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Increased opioid synthesis and release, and enhanced alpha-2 adrenoceptor signaling have been suggested to mediate repeated oxytocin-induced long-lasting effects including elevated pain threshold in rats. This study evaluated whether oxytocin pretreatment would influence development of dependence and tolerance to the nociceptive and body temperature responses to morphine and enhance effects of alpha-2 adrenergic agonist clonidine on nociceptive threshold, body temperature and morphine withdrawal signs. Rats injected subcutaneously with saline or 1 mg/kg oxytocin for 5 days were implanted with placebo or morphine pellets 24 h after the treatment period. Body temperature and nociception were assessed, with nociception determined via by hot plate and tail immersion tests, before and 4, 24 and 48 h after pellet implantation, and following a challenge dose of morphine. Withdrawal signs were determined after naloxone administration. Oxytocin produced analgesia, as evidenced by increased paw withdrawal latency in the hot plate test. Morphine increased body temperature and nociceptive threshold which declined over time. Morphine challenge could not demonstrate tolerance to the body temperature response. Analgesic tolerance was observed in the hot plate test in saline and in both tests in oxytocin pretreated rats. Naloxone-precipitated withdrawal appeared to be less severe in oxytocin pretreatment. Clonidine was ineffective on the withdrawal signs but decreased body temperature and increased tail flick latency in the tail immersion test in oxytocin pretreated animals. These results, while producing evidence for a hyperresponsiveness in alpha-2 adrenoceptors, provide contrasting effects on morphine tolerance and dependence, and their partial mediation by opioidergic and adrenergic activation in repeated oxytocin treatment.

Locus coeruleus (LC)-spinal noradrenergic projections are important to endogenous analgesic mechanisms and can be activated by local glutamate signaling in the LC. The current study examined the local glutamatergic, GABAergic, and noradrenergic influences on glutamate release in the LC and noradrenergic descending inhibition in rats 6 weeks after spinal nerve ligation (SNL). Intra-LC injection of the α2 adrenoceptor antagonist idazoxan or the group 2 metabotropic glutamate receptor (mGluR) antagonist (RS)-α-Methyl-4-tetrazolylphenylglycine (MTPG) increased withdrawal thresholds in SNL animals and this was reversed by the blockade of α-amino-3-hydroxy-5-methyl- 4-isoxazolepropionic acid (AMPA) receptors in the LC or α2-adrenoceptors in the spinal cord, but not in normal animals. Neither blockade of GABA-A nor GABA-B receptors in the LC affected withdrawal thresholds in normal and SNL animals. Intra-LC perfusion of idazoxan increased extracellular glutamate in the LC in SNL animals but not in normal animals. Intra-LC perfusion of MTPG increased extracellular glutamate in the LC in both normal and SNL animals. These results suggest that local noradrenaline and glutamate tonically inhibit glutamate release in the LC after peripheral nerve injury and this may contribute to reduced descending inhibition in response to noxious input during chronic neuropathic pain.

It is general acknowledged that genes play a vital role in the etiology of attention deficit/hyperactivity disorder (ADHD). The relationship between the genes involved in catecholamine (dopamine, noradrenaline)/serotonin transmissions and ADHD has been widely described in medical literature. A pathway-based study was conducted in this study to test the association of gene-gene interaction and the cumulative effect of genetic polymorphisms within the dopamine, norepinephrine, and serotonin neurotransmitter pathways with ADHD susceptibility. A case-control study was conducted among Chinese children, and 168 ADHD patients and 233 controls were recruited using a combination diagnosis according to the DSM-IV ADHD rating scale. Classification and regression tree (CART) analysis was conducted to explore the gene-gene interaction, and logistic regression modal was applied to estimate the polygenic risk of the potential multiple genetic variants. The results of CART analyses indicated that the children carrying the combination of ADRA2A rs553668GG/GA and SLC6A4 rs6354 GG/GT genotypes displayed a 6.15-fold increased risk of ADHD, compared to those with the combination of ADRA2A rs553668 AA and ANKK1 rs1800497 AA genotypes. The unfavorable alleles of ADRA2A rs553668 G, DRD2 rs1124491 G and SLC6A4 rs6354 G showed cumulative effects on ADHD, and the OR for ADHD may increase by 1.42 times when the number of unfavorable allele number increased by one. Those findings reveal the importance of the gene-gene interactions and polygenic effects of many common variants to ADHD susceptibility, even the effect of each variant is very small.

Control of postoperative pain is far from satisfactory. Yet, non-steroidal anti-inflammatory drugs (NSAIDs) remain an important choice. The production of nitric oxide (NO), which plays an important role in the development and maintenance of inflammatory hyperalgesia, is inhibited by NSAIDs. Monoamines also play a key role in the modulation of nociception. The aim of the present work is to study the involvement of NO and monoamines in the antinociceptive mechanism of ibuprofen in postsurgical pain in mice. Surgical incision resulted in mechanical allodynia and increased spinal NO levels. The nitric oxide synthase inhibitor l-NAME (50mg/kg), administered intraperitoneally (i.p.), 30min before the incision decreased the development of postsurgical mechanical allodynia and reduced spinal NO levels. Ibuprofen (100 and 300mg/kg, i.p.), administered 30min before the incision, dose-dependently decreased both spinal NO levels and the development of mechanical allodynia. Administration of ibuprofen (100mg/kg i.p.), 20min following surgery, did not significantly reduce spinal NO level and resulted in a smaller antiallodynic effect. l-Arginine (600mg/kg i.p.), administered 20min before ibuprofen administration, restored both spinal NO level and mechanical allodynia in ibuprofen-treated mice. The selective alpha-2 adrenoceptor blocker yohimbine (4mg/kg i.p.), administered 30min before ibuprofen, also blocked ibuprofen effect on both mechanical allodynia and spinal NO level. These results suggest that inhibition of NO synthesis is involved in the analgesic activity of ibuprofen in post-surgical pain. Alpha-2 adrenoceptors are also involved in the analgesic activity of ibuprofen and NO may be involved in this mechanism.

