Role of giant cell nuclei of the reticular formation in mechanisms of analgesia during auricular electroacupuncture and action of morphine

The activity of antinociceptive mechanisms induced by auricular electroacupuncture (AEA) and intraperitoneal injection of morphine (M) was studied on rats subjected to bilateral lesion of gigantocellular nuclei (GCN). It was shown that under AEA hot-plate (HP) and tail-flick (TF) latencies (L) significantly increased as compared to the baseline level. A comparison of L shows that HP and TF in the experimental group were significantly shorter than in the control group. After M injection the rats of both the experimental and control groups showed a significant increase of HP L and TF L as compared to the baseline level, but there was no difference in L between the groups. It is concluded that neurochemical systems of GCN are involved in the mechanism of antinociception elicited in AEA while the mechanisms of antinociceptive effect of M do not involve GCN.     

Fatty acid amide hydrolase determines anandamide-induced cell death in the liver

The endocannabinoid anandamide (AEA) induces cell death in many cell types, but determinants of AEA-induced cell death remain unknown. In this study, we investigated the role of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) in AEA-induced cell death in the liver. Primary hepatocytes expressed high levels of FAAH and were completely resistant to AEA-induced cell death, whereas primary hepatic stellate cells (HSCs) expressed low levels of FAAH and were highly sensitive to AEA-induced cell death. Hepatocytes that were pretreated or with the FAAH inhibitor URB597 isolated from FAAH(-/-) mice displayed increased AEA-induced reactive oxygen species (ROS) formation and were susceptible to AEA-mediated death. Conversely, overexpression of FAAH in HSCs prevented AEA-induced death. Since FAAH inhibition conferred only partial AEA sensitivity in hepatocytes, we analyzed additional factors that might regulate AEA-induced death. Hepatocytes contained significantly higher levels of glutathione (GSH) than HSCs. Glutathione depletion by dl-buthionine-(S,R)-sulfoximine rendered hepatocytes susceptible to AEA-mediated ROS production and cell death, whereas GSH ethyl ester prevented ROS production and cell death in HSCs. FAAH inhibition and GSH depletion had additive effects on AEA-mediated hepatocyte cell death resulting in almost 70% death after 24 h at 50 microm AEA and lowering the threshold for cell death to 500 nm. Following bile duct ligation, FAAH(-/-) mice displayed increased hepatocellular injury, suggesting that FAAH protects hepatocytes from AEA-induced cell death in vivo. In conclusion, FAAH and GSH are determinants of AEA-mediated cell death in the liver.     

Effects of systemically administered monoamine reuptake blocking agents on patterns of buspirone-induced analgesia in rats

We have recently shown the serotonin 5-HT1A receptor agonist buspirone to produce analgesia in several pain tests in rats. As a 5-HT1A agonist, buspirone may change serotonergic (5-HT) tone to alter the balance of central monoaminergic (MA) systems that function in analgesia. MA-reuptake blocking drugs have been shown to produce analgesia, both when given alone and in combination with a variety of other agents, presumably via their action upon MA neurochemistry. The present study was undertaken to examine the effect of systemic administration of the 5-HT-reuptake blocker amitriptyline (AMI: 10 mg/kg), NE-reuptake blocker desipramine (DMI: 10 mg/kg) or DA-reuptake blocker GBR-12909 (7.5 mg/kg) on patterns of analgesia produced by buspirone (1-5 mg/kg) in thermal and mechanical pain tests in rats. Neither reuptake blocking agents or buspirone, when administered alone or in combination, produced overt changes in motor activity at the doses tested. AMI alone was not analgesic in either thermal or mechanical pain tests. In both assays, AMI reduced the analgesic action of buspirone, with greater effects seen in the thermal test. When administered alone, DMI produced significant analgesia against thermal and mechanical pain. DMI significantly attenuated the analgesic action of all doses of buspirone in both pain tests. Alone, GBR-12909 did not affect nociception in thermal or mechanical tests. GBR-12909 decreased buspirone-induced analgesia at all doses in the thermal test, while having no effect on buspirone-induced analgesia against mechanical pain. These results demonstrate that facilitation of 5-HT, NE and DA function with reuptake blocking drugs did not enhance the analgesic action of buspirone. These data indicate against the adjuvant use of reuptake blocking compounds and buspirone as analgesics. Furthermore, such findings may suggest other possible non-MA substrates of buspirone-induced analgesia.     

