001) by the presence of either cycloheximide or puromycin during incubation with LP. The addition of these inhibitors to unstimulated hypothalamic tissue incubations did not reduce background levels of PGE2. It is concluded that the antipyretic effect of protein synthesis inhibitors results in a specific decrease in LP-induced levels of PGE2.     

Roles of prostaglandin synthesis in excitotoxic brain diseases

Cyclooxygenase (COX) is a rate-limiting enzyme in prostaglandin synthesis. COX consists of two isoforms, constitutive COX-1 and inducible COX-2. We have first found that COX-2 expression in the brain is tightly regulated by neuronal activity under physiological conditions, and electroconvulsive seizure robustly induces COX-2 mRNA in the brain. Our recent in-depth studies reveal COX-2 expression is divided into two phases, early in neurons and late in non-neuronal cells, such as endothelial cells or astrocytes. In this review, we present that early synthesized COX-2 facilitates the recurrence of hippocampal seizures in rapid kindling model, and late induced COX-2 stimulates hippocampal neuron loss after kainic acid treatment. Hence, we consider the potential role of COX-2 inhibitors as a new therapeutic drug for a neuronal loss after seizure or focal cerebral ischemia. The short-term and sub-acute medication of selective COX-2 inhibitors that suppresses an elevation of prostaglandin E(2) (PGE(2)) may be an effective treatment to prevent neuronal loss after onset of neuronal excitatory diseases. This review also discusses a novel role of vascular endothelial cells in brain diseases. We found that these cells produce PGE(2) by synthesizing COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) in response to excitotoxicity and neuroinflammation. We also show a possible mechanisms of neuronal damage associated with seizure via astrocytes and endothelial cells. Further analysis of the interaction among neurons, astrocytes and endothelial cells may provide a better understanding of the processes of neuropathological disorders, as well as facilitating the development of new treatments.     

Primary explants as a model of the hypothalamus in situ

Anatomical and electrophysiological studies with neurotensin and somatostatin were performed to assess the integrity of primary explants of the preoptic anterior hypothalamus (POAH) maintained in organ culture as a model to study peptidergic neurons in vitro. POAH neurons maintained in vitro were found to be morphologically similar to those visualized histologically in situ. Somatostatin- and neurotensin-immunoreactive neurons were also present in both preparations. Additionally, electrophysiological responses of neurons in vitro to neurotensin and somatostatin were qualitatively similar to neuronal responses in situ. The striking morphological and pharmacological similarity between POAH neurons in vitro and in situ suggests that primary explants of the POAH are organotypic.     

Characterization of the major phosphoinositide-specific phospholipase C of human amnion

Most of the phosphoinositide-specific phospholipase C activity in human amnion at term was found to be attributable to a single isoform (Mr 85,000). Phospholipase C purified from amnion catalyzed the calcium-dependent hydrolysis of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate. The high phospholipase C activity of amnion cells isolated at 38-41 weeks of gestation declined greater than 80% during the initial 2-5 days of culture to values characteristic of amnion tissue in early gestation. Activities of phospholipase A2 and phosphatidylinositol synthase remained essentially unaltered during this period of culture. Loss of phospholipase C activity was apparently due neither to the appearance of an inhibitor nor to the loss of an activator and most likely reflected a decrease in the amount of enzyme in amnion cells. Basal production of prostaglandin E2 (PGE2) by amnion cells also declined greatly during the period of loss of phospholipase C activity. Involvement of phospholipase C in the regulation of amnion prostaglandin production was also supported by the finding that the phospholipase C inhibitor, U-73122, potently inhibited amnion cell PGE2 production. In contrast, vasopressin, which appears to stimulate prostaglandin production in amnion cells by a phospholipase C-dependent mechanism, was equipotent in stimulating PGE2 production by amnion cells on Day 2 and Day 5 of culture, even though phospholipase C activity had declined by more than 75%. Furthermore, epidermal growth factor stimulation of PGE2 production by amnion cells appeared to be largely attributable to an increase in prostaglandin H synthase activity and did not involve an increase in phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin E(2) stimulates glutamate receptor-dependent astrocyte neuromodulation in cultured hippocampal cells.

Recent Ca(2+) imaging studies in cell culture and in situ have shown that Ca(2+) elevations in astrocytes stimulate glutamate release and increase neuronal Ca(2+) levels, and that this astrocyte-neuron signaling can be stimulated by prostaglandin E(2) (PGE(2)). We investigated the electrophysiological consequences of the PGE(2)-mediated astrocyte-neuron signaling using whole-cell recordings on cultured rat hippocampal cells. Focal application of PGE(2) to astrocytes evoked a Ca(2+) elevation in the stimulated cell by mobilizing internal Ca(2+) stores, which further propagated as a Ca(2+) wave to neighboring astrocytes. Whole-cell recordings from neurons revealed that PGE(2) evoked a slow inward current in neurons adjacent to astrocytes. This neuronal response required the presence of an astrocyte Ca(2+) wave and was mediated through both N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors. Taken together with previous studies, these data demonstrate that PGE(2)-evoked Ca(2+) elevations in astrocyte cause the release of glutamate which activates neuronal ionotropic receptors.     