Dexmedetomidine, a selective alpha 2-adrenoceptor (α2AR) agonist, has provided significant analgesia in neuropathic pain. However, its underlying molecular mechanism has not been fully elucidated. In the present study, we found that intrathecal administration of dexmedetomidine alleviated mechanical allodynia induced by chronic constriction injury (CCI), and pretreatment with BRL44408 significantly reversed the dexmedetomidine-induced anti-nociceptive effect. Western blotting revealed that dexmedetomidine reduced the activation of microglia and the upregulation of interleukin-18 (IL-18) protein expression in the ipsilateral lumbar spinal dorsal horn, while BRL44408 pretreatment significantly blocked these effects of dexmedetomidine. Immunocytochemistry/immunohistochemistry indicated that the α2A-adrenoceptor was localised to microglia in primary culture, and IL-18 predominantly colocalised with the microglial marker Iba-1 in the dorsal horn of the spinal cord. These results suggest that the IL-18 signalling pathway in microglia may be involved in the anti-nociceptive effect of dexmedetomidine in rats subjected to CCI.

Previous studies have revealed that the central dopaminergic system may participate in regulating sleep/wakefulness. In particular, rapid eye movement (REM) sleep behavior disorder (RBD) occurs in patients with Parkinson's disease (PD), highlighting the possible connection between dopamine and REM sleep-related neural structures. The dorsal subcoeruleus nucleus (SubCD) is a critical structure for the generation and maintenance of REM sleep. Thus, the present study investigated the modulatory effects of dopamine on SubCD neurons. Using whole-cell patch clamp recordings, we first observed that dopamine induced a hyperpolarization of the membrane potentials in SubCD neurons and thus inhibited their firing. We determined that a dose-dependent and tetrodotoxin-resistant postsynaptic outward current underpinned this inhibitory effect on SubCD neurons induced by dopamine. Finally, using pharmacological agents, we revealed that the dopamine-elicited outward current in SubCD neurons was mediated by α2-adrenergic receptors, but not by the dopamine receptors, including D1-like and D2-like receptors. These results suggest that the central dopaminergic system may play a role in the regulation of REM sleep through the effect of dopamine on SubCD neurons. The relationship between the loss of this effect and the RBD in PD is discussed.

The role played by spinal adrenergic and cholinergic receptors in the antinociceptive effects of intrathecal sildenafil in formalin-induced nociception was examined. Intrathecal catheters were inserted into the subarachnoid space of male Sprague-Dawley rats, and nociception was assessed using the formalin test, consisting of a subcutaneous injection of 50 μL of 5% formalin solution into the hind paw. We examined the effects of an alpha 1 adrenergic receptor antagonist (prazosin), an alpha 2 adrenergic receptor antagonist (yohimbine), a muscarinic acetylcholine receptor antagonist (atropine), and a nicotinic acetylcholine receptor antagonist (mecamylamine) on sildenafil-induced antinociception. Intrathecal sildenafil (3, 10, and 30 μg) suppressed, in a dose-dependent manner, formalin-induced flinching during phases 1 and 2 of the test. Intrathecal sildenafil (30 μg) could not show any effects against intrathecal prazosin (3 μg), yohimbine (10 μg), atropine (10 μg), and mecamylamine (10 μg) pretreatment during both phases of the formalin test. These results suggest that intrathecal sildenafil effectively attenuated the pain evoked by formalin injection. Additionally, spinal alpha 1, alpha 2, muscarinic and nicotinic receptors might play a role in sildenafil-induced antinociception.

In this study we provide evidence that animals of the same population, although identical in age and sex, have individual reactions to the prefrontal modulation of adrenoceptors. We have examined the dose-dependent action of α(2)-adrenoceptor agents on the anxiety of rats with different response to novelty in the elevated plus maze (EPM) apparatus. Rats were divided into high (HR) and low responder (LR) groups based on their locomotor activity in a novel open field environment. HR rats also showed increased locomotion and low anxiety in the EPM. Prefrontal injection of α(2)-receptor antagonist yohimbine, BRL44408 or imiloxan caused anxiety only in HR rats. The α(2A/D)-receptor agonist guanfacine increased anxiety levels of both groups. However, the effective dose was lower in HR rats. The present results propose different prefrontal adrenoceptor sensitivity of rats showing distinct baseline activity levels.

The authors examined the antinociceptive effect of melatonin in a nociceptive state and investigated a possible interaction with adrenergic or cholinergic receptors underlying this effect at the spinal level. Nociception was induced by a subcutaneous injection of 50 μl of a 5% formalin solution to the hindpaw of male Sprague-Dawley rats. The reversal effects of alpha-1 adrenoceptor antagonist (prazosin), alpha-2 adrenoceptor antagonist (yohimbine), muscarinic receptor antagonist (atropine) and nicotinic receptor antagonist (mecamylamine) on the activity of melatonin were assessed. Intrathecal melatonin reduced the flinching response during phase 1 and phase 2 in the formalin test. Intrathecal prazosin, yohimbine, atropine and mecamylamine increased the attenuating flinching response in both phases observed by intrathecal melatonin. Collectively, the present data suggest that intrathecal melatonin attenuates the facilitated state and acute pain evoked by formalin injection. Furthermore, the antinociception of melatonin is mediated through the alpha-1 adrenoceptor, alpha-2 adrenoceptor, muscarinic and nicotinic receptors in the spinal cord.

Activation of the alpha(2)-adrenoceptor has been shown to produce antinociception. We have previously shown that the antinociceptive effect of clonidine, an alpha(2)-adrenoceptor agonist, is sex-specific and is abolished by exogenous estrogen in ovariectomized rats or high level of endogenous estrogen in proestrous females. Here, we investigated whether testosterone mediates the antinociceptive effect of clonidine in the trigeminal region of the male rat. Clonidine (7 microg/5 microl) was injected intracisternally through a PE-10 cannula implanted dorsal to the trigeminal region in orchidectomized (GDX) male Sprague-Dawley rats. In separate groups, testosterone propionate (250 microg/100 microl; GDX+T) or beta-estradiol benzoate (100 microg/100 microl; GDX+E) were injected subcutaneously 24 and 48 h respectively prior to the N-methyl-D-aspartic acid (NMDA)--or heat-evoked nociceptive test. NMDA-induced number of scratches or duration of scratching behavior did not change significantly in control groups with or without hormonal replacement. Clonidine significantly reduced both measures only in the GDX+T group but not in GDX or GDX+E group. Clonidine also significantly increased head withdrawal latency (HWL) in the GDX+T group, but not in GDX or GDX+E group. The antinociceptive effect of clonidine was reversed by yohimbine, an alpha(2)-adrenoceptor antagonist, in GDX+T group. We conclude that testosterone is required for the expression of antinociception produced by selective activation of the alpha(2)-adrenoceptor in the trigeminal region of the male rat. These findings further our understanding of sex-related differences in the modulation of nociception and may provide insight into development and administration of analgesic agents in young vs. aging men.