Nicotene-induced analgesia in rats: The role of calcium and the diversity of responders and nonresponders

Following a single dose of nicotine, (NIC, 1 mg/kg s.c.), 60% of tested rats revealed significant anticociception as measured by the tail-flick (TF) test, and were classified as responders, with those in which TF latencies did not change, nonresponders. The following experiments were carried out one week later. In nonresponders, pretreatment with ethylenediaminetetraacetic acid (EDTA, 250 μM/kg s.c. four times every 15 min) followed by 1 mg NIC, produced significant analgesia in 50% of rats, to the same magnitude as did nicotene alone (1 mg) in responders. The other 50% of rats which failed to respond to EDTA pretreatment, all revealed similar analgesia following the higher dose of NIC (1.5 mg/kg s.c.), with similar side effects, as generally observed in responders. In responders, pretreatment with CaCl₂ (1.5 mM/kg s.c.) completely abolished NIC (1 mg/kg s.c.) - induced analgesia in all rats. Our data provide stronger evidence and a further verification that EDTA potentiates, whereas CaCl₂ completely abolishes, nicotene-induced analgesia in rats; supporting our hypothesis of the involvement of calcium ions in this effect.     

Effects of repeated acupuncture action on pain sensitivity and beta-endorphin contents of the hypothalamus and midbrain of the rat

Changes in latencies (L) of nociceptive (NC) reactions of hot plate (HP) tail flick (TF) tests and dynamics of beta-endorphin (beta-ED) in hypothalamus and midbrain were studied in albino rats receiving 15 sessions of acupuncture (A) (20 min daily in bilateral Ho-Ku). It was shown that repeated A facilitated prolongation of LHP and LTF as compared to baseline and control levels. A decrease of the anti-NC systems capacity for activation in response to each A stimulation was observed. The dynamics of beta-endorphin in hypothalamus showed an opposite tendency as compared to that in midbrain. It is suggested that such a correlation of beta-ED activities in midbrain and hypothalamus might be one of the reasons for anti-NC mechanisms adaptation to A.     

Role of catecholamine neurons in the reticular lateral nuclei in regulating sensitivity to pain during exposure to reflex stimuli

Experiments were made on rats in which the effects of catecholaminergic neuronal systems of lateral reticular A-1 nuclei were eliminated with 6-OHDA. The latency of pain reactions tested by the hot-plate and tail-flick tests remained unchanged after operation. After auricular electric acupuncture the rats manifested no changes in the above reactions as compared with the initial level, which evidences that A-1 nuclei play an important role in the mechanisms of analgesia under consideration. Stimulation of the small pelvis organs (SSPO) entailed a short-term and significant inhibition of the analgetic effect as regards the control which also points to the involvement of A-1 nuclei into activation of antinociceptive processes. Besides, during SSPO, there was a significant elevation of the response measured by the tail-flick test as compared to the initial level of the pain reaction.     

The selective role of the nucleus raphe magnus in the mechanisms of analgesia in electrocutaneous pain stimulation, cold stress and the action of morphine

Inhibition of the analgetic activity of systemic morphine and inescapable foot shock in certain moments of the experiment was shown on rats subjected to electrolytic destruction of nucleus raphe magnus. Cold swim stress increased analgesia as compared to the control animals. It is concluded that this formation of the brain is selectively and dynamically involved in mechanisms of various types of analgesia.     

Role of FAAH-Like Anandamide Transporter in Anandamide Inactivation

The endocannabinoid system modulates numerous physiological processes including nociception and reproduction. Anandamide (AEA) is an endocannabinoid that is inactivated by cellular uptake followed by intracellular hydrolysis by fatty acid amide hydrolase (FAAH). Recently, FAAH-like anandamide transporter (FLAT), a truncated and catalytically-inactive variant of FAAH, was proposed to function as an intracellular AEA carrier and mediate its delivery to FAAH for hydrolysis. Pharmacological inhibition of FLAT potentiated AEA signaling and produced antinociceptive effects. Given that endocannabinoids produce analgesia through central and peripheral mechanisms, the goal of the current work was to examine the expression of FLAT in the central and peripheral nervous systems. In contrast to the original report characterizing FLAT, expression of FLAT was not observed in any of the tissues examined. To investigate the role of FLAT as a putative AEA binding protein, FLAT was generated from FAAH using polymerase chain reaction and further analyzed. Despite its low cellular expression, FLAT displayed residual catalytic activity that was sensitive to FAAH inhibitors and abolished following mutation of its catalytic serine. Overexpression of FLAT potentiated AEA cellular uptake and this appeared to be dependent upon its catalytic activity. Immunofluorescence revealed that FLAT localizes primarily to intracellular membranes and does not contact the plasma membrane, suggesting that its capability to potentiate AEA uptake may stem from its enzymatic rather than transport activity. Collectively, our data demonstrate that FLAT does not serve as a global intracellular AEA carrier, although a role in mediating localized AEA inactivation in mammalian tissues cannot be ruled out.     

