Endogenous opioids: role in prostaglandin-dependent and -independent fever

This study evaluated the participation of mu-opioid-receptor activation in body temperature (T-b) during normal and febrile conditions (including activation of heat conservation mechanisms) and in different pathways of LPS-induced fever. The intracerebroventricular treatment of male Wistar rats with the selective opioid mu-receptor-antagonist cyclic D-Phe-Cys-Try-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; 0.1-1.0 mu g) reduced fever induced by LPS (5.0 mu g/kg) but did not change Tb at ambient temperatures of either 20 C or 28 C. The subcutaneous, intracerebroventricular, and intrahypothalamic injection of morphine (1.0 -10.0 mg/kg, 3.0 -30.0 mu g, and 1 -100 ng, respectively) produced a dose-dependent increase in Tb. Intracerebroventricular morphine also produced a peripheral vasoconstriction. Both effects were abolished by CTAP. CTAP (1.0 mu g icv) reduced the fever induced by intracerebroventricular administration of TNF-alpha (250 ng), IL-6 (300 ng), CRF (2.5 mu g), endothelin-1 (1.0 pmol), and macrophage inflammatory protein (500 pg) and the first phase of the fever induced by PGF(2 alpha) (500.0 ng) but not the fever induced by IL-1 beta (3.12 ng) or PGE(2) (125.0 ng) or the second phase of the fever induced by PGF(2 alpha). Morphine-induced fever was not modified by the cyclooxygenase (COX) inhibitor indomethacin (2.0 mg/kg). In addition, morphine injection did not induce the expression of COX-2 in the hypothalamus, and CTAP did not modify PGE2 levels in cerebrospinal fluid or COX-2 expression in the hypothalamus after LPS injection. In conclusion, our results suggest that LPS and endogenous pyrogens (except IL-1 beta and prostaglandins) recruit the opioid system to cause a mu-receptor-mediated fever.

The antipyretic effect of dipyrone is unrelated to inhibition of PGE(2) synthesis in the hypothalamus

BACKGROUND AND PURPOSE Bacterial lipopolysaccharide (LPS) induces fever through two parallel pathways; one, prostaglandin (PG)-dependent and the other, PG-independent and involving endothelin-1 (ET-1). For a better understanding of the mechanisms by which dipyrone exerts antipyresis, we have investigated its effects on fever and changes in PGE(2) content in plasma, CSF and hypothalamus induced by either LPS or ET-1. EXPERIMENTAL APPROACH Rats were given (i.p.) dipyrone (120 mg center dot kg-1) or indomethacin (2 mg center dot kg-1) 30 min before injection of LPS (5 mu g center dot kg-1, i.v.) or ET-1 (1 pmol, i.c.v.). Rectal temperature was measured by tele-thermometry. PGE(2) levels were determined in the plasma, CSF and hypothalamus by elisa. KEY RESULTS LPS or ET-1 induced fever and increased CSF and hypothalamic PGE(2) levels. Two hours after LPS, indomethacin reduced CSF and hypothalamic PGE(2) but did not inhibit fever, while at 3 h it reduced all three parameters. Three hours after ET-1, indomethacin inhibited the increase in CSF and hypothalamic PGE(2) levels but did not affect fever. Dipyrone abolished both the fever and the increased CSF PGE(2) levels induced by LPS or ET-1 but did not affect the increased hypothalamic PGE(2) levels. Dipyrone also reduced the increase in the venous plasma PGE(2) concentration induced by LPS. CONCLUSIONS AND IMPLICATIONS These findings confirm that PGE(2) does not play a relevant role in ET-1-induced fever. They also demonstrate for the first time that the antipyretic effect of dipyrone was not mechanistically linked to the inhibition of hypothalamic PGE(2) synthesis.