Anti-hypertensive drugs that act on central alpha(2)-adrenoceptors and imidazoline receptors usually cause dry mouth in patients. A central area important for the control of salivary secretion and also for the effects of alpha(2)-adrenoceptor activation is the lateral hypothalamus (LH). Therefore, in the present study we investigated the effects of the injections of moxonidine (an alpha(2)-adrenoceptor and imidazoline agonist) alone or combined with RX 821002 (alpha(2)-adrenoceptor antagonist) into the LH on the salivation induced by intraperitoneal (i.p.) pilocarpine (cholinergic muscarinic agonist). Male Holtzman rats with stainless steel cannula implanted into the LH were used. Saliva was collected using pre-weighted small cotton balls inserted into the animal's mouth under ketamine anesthesia. Salivation induced by i.p. pilorcarpine (4micromol/kg of body weight) was reduced by the injection of moxonidine (10 and 20nmol/0.5microl) into the LH (222+/-46 and 183+/-19mg/7min, vs. vehicle: 480+/-30mg/7min). The inhibitory effect of moxonidine on pilocarpine-induced salivation was abolished by prior injections of RX 821002 (160 and 320nmol/0.5microl) into the LH (357+/-25 and 446+/-38mg/7min). Injections of the alpha(1)-adrenoceptor antagonist prazosin (320nmol/0.5microl) into the LH did not change the effects of moxonidine. The results show that activation of alpha(2)-adrenoceptors in the LH inhibits pilocarpine-induced salivation, suggesting that LH is one of the possible central sites involved in the anti-salivatory effects produced by the treatment with alpha(2)-adrenoceptor agonists.

Studies on the neurotransmitter substrate of locomotion and place navigation occupy a central position in behavioral neuroscience. Active allothetic place avoidance (AAPA) is a task, in which animals are trained to avoid a room frame defined stable sector on a continuously rotating arena. The aim of the present study was to test the effect of the blockage of alpha1- and alpha2-adrenoceptors, using specific antagonists prazosin and idazoxan, on the locomotor activity and spatial behavior in the AAPA task. Both prazosin and idazoxan at the highest doses (4 and 6 mg/kg, respectively) were found to decrease the locomotor activity in the AAPA and they also impaired navigational performance. The results suggest that antagonizing alpha-adrenoceptors with systemically administered drugs affects locomotor activity together with avoidance behavior and does not cause a purely cognitive deficit in the AAPA task.

In the present study we sought to investigate the signal transduction mechanisms that underlie the alpha2-adrenergic receptor (AR)-induced, subtype-specific neuronal differentiation of PC12 cells. Alpha2-ARs induced NF-kappaB transcriptional activity and p21(waf-1) gene transcription in the same subtype-specific manner (alpha2Aalpha2Balpha2C) as the neuronal differentiation induced by these receptors. Following pretreatment with the MEK1 inhibitor PD98059, the NF-kappaB response to epinephrine becomes uniform with loss of subtype specificity. In contrast, there is complete abolishment of epinephrine-induced transcription by NF-kappaB after pretreatment with the PI3-K inhibitor LY294002. These data imply that induction of transcription by NF-kappaB is PI-3K-dependent; however, the subtype-specific induction of transcription by NF-kappaB is ERK1/2-dependent. Taken together, these results suggest that the three alpha2-adrenoceptors promote an interaction between PI-3K and ERK1/2 in a subtype-specific way, leading to subtype-specific induction of transcription by NF-kappaB and p21(waf-1) gene transcription, which might underlie the subtype-specific differentiation of PC12 cells induced by these receptors.

Yohimbine has been widely used as alpha2-adrenergic receptor antagonist in neurophysiological research and in clinical therapy. In this study, we provide in vivo electrophysiological evidence, that microiontophoretic application of yohimbine (YOH) inhibits spontaneous activity of prefrontal neurons of the rat. By microiontophoretic application of the alpha2A-receptor antagonist BRL44408 (BRL), the effects of YOH could be mimicked, indicating that the action of YOH is manifested through alpha2A/D-receptor mechanisms. Furthermore, the inhibiting effects of YOH or BRL were blocked by alpha2B-receptor antagonist imiloxan. In concert with previous microiontophoretic data, the present findings suggest that alpha2-receptor antagonist YOH predominantly acts on the alpha2A/D-receptor subtype in vivo. Furthermore, we hypothesize that this action is manifested via deactivation of autoreceptors causing increased norepinephrine release, finally inhibiting postsynaptic neurons through the activation of alpha2B-receptors.

Since running accompanied with blood lactate accumulation stimulates the release of adrenocorticotropic hormone (ACTH), running above the lactate threshold (LT) acts as stress (running stress). To examine whether A1/A2 noradrenergic neurons that project to the hypothalamus activate under running stress, c-Fos immunohistochemistry was used to compare the effects of running with or without stress response on A1/A2 noradrenergic neurons. Blood lactate and plasma ACTH concentrations significantly increased in the running stress group, but not in the running without stress response and control groups, confirming different physiological impacts between different intensity of running with or without stress. Running stress markedly increased c-Fos accumulation in the A1/A2 noradrenergic neurons. Running without stress response also induced a significant increase in c-Fos expression in the A1/A2 noradrenergic neurons, and the percentage of the increase was smaller than that of running stress. The extent of c-Fos expression in the A1/A2 noradrenergic neurons correlates with exercise intensity, signifying that this neuronal activation is running speed-dependent. We thus suggest that A1/A2 noradrenergic neurons are activated in response to not only running stress, but also to other physiological running, enhanced by non-stressful running. These findings will be helpful in studies of specific neurocircuits and in identifying their functions in response to running at different intensities.

Although nerve injury is known to up- and down-regulate some metabotropic receptors in vagal afferent neurons of the nodose ganglia (NG), the functional significance has not been elucidated. In the present study, thus, we examined whether nerve injury affected receptor-mediated Ca2+ channel modulation in the NG neurons. In this regard, unilateral vagotomy was performed using male Sprague-Dawley rats. One week after vagotomy, Ca2+ currents were recorded using the whole-cell variant of patch-clamp technique in enzymatically dissociated NG neurons. In sham controls, norepinephrine (NE)-induced Ca2+ current inhibition was negligible. Following vagotomy, however, the NE responses were dramatically increased. This phenomenon was in accordance with up-regulation of alpha2A/B-adrenergic receptor mRNAs as quantified using real-time RT-PCR analysis. In addition, neuropeptide Y (NPY) and prostaglandin E2 responses were moderately augmented in vagotomized NG neurons. The altered NPY response appears to be caused by up-regulation of Y2 receptors negatively coupled to Ca2+ channels. In contrast, nerve injury significantly suppressed opioid (tested with DAMGO)-induced Ca2+ current inhibition with down-regulation of micro-receptors. Taken together, these results demonstrated for the first time that the profile of neurotransmitter-induced Ca2+ channel modulation is significantly altered in the NG neurons under pathophysiological state of nerve injury.