ICV-CRH potently affects behavior without altering antinociceptive responding

To explore the hypothesized integrative function of corticotropin releasing hormone (CRH) in the stress response, stress-related behaviors including antinociception were studied in rats after either intracerebroventricular (ICV) or peripheral administration of CRH. The effects of low-dose (0.3 microgram) and high-dose (3.0 micrograms) ICV-CRH were compared to those of vehicle, employing a within-S design. The two doses yielded comparable behavioral changes suggestive of increased arousal and stress. These changes were characterized by significant increases in grooming, walking, burrowing, self-gnawing, and pica, and decreases in rearing and sleeping. None of these effects of ICV-CRH were obtained with peripheral administration of the same doses. The hot-plate test of analgesia failed to show a significant effect of ICV-CRH or peripherally administered CRH. A between-S experiment incorporating both the tail-flick and the hot-plate tests of analgesia compared ICV-CRH (3.0 micrograms) with vehicle. ICV-CRH did not affect antinociceptive responding in either of these tests. In contrast, ICV morphine (10 micrograms) yielded potent analgesia in both tests. Thus, with doses of ICV-CRH yielding clear evidence of stress-related behavior, no evidence of analgesia was obtained. These findings question the possible role of central CRH systems in antinociceptive processes.     

Age-dependent differences in the effect of atrial natriuretic factor

Replacement of drinking water by 1.8% NaCl solution produces a severe hypernatremia in prepubertal rats and only a moderate elevation of the plasma sodium concentration (pNa) in adults. Hypernatremia in prepubertal rats was reduced by infusion of blood from adult rats, whereas blood from young rats was without this effect. The aim of this study was to find out if the natriuretic factor containing atrial extracts from prepubertal (AEP) and adult (AEA) rats also differ in their ability to reduce hypernatremia in prepubertal rats. A decrease of pNa was observed 10 min after the administration of AEA (delta = -5.43 mmol/l). AEP failed to show this effect (delta = +0.92 mmol/l). No significant effect of AEA on pNa was observed in adult rats with moderate hypernatremia (delta = -1.88 mmol/l). Natriuretic and diuretic effects of AEP were also significantly lower than those of AEA if they were tested in prepubertal recipients, whereas no difference in activities of AEP and AEA was found in adult recipients. Possible mechanisms of the hypernatremia suppressing activity of AEA, causes of the differences in activities between AEA and AEP, and consequences of the low AEP activity for prepubertal rats are discussed.     

Threshold of pain in chronic magnetic field- (50 Hz, 17.9 microT) exposed rats: effect of sucrose ingestion

Both magnetic field (MF) exposure and sucrose ingestion produce opioid mediated analgesia, independently. This article addresses the query whether or not sucrose ingestion potentiates the analgesia of MF exposure. The pain threshold, as reflected by withdrawal of tail (thresholds of tail flick, TF), vocalization during stimulus (VD), and vocalization after stimulus (VA) were determined in 7 sessions at 0, 5, 10, 15, 30, 45, and 60 min, respectively (control rats). After an interval of 24 h the rats were provided with sucrose solution and were allowed to ingest it for 10 min. The pain thresholds were determined (Control sucrose-fed rats). The rats then received exposure to magnetic field (50 Hz, 17.9 microT) for 7 d (8 h/d) (MF-exposed rats) and the effect of sucrose ingestion was repeated (MF-Sucrose-fed rats). The basal threshold of pain did not vary in between the control and MF-exposed rats. Pain threshold during sessions II-VII did not vary in control rats but it increased in MF rats. Moreover, the pain thresholds were elevated in MF rats as compared to control rats. Sucrose-ingestion elevated the threshold of TF in controls but not in MF-exposed rats; while the thresholds of VA and VD were elevated more in control than MF rats. The results suggest that the MF exposure (50 Hz, 17.9 microT, 8 h/d) for 7 d did not affect the basal thresholds of pain, increased stress induced analgesia, and attenuated the sucrose ingestion-induced analgesia.     