Endogenous cannabinoids induce fever through the activation of CB(1) receptors

Background and purpose: The effects of centrally administered cannabinoids on body core temperature (Tc) and the contribution of endogenous cannabinoids to thermoregulation and fever induced by lipopolysaccharide (LPS) (Sigma Chem. Co., St. Louis, MO, USA) were investigated. Experimental approach: Drug-induced changes in Tc of male Wistar rats were recorded over 6 h using a thermistor probe (Yellow Springs Instruments 402, Dayton, OH, USA) inserted into the rectum. Key results: Injection of anandamide [(arachidonoylethanolamide (AEA); Tocris, Ellisville, MO, USA], 0.01-1 mu g i.c.v. or 0.1-100 ng intra-hypothalamic (i.h.), induced graded increases in Tc (peaks 1.5 and 1.6 degrees C at 4 h after 1 mu g i.c.v. or 10 ng i.h.). The effect of AEA (1 mu g, i.c.v.) was preceded by decreases in tail skin temperature and heat loss index (values at 1.5 h: vehicle 0.62, AEA 0.48). Bell-shaped curves were obtained for the increase in Tc induced by the fatty acid amide hydrolase inhibitor [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate (Cayman Chemical Co., Ann Arbor, MI, USA) (0.001-1 ng i.c.v.; peak 1.9 degrees C at 5 h after 0.1 ng) and arachidonyl-2-chloroethylamide (ACEA; Tocris) (selective CB(1) agonist; 0.001-1 mu g i.c.v.; peak 1.4 degrees C 5 h after 0.01 mu g), but (R,S)-(+)-(2-Iodo-5-nitrobenzoyl)-[1-(1-methyl-piperidin-2-ylmethyl)-1H-indole-3-yl] methanone (Tocris) (selective CB(2) agonist) had no effect on Tc. AEA-induced fever was unaffected by i.c.v. pretreatment with 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indole-3-yl](4-methoxyphenyl) methanone (Tocris) (selective CB(2) antagonist), but reduced by i.c.v. pretreatment with N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251; Tocris) (selective CB(1) antagonist). AM251 also reduced the fever induced by ACEA or LPS. Conclusions and implications: The endogenous cannabinoid AEA induces an integrated febrile response through activation of CB(1) receptors. Endocannabinoids participate in the development of the febrile response to LPS constituting a target for antipyretic therapy.

Fluorescent indication that nitric oxide formation in NTS neurons is modulated by glutamate and GABA

Nitric oxide (NO) in NTS plays an important role in regulating autonomic function to the cardiovascular system. Using the fluorescent dye DAF-2 DA, we evaluated the NO concentration in NTS. Brainstem slices of rats were loaded with DAF-2 DA, washed, fixed in paraformaldehyde and examined under fluorescent light. In different experimental groups, NTS slices were pre-incubated with 1 mM L-NAME (a non-selective NOS inhibitor), 1 MM D-NAME (an inactive enantiomere of L-NAME), 1 mM kynurenic acid (a nonselective ionotropic receptors antagonist) or 20 mu M bicuculline (a selective GABA(A) receptors antagonist) before and during DAF-2 DA loading. Images were acquired using a confocal microscope and the intensity of fluorescence was quantified in three antero-posterior NTS regions. In addition, slices previously loaded with DAF-2 DA were incubated with NeuN or GFAP antibody. A semi-quantitative analysis of the fluorescence intensity showed that the basal NO concentration was similar in all antero-posterior aspects of the NTS (rostral intermediate, 15.5 +/- 0.8 AU: caudal intermediate, 13.2 +/- 1.4 AU; caudal commissural, 13.8 +/- 1.4 AU, n = 10). In addition, the inhibition of NOS and the antagonism of glutamatergic receptors decreased the NO fluorescence in the NTS. On the other hand, D-NAME did not affect the NO fluorescence and the antagonism of GABAA receptors increased the NO fluorescence in the NTS. It is important to note that the fluorescence for NO was detected mainly in neurons. These data show that the fluorescence observed after NTS loading with DAF-2 DA is a result of NO present in the NTS and support the concept that NTS neurons have basal NO production which is modulated by L-glutamate and GABA. (C) 2009 Elsevier Inc. All rights reserved.