The effect of reboxetine on norepinephrine (NE) efflux in the frontal cortex of freely moving rats has been studied using in vivo microdialysis. Reboxetine was administered either by injection (10 and 30 mg/kg i.p.) or directly to the frontal cortex via the dialysis probe (10 and 100 microM). To further elucidate the mechanism of action of reboxetine in this region yohimbine (10 microM) was co-infused with reboxetine via the frontal cortex probe. Both routes of administration of reboxetine resulted in a drug-induced, dose-dependent decrease in frontal cortex NE efflux. This effect was reversed following co-infusion of yohimbine, which has been shown to possess both alpha(2)-adrenoceptor antagonist and 5-HT(1A) agonist properties.

This study was undertaken to investigate the effects of the administration of 3,4-methylenedioxymethamphetamine (MDMA) on the locus coeruleus firing rate, on the sensitivity of the alpha(2)-adrenoceptors which regulate neuronal activity and on the in vivo tyrosine hydroxylase activity in hippocampus. The basal firing rate was not modified by either a single dose or repeated doses of MDMA, although the latter produced a shift to the right in the dose-response curve for clonidine-induced inhibition of the firing rate (ED(50) increased by 59%) and a reduction in tyrosine hydroxylase activity (20%) in the hippocampus. However, 8 days after the final dose alpha(2)-adrenoceptor sensitivity and tyrosine hydroxylase activity had returned to control values. Our results show a desensitization of alpha(2)-adrenoceptors in locus coeruleus and the existence of short-term changes in the noradrenergic system.

In decerebrated rabbits, reflexes were evoked in medial gastrocnemius motoneurones by mechanical stimulation of the heel, using four pinch strengths from 183 (innocuous) to 4577 (noxious) mN. The alpha(2)-adrenoceptor antagonist idazoxan (1-156 microg intrathecal (i.th.) significantly increased responses to pinch strengths of 607 mN and above. Subsequent administration of the alpha(1) adrenoceptor selective antagonist prazosin (200 microg i.th.) decreased reflexes to 4577 mN pinches but had no other significant effects. The opioid antagonist (-)-quadazocine (25 microg i.th.) caused no further changes in reflexes. Spinal section at L1 in the presence of this drug combination enhanced gastrocnemius responses to 183 and 607 mN stimuli, had no effect on reflexes to 1866 mN and significantly decreased responses to 4577 mN pinches. These data confirm that reflexes evoked by 'natural' stimulation of heel mechanoreceptors are subject to powerful tonic descending inhibition mediated by alpha(2)-adrenoceptors. For the highest strength stimulus, the results of alpha(2) blockade involved enabling of descending facilitation as well as reduction of descending inhibition.

The goal of the present study was to determine whether bee venom (BV) injection into the Zhongwan acupoint (CV12), compared to injection into a non-acupoint, produced antinociception in an acetic acid-induced visceral pain model. This was accomplished by injecting BV subcutaneously into the Zhongwan acupoint or into a non-acupoint 30 min before intraperitoneal injection of acetic acid in ICR mice. BV injection into the acupoint produced a dose dependent suppression of acetic acid-induced abdominal stretches and of acetic acid-induced Fos expression in the spinal cord and the nucleus tractus solitarii. In contrast BV injection into the non-acupoint only produced antinociception at the highest dose of BV tested. Naloxone pretreatment did not alter the antinociceptive effect of BV acupoint injection on the abdominal stretch reflex. On the other hand, pretreatment with the alpha 2-adrenoceptor antagonist, yohimbine completely blocked the antinociceptive effect of BV acupoint injection. These results imply that BV acupoint stimulation can produce visceral antinociception that is associated with activation of alpha 2-adrenoceptors, but not with naloxone-sensitive opioid receptors.

The effects of α 2 -adrenoceptor activation on N -methyl- d -aspartic acid (NMDA)-induced perturbation of neurotransmission and normal NMDA-receptor dependent function (long-term potentiation, (LTP)) were investigated in the hippocampal CA1 field in vitro. Bath perfusion of dexmedetomidine hydrochloride (50 nM), which was initiated before NMDA (100 μM) exposure, enhanced the extent of recovery of extracellular field excitatory postsynaptic potentials after NMDA infusion. On the other hand, the induction and early maintenance of LTP was normal in the presence of dexmedetomidine. Thus, dexmedetomidine can diminish acute NMDA-induced perturbation of neurotransmission while the same dose of this drug does not influence the normal activation of NMDA receptors.

7-Methoxy-6-[3-[4-(2-methoxyphenyl)piperazin-1-yl]propoxy]-3,4-dim ethyl-2H-1-benzopyran-2-one hydro-chloride (KA-672), structurally related to naturally occurring coumarins, has been described as a potential drug for enhancing cognitive functions. However, a detailed characterization of the pharmacological profile of KA-672 in vivo is still lacking. Quantitative neurotransmitter receptor autoradiography was used as a tool to screen for KA-672-induced changes in a number of transmitter receptors including cholinergic, noradrenergic, glutamatergic, GABAergic, and serotonergic subtypes throughout the brain. Two hours following treatment of mice with 1 mg/kg KA-672 per os, slight increases of nicotinic and M1-muscarinic cholinergic receptor binding, of alpha2-and beta-adrenoceptor as well as 5-HT1A receptors in various cerebral cortical regions were observed, whereas 5-HT2A binding sites were strikingly increased throughout the brain following KA-672 treatment. In contrast, (+/-)-alphaamino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor binding was significantly decreased in some cortical regions after drug treatment. No effects of KA-672 treatment on N-methyl-D-aspartate, kainate, GABA(A) and benzodiazepine receptor as well as M2-muscarinic cholinergic and high-affinity choline uptake binding were observed. As interactions between the cholinergic, noradrenergic and serotonergic neurotransmission have been stressed to play important roles in realizing learning and memory events, the cognition-enhancing effects of KA-672 may be due to this complex in vivo pharmacological profile of KA-672.