Study of Dopamine Turnover by Monitoring the Decline of Dopamine Metabolites in Rat CSF After α-Methyl-p-Tyrosine

CSF was continuously withdrawn from the third ventricle of anesthetized rats. CSF 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid concentrations were determined every 15 min by liquid chromatography coupled with electrochemical detection. Acute tyrosine hydroxylase inhibition [with alpha-methyl-p-tyrosine (alpha-MPT)] induced an exponential decline in levels of DOPAC and HVA in CSF. The decline in DOPAC and HVA concentrations was identical in CSF and forebrain but was much slower in the striatum, suggesting that CSF metabolites of 3,4-dihydroxyphenylethylamine (dopamine) reflect whole forebrain metabolites. The decay in CSF DOPAC and HVA levels after dopamine synthesis inhibition was also used as an in vivo index of forebrain dopamine turnover after various pharmacological treatments. Haloperidol pretreatment accelerated this decay, confirming the increase in brain dopamine turnover induced by neuroleptics. After reserpine pretreatment (15 h before), alpha-MPT produced a very sharp decay in levels of DOPAC and HVA. This result indicates that the residual dopamine that cannot be stored after reserpine treatment is very rapidly renewed and metabolized. Nomifensine strongly diminished the slope of DOPAC and HVA level decreases after alpha-MPT, a result which can be explained either by a slower dopamine turnover or by the involvement of storage dopamine pools. These results exemplify the use of monitoring the decay of dopamine metabolites after alpha-MPT administration in the study of the pharmacological action of drugs on the central nervous system of the rat.     

The vanilloid receptor (VR1)-mediated effects of anandamide are potently enhanced by the cAMP-dependent protein kinase

The endogenous cannabinoid receptor ligand, anandamide (AEA), is a full agonist of the vanilloid receptor type 1 (VR1) for capsaicin. Here, we demonstrate that the potency and efficacy of AEA at VR1 receptors can be significantly increased by the concomitant activation of protein kinase A (PKA). In human embryonic kidney (HEK) cells over-expressing human VR1, AEA induces a rise in cytosolic Ca(2+) concentration that is mediated by this receptor. The EC(50) for this effect was decreased five-fold in the presence of forskolin (FRSK, 1-5 microM) or the cAMP analogue, 8-Br-cAMP (10-100 microM). The effects of 8-Br-cAMP and FRSK were blocked by a selective PKA inhibitor. The FRSK (10 nM) also potently enhanced the sensory neurone- and VR1-mediated constriction by AEA of isolated guinea-pig bronchi, and this effect was abolished by a PKA inhibitor. In rat dorsal root ganglia slices, AEA-induced release of substance P, an effect mediated by VR1 activation, was enhanced three-fold by FRSK (10 nM). Thus, the ability of AEA to stimulate sensory VR1, with subsequent neuropeptide release, appears to be regulated by the state of activation of PKA. This observation supports the hypothesis that endogenous AEA might stimulate VR1 under certain pathophysiological conditions.     

Opioid antinociception and positive reinforcement are mediated by different types of opioid receptors

Fentanyl (FEN) and diprenorphine's (DIPR) potentials for analgesia and reinforcement were assayed using rats. Analgesia was measured by the classic tail-flick test. The test germane to opioid reinforcement involved measuring pressing rates for direct electrical stimulation of the lateral hypothalamus and ventral tegmental area. FEN, as does morphine and heroin, produced strong analgesia and enhanced pressing rates for brain stimulation. DIPR produced no analgesia and antagonized FEN's analgesia. DIPR, at doses antagonizing FEN's analgesia, enhanced pressing for brain stimulation. DIPR's enhancement of pressing was antagonized by naloxone (100 micrograms/kg). When FEN and DIPR were given concurrently, pressing for brain stimulation was not reduced and was greater than after FEN alone was given. These data support a conclusion that different types of receptors are associated with opioid analgesia and reinforcement.     

Effects of <Emphasis Type="SmallCaps">d</Emphasis>-kyotorphin on nociception and NADPH-d neurons in rat’s periaqueductal gray after immobilization stress