Ccl22/MDC, is a prostaglandin dependent pyrogen, acting in the anterior hypothalamus to induce hyperthermia via activation of brown adipose tissue

CC Chemokine ligand 22 (Ccl22) is a selective, high affinity ligand at the CC chemokine receptor 4 (Ccr4). We have identified cDNAs encoding both ligand and receptor of the Ccl22–Ccr4 pair in cDNA libraries of the anterior hypothalamus/pre-optic area (AH/POA) by PCR. The AH/POA is the key brain region where endogenous pyrogens have been shown to act on warm sensitive neurons to affect thermogenesis in brown adipose tissue (BAT) and other thermogenically responsive tissues. We show that functional Ccr4 receptors are present in the AH/POA neurons as injection of Ccl22 into the POA but not to other hypothalamic nuclei induces an increase in core body temperature as measured by radiotelemetry. Indomethacin (5 mg/kg s.c) pre-treatment markedly reduced the hyperthermia evoked by POA injection of Ccl22 (10 ng/0.5 ul) and thus suggests that this hyperthermia is mediated through cyclooxygenase activation and thus likely through the formation and action of the pyrogen prostaglandin E2. The temperature elevation involves a decrease in the respiratory exchange ratio and increased activation of the brown adipose tissue as demonstrated by ¹⁸F-FDG–PET imaging. We describe a novel role to the ligand Ccl22 and its receptor Ccr4 in the anterior hypothalamus in temperature regulation that depends on the synthesis of the endogenous pyrogen, prostaglandin E2.     

Effect of a continuously applied compressive pressure on mouse osteoblast-like cells (MC3T3-E1) in vitro

Bone metabolism is often affected by a variety of mechanical forces, but the cytological basis of their action is not known. In this study, we examined the effect of a continuously applied compressive pressure (CCP) on the growth and differentiation of clonal mouse osteoblast-like cells (MC3T3-E1) cultured in a specifically devised culture chamber. The gas phase of the chamber was maintained at a pressure of 2 atmospheres (atm) above ambient (3 atm total, 3.1 kg/cm2; 3.0 x 10(5) Pa) by continuously infusing a compressed mixed gas (O2: N2:CO2 = 7.0%:91.3%:1.7%). The pO2, pCO2, and pH in the culture medium at 37 degrees C under 3 atm were maintained at the same levels as those under 1 atm. MC3T3-E1 cells were cultured in alpha-minimal essential medium containing 10% fetal bovine serum under either 3 atm in the CCP culture chamber or 1 atm in an ordinary CO2 incubator. Alkaline phosphatase activity, a marker of osteoblasts, was greatly suppressed by the CCP treatment. The inhibition of alkaline phosphatase activity was rapidly restored when the cells were transferred to an ordinary CO2 incubator under 1 atm, indicating that the inhibition of alkaline phosphatase activity by CCP is reversible. Cell growth was not altered under CCP. The CCP treatment greatly increased the production and secretion of prostaglandin E2 (PGE2). Adding either conditioned medium from the CCP culture or exogenous PGE2 to the control culture under 1 atm suppressed alkaline phosphatase activity dose-dependently. The CCP treatment also suppressed collagen synthesis and calcification. These results suggest that CCP causes the cells to produce and secrete PGE2, which, in turn, inhibits differentiation of osteoblasts and the concomitant calcification.     

Role of cyclic adenosine 3',5'-monophosphate in mediating the effect of prostaglandin E2 on decidualization in vitro.

When rat endometrial stromal cells from uteri sensitized for decidualization are cultured in vitro, there is an increase in alkaline phosphatase (ALP) activity paralleling that seen in vivo during decidualization. The addition of indomethacin to the culture medium decreases the endogenous production of prostaglandin E2 (PGE2) to below detectable levels and substantially reduces the increase in ALP activity. The addition of either PGE2 or its analog 16,16-dimethyl-PGE2, but not PGF2 alpha or its analog 15(S),15-methyl-PGF2 alpha, overrides this inhibitory effect, suggesting that PGE2 has a specific stimulatory effect upon ALP activity. This in vitro system was used to investigate the role of the cAMP pathway in mediating the stimulatory effect of PGE2 on ALP activity. The data indicate that PGE2 causes an increase in cAMP accumulation by the cells and that the addition of an analog of cAMP or substances which increase the level of cAMP in the cells (1-methyl-3-isobutyl xanthine, cholera toxin, forskolin) causes an increase in ALP activity. Collectively, the results suggest that the stimulatory effect of PGE2 is at least partially mediated by the cAMP pathway.