It is known that electrical and l -glutamate stimulation of the locus coeruleus (LC) reduce the multiunit activity evoked in the nucleus of the tractus solitarius (NTS) by cyanoboro-hydride. In the present study, rats anaesthetised with urethane were microinjected with noradrenergic antagonists into the NTS, to test whether the inhibitory effects of LC stimulation on cyanide-evoked discharge in the NTS were mediated by noradrenaline. Microinjection of yohimbine into the NTS (0.2, 0.6, 1.8, 5.4 nmol) induced dose-dependent reduction of the inhibitory effect of LC stimulation on NTS neurones, while prazosin produced no effect. The results indicate that LC-induced inhibition of the cyanide-evoked discharge in the NTS is mediated by endogenous noradrenaline release acting on α 2 -adrenoreceptors.

The present study examined the effects of sympathetic stimulation on the activity of primary afferent neurons that had peripheral axons being injured previously by a spinal nerve ligation. About 22% of afferents with injured fibers that showed spontaneous discharge were excited by sympathetic stimulation or systemic injection of adrenaline. Most sympathetically-excited afferent neurons had axons that conducted in the A-fiber range. This sympathetically-evoked afferent excitation was not affected by cutting the spinal nerve at a place close to the dorsal root ganglion (DRG). Yohimbine, alpha2-antagonist, suppressed sympathetically-evoked afferent excitation which was not affected by alpha1-antagonist prazosin. Clonidine, alpha2-agonist, exerted an excitatory effect, whereas alpha1-agonist phenylephrine had no effect on the activity of afferents with injured fibers. No afferent fibers in control preparations responded to sympathetic stimulation. The results suggest that after a spinal nerve ligation, injured DRG neurons with fast-conducting fibers become sensitive to sympathetic activity via activation of alpha2-adrenoceptors.

We have studied the effect of [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO, opioid mu-receptor agonist) and ICI-204,448 (kappa-receptor agonist) on endogenous noradrenaline release in the guinea-pig isolated distal colon. DAMGO enhances noradrenaline over-flow and this effect is antagonized by naloxone (pIC50 = 10.27) and nor-binaltorphimine (pIC50 = 7.97), and concentration-dependently turned into inhibition by yohimbine. ICI-204,448 inhibits noradrenaline overflow and is antagonized by naloxone (pIC50 = 9.38) and nor-binaltorphimine (pIC50 = 10.48), but is not affected by yohimbine. Evidence is thus given that mu- and kappa-opioid receptors modulate noradrenaline release in the guinea-pig colon. Modifications by yohimbine of the effect of DAMGO indicate the existence of a functional relationship between mu-receptors and alpha(2)-autoreceptors in this model.

α2 Adrenoceptors (α2-ARs) are important in regulating the central control of blood pressure in medulla oblongata. However, it is unclear how this receptor is modulated by different receptors, especially the glutamatergic. In the present study, we studied the influence of ionotropic glutamatergic receptors over the α2-ARs in cultured cells of the medulla oblongata of newborn rats. For this purpose, the protein level of the α2-ARs was assessed after administration to the cultured cells of glutamate (glu), the agonists NMDA and kainate (KA), the NMDA receptor antagonist MK801 and the KA receptor antagonist DNQX. Results indicate that the α2-AR protein levels were increased after the treatments with glu and NMDA, and the addition of MK801 to this treatment thwarted this increase. Notwithstanding the fact that KA did not alter the receptor protein level, the combined treatment of DNQX with glu prevented the α2-AR protein modulation. In conclusion, the present study suggests that ionotropic glutamatergic receptors could be related to the α2-AR protein regulation in the medulla oblongata.

The purpose of this study was to examine the effects of intraplantar dexmedetomidine to relieve neuropathic pain and determine the role of peripheral α2-adrenoceptors. Neuropathic pain was induced by ligating the L5 and L6 spinal nerves in male Sprague-Dawley rats, and mechanical allodynia was assessed using von Frey filaments. Several antagonists were injected into the hindpaws to evaluate the mechanisms of action of dexmedetomidine, a nonselective α2-adrenoceptor antagonist yohimbine, an α2A-adrenoceptor antagonist BRL 44408, an α2B-adrenoceptor antagonist ARC 239, and a α2C-adrenoceptor antagonist JP 1302. The expression of α2A-adrenoceptor, α2B-adrenoceptor, and α2C-adrenoceptor genes in the lumbar segment of the spinal cord and the plantar skin of the nerve-injured leg was detected by reverse transcription-polymerase chain reaction. Ipsilateral intraplantar injection of dexmedetomidine produced dose-dependent antiallodynia. Ipsilateral, but not contraleral, intraplantar injection of yohimbine reversed the antinociception of dexmedetomidine. Intraplantar BRL 44408, ARC 239, and JP 1302 reversed the antinociception of dexmedetomidine. The expression levels of α2-adrenoceptor genes in the lumbar spinal cord did not differ between rats with neuropathic pain and naive rats. The expression levels of α2B-adrenoceptor and α2C-adrenoceptor genes of plantar skin were upregulated significantly in the model group, whereas α2A-adrenoceptor expression was unchanged. These results suggest that intraplantar injection of dexmedetomidine produced an antiallodynic effect in spinal nerve ligation-induced neuropathic pain. All three types of peripheral α2A, α2B, and α2C-adrenoceptors were involved in the antiallodynic mechanism of dexmedetomidine.

α2-Adrenergic agonists simulate norepinephrine (NE) action on α2 receptors of sympathetic neurons to mediate feedback inhibition of NE release. These agents are used as valuable adjuncts for management of hypertension and for anesthesia. Their action, equivalent to NE on α2 adrenergic receptors, raises the question whether α2 agonists may also target NE transporters (NETs), another major control mechanism for noradrenergic neurotransmission. We thus investigated the effect of α2 agonists on transport of the NE analog, (131)I-metaiodobenzylguanidine (MIBG). Results from this investigation showed that xylazine and dexmedetomidine dose-dependently blocked [(3)H]nisoxetine binding in neuron-like SK-N-SH cells. Furthermore, the agents acutely suppressed cellular MIBG uptake in a dose-dependent manner. This effect was uninfluenced by the α2 antagonist yohimbine, but was completely reversed by drug removal. There was no change in membrane NET density by the agents. Moreover, saturation analysis showed that xylazine and dexmedetomidine significantly increased Km without affecting Vmax, indicating competitive inhibition of MIBG transport. Thus, the α2 adrenergic agonists xylazine and dexmedetomidine, acutely suppress NET function through competitive inhibition of substrate transport, likely by direct interaction on a region that over-laps with the nisoxetine binding site.