The Aspergillus nidulans nucA(EndoG) Homologue Is Not Involved in Cell Death

Upon apoptosis induction, translocation of mammalian mitochondrial endonuclease G (EndoG) to the nucleus coincides with large-scale DNA fragmentation. Here, we describe for the first time a homologue of EndoG in filamentous fungi by investigating if the Aspergillus nidulans homologue of the EndoG gene, named nucA(EndoG), is being activated during farnesol-induced cell death. Our results suggest that NucA is not involved in cell death, but it plays a role in the DNA-damaging response in A. nidulans.

BLOCKING CENTRAL LEUKOTRIENES SYNTHESIS AFFECTS VASOPRESSIN RELEASE DURING SEPSIS

Recent studies revealed that vasopressinergic neurons have a high content of cys-leukotriene C(4) (LTC(4)) synthase, a critical enzyme in cys-leukotriene synthesis that may play a role in regulating vasopressin secretion. This study investigates the role of this enzyme in arginine vasopressin (AVP) release during experimentally induced sepsis. Male Wistar rats received an i.c.v. injection of 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2-dimethylpropanoic acid (MK-886) (1.0 mu g/kg), a leukotrienes (LTs) synthesis inhibitor, or vehicle, 1 h before cecal ligation and puncture (CLP) or sham operation. In one group of animals the survival rate was monitored for 3 days. In another group, the animals were decapitated at 0, 4, 6, 18 and 24 h after CLP or sham operation, and blood was collected for hematocrit, serum sodium and nitrate, plasma osmolality, protein and AVP determination. A third group was used for blood pressure measurements. The neurohypophysis was removed for quantification of AVP content, and the hypothalamus was dissected for LTC4 synthase analysis by Western blot. Mortality after CLP was reduced by the central administration of MK-886. The increase in plasma AVP levels and hypothalamus LTC4 synthase content in the initial phase of sepsis was blocked, whereas the decrease in neurohypophyseal AVP content was partially reversed. Also the blood pressure drop was abolished in this phase. The increase of serum nitric oxide and hematocrit was reduced, and the decrease in plasma protein and osmolality was not affected by the LTs blocker. In the final phase of sepsis, the plasma AVID level and the hypothalamic LTC4 synthase content were at basal levels. The central administration of MK-886 increased the hypothalamic LTC4 synthase content but did not alter the plasma and neurohypophysis AVID levels observed, or the blood pressure during this phase. These results suggest that the central LTs are involved in the vasopressin release observed during sepsis. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Reproductive system of the male hermit crab Clibanarius sclopetarius: gonopore, spermatophore, and spermatozoal morphologies

In the present study, the morphology and biometry of the spermatophores of the western Atlantic hermit crab Clibanarius sclopetarius (Herbst, 1796) are described, and the results are placed in the context of the Paguroidea, in particular the Diogenidae. Individuals of C. sclopetarius were sampled from a human-impacted mangrove area of southern Brazil. The male reproductive system was removed, measured and analyzed using stereoscopic, light, transmission-electron and scanning-electron microscopy. This system is composed of lobular testes connected to the vas deferens, and gonopores with membranous coverage. The mature spermatophore consists of a spherical pack that stores sperm. These cells consist of a spherical acrosomal vesicle, an amorphous cytoplasm and a distal nucleus. The results revealed that the gonopores, testis and vas deferens have the expected characteristics of the family Diogenidae, while the non-tripartite morphology of the spermatophores and the sperm follow the patterns found only in the genus Clibanarius, and the presence of the dense perforatorial ring is, to date, unique in the species of the genus, being a possible apomorphic characteristic.