d-kyotorphin (d-Kyo) is a synthetic analogue of the neuropeptide kyotorphin and produces naloxone reversible analgesia. Stress-induced analgesia (SIA) is an in-built mammalian pain-suppression response that occurs during or following exposure to a stressful stimulus. The periaqueductal gray (PAG) is implicated as a critical site for processing strategies for coping with different types of stress and pain and NO affects its activity. The objectives of the present study were twofold: (1) to examine the effects of d-Kyo (5 mg/kg) on acute immobilization SIA; (2) to investigate the effect of peptide on NO activity in rat PAG after the stress procedure mentioned above. All drugs were injected intraperitoneally in male Wistar rats. The nociception was measured by the paw pressure and hot plate tests. A histochemical procedure for nicotinamide adenine dinucleotide phosphate–diaphorase (NADPH-d)-reactive neurons was used as indirect marker of NO activity. Our results revealed that d-Kyo has modulating effects on acute immobilization stress-induced analgesia in rats may be by opioid and non-opioid systems. Although d-Kyo is incapable of crossing the blood–brain barrier it showed an increased number of NADPH-d reactive neurons in dorsolateral periaqueductal gray (dlPAG) in control but not in stressed groups. We may speculate that the effect of d-Kyo in the brain is due to structural and functional interaction between opioidergic and NO-ergic systems or d-Kyo appears itself as a stressor. Further studies are needed to clarify the exact mechanisms of its action.     

Role of the brain neurochemical systems in altering the reactivity of the hypophyseal-adrenal system in the gray rat selected for behavior

Studies have been made of the interrelationship between brain monoaminergic system and pituitary-adrenal function in two groups of the grey rat Rattus norvegicus Berk. One group consisted of non-aggressive rats selected for lack of agonism towards experimentator, the other one included aggressive animals. Domesticated animals exhibited the decreased reaction of the pituitary-adrenocortical complex to emotional stress as well as the decreased response of the endocrine system during stimulation of noradrenaline or serotonin mechanisms of the brain. In addition, noradrenaline and serotonin content of the brain decreased in domesticated rats during emotional stress to a lower extent, as compared to that in aggressive ones. Therefore, one of the sources of correlational changes in the activity of the pituitary-adrenocortical complex during selection for domesticated behaviour is the effect of selection on neurochemical, in particular monoaminergic systems of the brain.     

Naloxone hyperalgesia and stress-induced analgesia in rats

Since past studies concerning the effects of naloxone on nociception have yielded inconclusive findings, the variables of pain test, baseline sensitivity, and stress condition were examined. Within a pure-bred strain of rats, consistent individual differences did not occur. All three measures of pain responsiveness demonstrated hyperalgesic effects of naloxone, but they differed in their capacity to reflect the effects of analgesia produced by continuous or intermittent electrical shock. By some measures, naloxone reversed the stress-induced analgesia due to intermittent shock; it did not influence the analgesia produced by continuous stress. The data support a model of pain inhibition involving both opioid and non-opioid systems and suggest that the hyperalgesic effects of naloxone can sometime gives rise to erroneous conclusions concerning apparent naloxone-reversability of putative analgesic procedures.     

Anandamide content and interaction of endocannabinoid/GABA modulatory effects in the NTS on baroreflex-evoked sympathoinhibition

Cannabinoids have been shown to modulate central autonomic regulation and baroreflex control of blood pressure (BP). The presence of cannabinoid CB(1) receptors on fibers in the nucleus tractus solitarius (NTS) suggests that some presynaptic modulation of transmitter release could occur in this region, which receives direct afferent projections from arterial baroreceptors and cardiac mechanoreceptors. This study, therefore, was performed to determine the mechanism(s) of effects of microinjection of an endocannabinoid, arachidonylethanolamide (anandamide, AEA), into the NTS on baroreflex sympathetic nerve responses produced by phenylephrine-induced pressure changes in anesthetized rats. AEA prolonged reflex inhibition of renal sympathetic nerve activity (RSNA), suggesting an increase in baroreflex sensitivity. This effect of AEA was blocked by prior microinjection of SR-141716 to block cannabinoid CB(1) receptors. To determine whether this baroreflex enhancement by AEA involved a GABA(A) mechanism, the baroreflex response to AEA was tested after prior blockade of postsynaptic GABA(A) receptors by bicuculline, which would eliminate any effects due to modulation of GABA activity. After bicuculline, which alone prolonged the baroreflex inhibition of RSNA, AEA shortened the duration of RSNA inhibition, suggesting a possible presynaptic inhibition of glutamate release previously obscured by a more dominant GABA(A) effect. To support a possible physiological role for AEA, AEA concentration in the NTS was measured after a phenylephrine-induced increase in BP. AEA content in the NTS was increased significantly over that in normotensive animals. These results support the hypothesis that AEA content is increased by brief periods of hypertension and suggest that AEA can modulate the baroreflex through activation of CB(1) receptors within the NTS, possibly modulating effectiveness of GABA and/or glutamate neurotransmission.