The effects of noradrenaline (NA) on synaptic responses in layer II of the entorhinal cortex (EC) were studied in normal and spontaneously epileptic mutant mice tottering using intracellular recording in a slice preparation. Neither the membrane properties of neurones nor the responses to NA differed between normal and tottering mice. NA (50 microM) hyperpolarized most (29/54) of the neurones via alpha 2 adrenergic receptors. Synaptic responses of EC neurones were complex. NA (10-100 microM) reduced all the components of the synaptic response in a concentration-dependent and reversible manner. The pharmacological properties of the inhibitory effect of NA were characterised and quantified on one component of the complex synaptic response, the fast excitatory postsynaptic potential. The effect of NA was mimicked by the alpha 2 agonist UK 14,304 and blocked by the alpha 2 antagonist yohimbine. It is concluded that NA can inhibit via an alpha 2 receptor-mediated action synaptic responses in the superficial layers of the EC.

To test the contribution of sympathetic postganglionic neurons (SPGNs) to adrenergic excitation of cutaneous nociceptor activities in adjuvant-inflamed rats (AI rats), we studied the effects of norepinephrine (NE) in a group of sympathectomized AI rats. Sympathectomy was complete in 4–6 weeks by guanethidine (30–50 mg/kg). NE excited polymodal receptor units in the saphenous nerve at an incidence (25–30%) similar to that in AI rats, while CH-38083 blocked such excitation. These results show that α 2 -adrenoceptors in SPGNs are not involved in the norepinephrine excitation of cutaneous nociceptors in AI rats.

Previously, it was proposed that sedative and anesthetic effects of alpha2-adrenergic receptor (alpha2-AR) agonists may be exerted via neuronal networks normally implicated in the regulation of wakefulness. The aim of this study was to evaluate the role of A subtype of alpha2-ARs in the development of drug-independent anesthetic state induced by hypothermia in newborn rats. Using short interfering RNA (siRNA) gene-targeting strategy, we found that down-regulation of the brainstem alpha2A-AR expression resulted in a delay in the onset of hypothermia-induced anesthesia assessed by loss of righting reflex. Involvement of the brain alpha2A-ARs in this delay was confirmed by inability of clonidine, a subtype-nonselective alpha2-AR agonist, to prolong duration of hypothermia-induced anesthesia in siRNA-treated animals, while significant prolongation of this anesthetic state by the alpha2A-AR agonist was observed in control pups. The data suggest that negative regulation of the animal's waking state is an intrinsic function of the brainstem alpha2A-ARs activated by exogenous agonists, as well as by endogenous noradrenaline, also.

Leptin binding to its functional receptor stimulates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signalling pathway, finally resulting in nuclear translocation of the phosphorylated STAT3 (P-STAT3). Systemic treatment with leptin (3mg/kg; intraperitoneal injection) induced the appearance of P-STAT3-immunoreactive cells in adult mouse preoptic area (POA). Here we show that the vast majority of leptin-responsive cells were located in medial POA (mPOA), followed by the median preoptic nucleus. Rare, scattered and weakly stained cells were found in ventromedial preoptic nucleus and lateral preoptic area. Co-localization studies disclosed that mPOA leptin-responsive cells were neurons, and that a large proportion expressed the alpha(1A)- and/or alpha(2A)-adrenergic receptor (AR) isoforms. Although understanding the functional relevance of leptin-responsive POA neurons requires further investigation, the finding that they bear alpha-ARs suggests that they may be targeted by the ascending noradrenergic system, which densely innervates the mPOA, and thus be involved in thermoregulation, arousal and/or the sleep-wake cycle.

Findings from preclinical and clinical research support the involvement of central noradrenergic dysregulation in the etiology of attention deficit hyperactivity disorder (ADHD). Previous studies have suggested that the alpha-2C-adrenergic receptor gene (ADRA2C) is associated with ADHD. The aims of this study were to examine the association between the ADRA2C (GT)n repeat polymorphism (STR marker adra2c1) and ADHD in a Korean sample. In this case-control and family-based association study, we assessed 184 ADHD probands, 150 normal controls, and 98 trios. There were no significant differences in the allele frequencies of the ADRA2C polymorphism between the ADHD and control groups (p > 0.05). The overall allele-wise transmission disequilibrium test (TDT) analysis showed statistical significance (chi2 = 19.07, p = 0.025). We found a trend for preferential transmission of the 183-bp allele (chi2 = 3.72, p = 0.054), and a significantly lower-than-expected rate of transmission of the 187-bp allele (chi2 = 6.26, p = 0.012). With regard to the temperament profiles of the Junior Temperament and Character Inventory (JTCI), the ADHD subjects with the 183/183 genotype at the ADRA2C polymorphism showed a trend toward a lower score in the Novelty Seeking (p = 0.020) profile than did those with the other genotypes. Our findings provide important evidence that the ADRA2C polymorphism is involved in the etiology of ADHD in Korean subjects. In addition, our results provide evidence that the temperament of Novelty Seeking and ADHD might share molecular genetic characteristics related to the noradrenergic system.

Some intravenous anesthetic agents such as midazolam are known to induce anterograde and retrograde amnesia. We analyzed the effect of midazolam by the conditioned taste aversion (CTA) acquisition and retention. After the rats were offered 0.1% sodium saccharin (Sac) as conditioned stimulus (CS), an intraperitoneal (i.p.) injection of several concentrations (5-30mg/kg) of midazolam was followed by an i.p. injection of 0.15M LiCl (2% of body weight) as unconditioned stimulus (US). The rats, which acquired CTA by every CS-US paradigm, strongly avoided Sac on the 1st test day after conditioning and maintained the avoidance for 3 days. We have already reported that Sac intake abruptly increased on the 2nd test day and the almost complete extinction occurred on the 3rd test day after conditioning by injection of subhypnotic dose of propofol before LiCl-injection. In contrast, we found that subhypnotic dose of midazolam suppressed not only CTA acquisition, but also CTA retention. On the other hand, an alpha2-adrenergic blocker, yohimbin (1mg/kg) suppressed only the CTA retention. These results suggest that the subhypnotic doses of midazolam firstly affect the acquisition mechanism of the CTA memory (CTAM), resulting the suppression of the retention of CTAM., Neuroscience Letters, 450(3), pp.270-274; 2009