Facilitation of 5-HT(1A)-mediated neurotransmission in dorsal periaqueductal grey matter accounts for the panicolytic-like effect of chronic fluoxetine

Chronic administration of antidepressants such as fluoxetine and imipramine increases the responsiveness of 5-HT(1A) receptors in dorsal periaqueductal grey matter (DPAG), a midbrain area consistently implicated in the pathogenesis of panic disorder. This effect has been related to the clinically relevant anti-panic action of these drugs. In this study we determined whether long-term administration of fluoxetine also affects 5-HT efflux in DPAG. As a comparison, the effect of chronic treatment with the anxiolytic 5-HT(1A) receptor agonist buspirone on DPAG 5-HT levels was assessed. We also investigated whether the inhibitory effect of chronic fluoxetine on escape behaviour in the rat elevated T-maze, considered as a panicolytic-like effect, is counteracted by intra-DPAG injection of the 5-HT(1A) receptor antagonist WAY 100635. Male Wistar rats were treated (1 or 21 d, i.p.) with fluoxetine, buspirone or vehicle, once daily. After treatment, 5-HT in DPAG was measured by in-vivo microdialysis coupled to HPLC. In another study, rats treated (21 d, i.p.) with either fluoxetine or vehicle also received intra-DPAG injection of WAY 100635 or saline 10 min before being tested in the elevated T-maze. Chronic, but not acute, administration of fluoxetine significantly raised extracellular levels of 5-HT in DPAG. Long-term treatment with buspirone was ineffective. In the elevated T-maze, intra-DPAG injection of WAY 100635 fully blocked the anti-escape effect of chronic administration of fluoxetine. Therefore, chronic fluoxetine facilitates 5-HT(1A)-mediated neurotransmission within DPAG and this effect accounts for the panicolytic-like effect of this antidepressant in the elevated T-maze.

Social separation and diazepam withdrawal increase anxiety in the elevated plus-maze and serotonin turnover in the median raphe and hippocampus

The present work aimed to evaluate the effects of social separation for 14 days (chronic stress) and of withdrawal from a 14-day treatment with diazepam (acute stress) on the exploratory behaviour of male rats in the elevated plus-maze and on serotonin (5-hydroxytryptamine) turnover in different brain structures. Social separation had an anxiogenic effect, evidenced by fewer entries into, and less time spent on the open arms of the elevated plus-maze. Separation also selectively increased 5-hydroxytryptamine turnover in the hippocampus and median raphe nucleus. Diazepam withdrawal had a similar anxiogenic effect in grouped animals and increased 5-hydroxytryptamine turnover in the same brain structures. Chronic treatment with imipramine during the 14 days of separation prevented the behavioural and neurochemical changes caused by social separation. It is suggested that the increase in anxiety determined by both acute and chronic stress is mediated by the activation of the median raphe nucleus-hippocampal 5-hydroxytryptamine pathway.

Antidepressant-Like Effects of Medial Prefrontal Cortex Deep Brain Stimulation in Rats

Background: Subcallosal cingulate gyrus (SCG) deep brain stimulation (DBS) is being investigated as a treatment for major depression. We report on the effects of ventromedial prefrontal cortex (vmPFC) DBS in rats, focusing on possible mechanisms involved in an antidepressant-like response in the forced swim test (FST). Methods: The outcome of vmPFC stimulation alone or combined with different types of lesions, including serotonin (5-HT) or nore-pineprhine (NE) depletion, was characterized in the FST. We also explored the effects of DBS on novelty-suppressed feeding, learned helplessness, and sucrose consumption in animals predisposed to helplessness. Results: Stimulation at parameters approximating those used in clinical practice induced a significant antidepressant-like response in the FST. Ventromedial PFC lesions or local muscimol injections did not lead to a similar outcome. However, animals treated with vmPFC ibotenic acid lesions still responded to DBS, suggesting that the modulation of fiber near the electrodes could play a role in the antidepressant-like effects of stimulation. Also important was the integrity of the serotonergic system, as the effects of DBS in the FST were completely abolished in animals bearing 5-HT, but not NE, depleting lesions. In addition, vmPFC stimulation induced a sustained increase in hippocampal 5-HT levels. Preliminary work with other models showed that DBS was also able to influence specific aspects of depressive-like states in rodents, including anxiety and anhedonia, but not helplessness. Conclusions: Our study suggests that vmPFC DES in rats maybe useful to investigate mechanisms involved in the antidepressant effects of SCG DBS.

The unconditioned fear produced by morphine withdrawal is regulated by mu- and kappa-opioid receptors in the midbrain tectum

We have recently shown that morphine withdrawal sensitizes the neural substrates of fear in the midbrain tectum structures-the dorsal periaqueductal gray (dPAG) and inferior colliculus (IC). In the present study, we investigated the role of mu- and kappa-opioid receptors in the mediation of these effects. Periadolescent rats chronically treated with morphine (10 mg/kg; s.c.) twice daily for 10 days were implanted with an electrode glued to a guide-cannula into the dPAG or the IC. Forty-eight hours after the interruption of this treatment, the effects of intra-dPAG or intra-IC microinjections of [D-Ala(2) N-Me-Phe(4) Gly(5)-ol]-enkephalin (DAMGO; 0.6 and 1 nmol/0.2 mu l) - a selective mu-receptor agonist - or nor-binaltorphimine (BNI; 2.5 and 5 mu g/0.2 mu l) - a selective K-receptor antagonist with tardive action - on the freezing and escape thresholds determined by electrical stimulation of the dPAG and the IC were examined. For both structures, morphine withdrawal produced pro-aversive effects. DAMGO and BNI had antiaversive effects when injected into the dPAG and IC of non-dependent rats. In morphine-withdrawn rats, only BNI continued to promote antiaversive effects in both structures. Whereas DAMGO lost its antiaversive efficacy when injected into the dPAG, only its highest dose promoted antiaversive effects in the IC of morphine-withdrawn rats, suggesting the development of an apparent tolerance. Thus, the enhanced reactivity of the midbrain tectum in morphine-withdrawn periadolescent rats may be due, at least partially, to an impairment of the inhibitory influence of mechanisms mediated by mu-receptors on the neural substrates of fear in this region. (C) 2009 Elsevier B.V. All rights reserved.

THE ANTERIOR CINGULATE CORTEX IS A TARGET STRUCTURE FOR THE ANXIOLYTIC-LIKE EFFECTS OF BENZODIAZEPINES ASSESSED BY REPEATED EXPOSURE TO THE ELEVATED PLUS MAZE AND FOS IMMUNOREACTIVITY

Prior experience with the elevated plus maze (EPM) increases the avoidance of rodents to the open arms and impairs the anxiolytic-like effects of benzodiazepines on the traditional behaviors evaluated upon re-exposure to the maze, a phenomenon known as one-trial tolerance. Risk assessment behaviors are also sensitive to benzodiazepines. During re-exposure to the maze, these behaviors reinstate the information-processing initiated during the first experience, and the detection of danger generates stronger open-arm avoidance. The present study investigated whether the benzodiazepine midazolam alters risk assessment behaviors and Fos protein distribution associated with test and retest sessions in the EPM. Naive or maze-experienced Wistar rats received either saline or midazolam (0.5 mg/kg i.p.) and were subjected to the EPM. Midazolam caused the usual effects on exploratory behavior, increasing exploratory activity of naive rats in the open arms and producing no effects on these conventional measures in rats re-exposed to the maze. Risk assessment behaviors, however, were sensitive to the benzodiazepine during both sessions, indicating anxiolytic-like effects of the drug in both conditions. Fos immunohistochemistry showed that midazolam injections were associated with a distinct pattern of action when administered before the test or retest session, and the anterior cingulate cortex, area 1 (Cg1), was the only structure targeted by the benzodiazepine in both situations. Bilateral infusions of midazolam into the Cg1 replicated the behavioral effects of the drug injected systemically, suggesting that this area is critically involved in the anxiolytic-like effects of benzodiazepines, although the behavioral strategy adopted by the animals appears to depend on the previous knowledge of the threatening environment. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Effects of microinjections of apomorphine and haloperidol into the inferior colliculus on the latent inhibition of the conditioned emotional response