Clinical studies have reported the beneficial outcome of addition of lower doses of risperidone to antidepressant therapy specifically with selective serotonin reuptake inhibitors (SSRIs) in the treatment of major depression. The present study, therefore, examined the beneficial effect, if any, of addition of risperidone (an atypical antipsychotic) to the antidepressant-like effect of venlafaxine (dual reuptake inhibitors of both serotonin and norepinephrine, SNRI) or fluoxetine (SSRI) in Porsolt's Forced Swim Test (FST) using male laca mice. Attempts have been made to study the involvement of alpha-2 adrenergic receptors in the mechanism of action. Immobility period was recorded for a period of 6 min. Venlafaxine (4 and 8 mg/kg, i.p.) or fluoxetine (10 and 20 mg/kg, i.p.) inhibited the immobility period in mice. Addition of risperidone (0.1 mg/kg, i.p.) potentiated the anti-immobility effect of either venlafaxine (4 and 8 mg/kg, i.p.) or fluoxetine (10 and 20 mg/kg, i.p.) in mouse FST. Furthermore, the anti-immobility effect of combination of risperidone (0.1 mg/kg, i.p) plus venlafaxine (4 mg/kg, i.p.) or fluoxetine (10 mg/kg, i.p.) was potentiated by the addition of yohimbine (2 mg/kg, i.p.), an alpha-2 adrenoceptors antagonist. The results of the present study suggest that the beneficial consequences of addition of risperidone with venlafaxine or fluoxetine in mouse forced swim test may involve alpha-2 adrenergic receptors.

The control of movement by the basal ganglia is influenced by inputs from diverse brain structures. Unfortunately, the mechanisms of modulation are poorly defined. Based on neuroanatomical evidence for alpha2A and alpha2C subtypes of alpha2 adrenergic receptors within this region, we hypothesize that noradrenergic alpha2-receptors can influence transmitter release in the SNr. To test this hypothesis we examined the effect of the alpha 2 adrenergic agonist, clonidine, and antagonist, rauwolscine, on the efflux of [3H]-GABA and [3H]-noradrenaline from brain slices of the rat substantia nigra pars reticulata. At low concentrations (10 nM), rauwolscine caused an 84.2 +/- 18.51% (p < 0.01) increase in KCl-evoked GABA release. At higher concentrations, rauwolscine caused a dose-dependent return to basal levels. Rauwolscine also enhanced basal GABA efflux after KCl washout with a similar biphasic concentration-dependence. Surprisingly, clonidine also enhanced [3H]-GABA release but had no effect on KCl-evoked [3H]-GABA release at concentrations which inhibited [3H]-NA efflux. These effects were potentiated by the GABA re-uptake inhibitor nipecotic acid. Together, our data indicate an important role for noradrenergic modulation in the SNr. The enhancing effect of both the alpha2 adrenoceptor agonist and antagonist on GABA release, while appearing paradoxical, can be rationalised by actions at distinct subsets of alpha2 adrenoceptors, using a simple model where alpha2A adrenoceptors are localized on the terminals of noradrenergic afferents impinging upon alpha2C adrenoceptor-containing GABAergic striato-nigral neurones.

Orexinergic and norepinephrinergic alpha2-adrenoceptor expressing neurons contribute to the regulation of the sleep-wakefulness cycle. In the present study, we have examined a possible interaction between orexinergic and alpha2-adrenergic systems in orexin-A (100 nM)- and K+ (25 mM)-evoked norepinephrine release from slices of rat cerebrocortex. In this tissue norepinephrinergic neurons are predominantly innervated via the locus coeruleus. Clonidine concentration-dependently inhibited K+-evoked norepinephrine release with pIC50 (Imax) of 6.44+/-0.38 (48.8+/-6.9%). A selective orexin-1 receptor antagonist, SB-334867 was ineffective. SB-334867 concentration-dependently inhibited orexin A-evoked norepinephrine release with pIC50 (Imax) of 6.05+/-0.14 (86.4+/-5.4%); clonidine (alpha2-agonist) was ineffective. In contrast, yohimbine reversed the inhibitory effects of clonidine (1 microM) on K+-evoked norepinephrine release with pIC50 (Imax) of 6.50+/-0.34 (77.6+/-10.9%); orexin A was ineffective. The present data suggest a lack of interaction between orexinergic and alpha2-adrenergic neurons in rat cerebral cortex.

Functional interactions between G protein-coupled receptors (GPCRs) are widely reported whereas the mechanisms underlying these interactions remain unclear. A rapidly growing literature suggests that many GPCRs form heteromeric complexes and that these complexes may possess pharmacological and functional properties that differ from their respective parent receptors. In order to investigate the functional relationship between co-localized GPCRs in intact, native CNS neurons, we used intracellular recording techniques in locus coeruleus (LC) slices. The LC consists of a homogenous population of cells, all of which co-express micro-opioid and alpha(2A)-adrenergic receptors (microOR and alpha(2A)ARs, respectively). Co-administration of microOR and alpha(2A)AR agonists in vivo results in synergistic interactions in analgesic assays and heteromeric microOR-alpha(2A)AR complexes have been detected in HEK-293 co-expressing the two receptors. The LC is therefore an excellent model system to explore these interactions. Dose-response and isobolographic analysis of agonist-induced hyperpolarization in individual LC neurons revealed that the interaction between these receptors was not synergistic but additive. This result suggests that co-localized microOR and alpha(2)ARs are either not physically associated in these neurons or that heteromer formation may not fully explain the microOR-alpha(2A)AR synergistic interactions reported following agonist administration in vivo.