Electrical or chemical stimulation of the inferior colliculus (IC) induces fear-like behaviors. More recently, consistent evidence has shown that electrical stimulation of the central nucleus of the IC supports Pavlovian conditioning and latent inhibition (Li). LI is characterized by retardation in conditioning and also by an impaired ability to ignore irrelevant stimuli, after a non-reinforced pre-exposure to the conditioned stimulus. LI has been proposed as a behavioral model of cognitive abnormalities seen in schizophrenia. The aim of the present study was to determine whether dopaminergic mechanisms in the IC are involved in LI of the conditioned emotional response (CER). To induce LI, a group of rats was pre-exposed (PE) to six tones in two sessions, while rats that were not pre-exposed (NPE) had two sessions without tone presentations. The conditioning consisted of two tone presentations to the animal, followed immediately by a foot shock. PE and NPE rats received IC microinjections of physiological saline, the dopaminergic agonist apomorphine (9.0 mu g/0.5 mu L/side), or the dopaminergic antagonist haloperidol (0.5 mu g/0.5 mu L/side) before both pre-exposure and conditioning. During the test, the PE rats that received saline or haloperidol had a lower suppression of the licking response compared to NPE rats that received vehicle or haloperidol, indicating that latent inhibition was induced. There was no significant difference in the suppression ratio in rats that received apomorphine injections into the IC, indicating reduced latent inhibition. These results suggest that dopamine-mediated mechanisms of the IC are involved in the development of LI. (C) 2008 Elsevier Inc. All rights reserved.

Involvement of the midbrain tectum in the unconditioned fear promoted by morphine withdrawal

The midbrain rectum structures, dorsal periaqueductal gray (dPAG) and inferior colliculus (IC), are involved in the organization of fear and anxiety states during the exposure to dangerous stimuli. Since opiate withdrawal is associated with increased anxiety in both humans and animals, this study aimed to investigate the possible sensitization of the neural substrates of fear in the midbrain tectum and its influence on the morphine withdrawal-induced anxiety. For the production of drug withdrawal, rats received morphine injections (10 mg/kg; s.c.) twice daily during 10 days. Forty-eight hours after the interruption of the chronic treatment, independent groups were probed in the elevated plus-maze and open-field tests. Additional groups of animals were implanted with a bipolar electrode into the dPAG OF the IC and submitted to the electrical stimulation of these structures for the determination of the freezing and escape thresholds after 48 h of withdrawal. Our results showed that the morphine withdrawal promoted clear-cut levels of anxiety without the somatic signs of opiate withdrawal. Moreover, morphine-withdrawn rats had an increase in the reactivity to the electrical stimulation of the dPAG and the IC. These findings suggest that the increased anxiety induced by morphine withdrawal is associated with the sensitization of the neural substrates of fear in the dPAG and the IC. So, the present results give support to the hypothesis that withdrawal from chronic treatment with morphine leads to fear states possibly engendered by activation of the dPAG and IC, regardless of the production of somatic symptoms. (C) 2008 Elsevier B.V. All rights reserved.

Serotonin-1A receptors in the dorsal periaqueductal gray matter mediate the panicolytic-like effect of pindolol and paroxetine combination in the elevated T-maze

The beta-adrenergic blocker and 5-HT(1A) receptor antagonist pindolol has been combined with selective serotonin reuptake inhibitors (SSRIs) in patients with depressive and anxiety disorders to shorten the onset of the clinical action and/or increase the proportion of responders. The results of a previous study have shown that pindolol potentiates the panicolytic effect of paroxetine in rats submitted to the elevated T-maze (ETM). Since reported evidence has implicated the 5-HT(1A) receptors of the dorsal periaqueductal gray matter (DPAG) in the panicolytic effect of antidepressants, rats treated with pindolol (5.0 mg/kg, i.p.) and paroxetine (1.5 mg/kg, i.p.) received a previous intra-DPAG injection of the selective 5-HT(1A) antagonist, WAY-100635 (0.4 mu g) and were submitted to the ETM. Pretreatment with WAY-100635 reversed the increase in escape latency, a panicolytic effect, determined by the pindolol-paroxetine combination. These results implicate the 5-HT(1A) receptors of the DPAG in the panicolytic effect of the pindolol-paroxetine combination administered systemically. They also give further preclinical support for the use of this drug combination in the treatment of panic disorder. (C) 2011 Elsevier Ireland Ltd. All rights reserved.