Northern blot analyses performed on the lumbar spinal cords (L4-L6) of rats with the Bennett and Xie chronic constriction injury (CCI) nerve ligation model of neuropathic pain were compared to non-ligation surgery and unoperated control rats to determine if there is a change in alpha2-adrenergic receptor mRNA expression with neuropathic pain. Compared to unoperated rats, CCI rats had bilaterally lower RG-20 mRNA, corresponding to the alpha2A-adrenergic receptor subtype (affected 62.4 +/- 22.1% and contralateral 69.5 +/- 16.6% of unoperated, P

The present study examined the possible contribution of endogenous opioids to inhibition of spinal reflexes by an alpha(2)-adrenoceptor agonist. In rabbits decerebrated and spinalised under halothane/nitrous oxide anaesthesia, the selective alpha(2)-adrenoceptor agonist dexmedetomidine (3-30 microg intrathecal) induced significant decreases in short- and long-latency reflex responses evoked in medial gastrocnemius (MG) motoneurones by stimulation of the sural nerve. After recovery from dexmedetomidine, the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA; 100 microg intrathecal) significantly enhanced short-latency but not long-latency MG reflex responses. After beta-FNA, inhibition of all reflexes by dexmedetomidine was significantly weaker than in the control state, whereas the cardiovascular actions of dexmedetomidine were unaffected. These data confirm that activation of spinal alpha(2)-adrenoceptors depresses MG reflexes evoked by all groups of sural nerve afferent fibres, and shows that endogenous opioid tone supports the inhibitory action of alpha(2) agonists, possibly by a synergistic interaction in the spinal cord.

This study investigates the involvement of alpha(2)-adrenergic receptors (AR) in mouse brain induced by a low dose of methamphetamine (METH, 2 mg/kg). Immunohistochemical studies show that alpha(2A)-AR increased in the dentate gyrus area of the hippocampus 24 h after five repeated administrations of METH. The hippocampal alpha(2A)-AR proteins rose 3.2-fold when compared to the saline-administered mice. The other adrenergic receptor, alpha(1D)-AR, were not changed by the treatment. Moreover, alpha(o)-subunits of GTP-binding proteins (Galpha(o)), one of the downstream molecules of alpha(2A)-AR, was also increased by the treatment. These suggest that the repeated administration of low-doses of METH causes quantitative changes of the signaling of alpha(2A)-AR in the mouse hippocampus.

Similar in vitro experiments were performed on brainstem – spinal cord preparations from mouse and rat neonates to compare the noradrenergic regulations of the respiratory network. In preparations retaining the pons, rhythmic phrenic bursts occurred in rats but not in mice. Transection of the pons, electrolytic lesions and noradrenaline applications showed that the pontine noradrenergic A5 group inhibited the respiratory rhythm generator in both species but the inhibition was especially potent in mice. After pons elimination, noradrenaline applications to the medulla decreased the respiratory frequency in rats but increased it in mice. Noradrenergic agent applications revealed that the frequency changes implicated medullary α1 and α2 noradrenergic receptors in mice and rats, respectively. Thus, interspecies differences seem to exist in the noradrenergic regulations of the rat and mouse medullary respiratory networks.

The objective of this study was to determine if yohimbine, a central norepinephrine enhancing drug, increases cortico-motoneuronal excitability in intact humans. Transcranial magnetic stimulation was used to assess excitability of the motor system reflected in the parameters motor threshold, recruitment curve, intracortical inhibition and intracortical facilitation before and after oral administration of 20 and 40 mg yohimbine. Oral intake of 40 but not 20 mg yohimbine increased slope and plateau of the recruitment curve and intracortical facilitation. Motor threshold and intracortical inhibition remained unchanged. The data show that pharmacological enhancement of central norepinephrine in humans is effective to increase the cortico-motoneuronal excitability. Since cortical excitability is closely linked to neuroplasticity, this observation might be of possible relevance for strategies to enhance rehabilitative processes after cortical lesions by pairing noradrenergic drugs with motor training.

We investigated whether there are any differences between the Lewis and Sprague-Dawley (SD) rats in chronic inflammation-induced norepinephrine (NE) sensitivity of nociceptors. Activities of C-fiber nociceptors innervating rat hairy hindpaw skin were recorded in an in vitro skin-nerve preparation. Sixty-five percent of C-fibers from inflamed Lewis rats were excited by NE (10 microM), against only 38% of C-fibers from inflamed SD rats. The average of the total impulses evoked in response to NE was also significantly higher in Lewis rats. The alpha2-adrenoceptor antagonist CH 38083 (10 microM) and yohimbine (10 microM) consistently blocked the NE-excitation of both strains. These results show that after chronic inflammation, C-fiber nociceptors of Lewis strain rats have a stronger sensitivity to NE, and that alpha2-adrenoceptors are predominately involved in the NE-sensitivity of inflamed rats in both strains.

The adrenergic system plays an important role in psychiatric disorders such as depression and schizophrenia. Antagonism of the adrenergic receptor subtypes α 1A and α 2A has been found to have an antipsychotic effect. Genetic mutations in these receptors could be related to the alterations in the adrenergic system observed in psychiatric patients and to failure to respond to drug antagonism. We have studied one polymorphism in the α 1A –adrenergic receptor (Arg492Cys) and two polymorphisms in the promoter region of the α 2A –adrenergic receptor (-1291-C/G and -261-G/A) in a sample of clozapine-treated schizophrenic patients and controls. No clear differences were observed between the different groups suggesting that these polymorphisms did not play an important role in the aetiology of the disorder or in determining antipsychotic response.

An antibody against a sequence from the divergent third intracellular loop of the human alpha2A-adrenoceptor, amino acids 262-276, was produced. The antiserum was tested, along with the preimmune serum, by means of ELISA and dot blot assays which demonstrated that the alpha2A-peptide used for the antibody production was recognized by the immune serum. The antibody also recognized the alpha2A-adrenoceptor protein in the human brain (immunoblot analysis). In cortical membranes a major immunoreactive peptide of approximately 70 kDa (mature glycosylated receptor) was detected and after treatment with N-glycosidase F only a approximately 50 kDa peptide (non-glycosylated receptor) was immunodetected. This antibody was used to demonstrate that a chemical modification of the alpha2A-adrenoceptor induced by the alkylating agent EEDQ facilitates the protease-mediated receptor degradation. Thus in vitro, normal receptor degradation (24-44% at 2-4 h) was enhanced by EEDQ (10(-6) M) (38-71% at 2-4 h) but in the presence of protease inhibitors this effect was almost abolished.

Moclobemide is an antidepressant which affects the monoaminergic neurotransmitter system through a reversible inhibition of monoamine oxidase (MAO), preferentially type A. We examined the antinociceptive effects of moclobemide alone and in conjunction with specific opioid, adrenergic and serotonergic antagonists, using the mouse-tail flick test. Intraperitoneal moclobemide produced a dose-dependent antinociceptive effect with an ED50 of 69.1 mg/kg. Tests with selective antagonists yielded positive results only for yohimbine (P0.001), implying a noradrenergic mechanism of action in the moclobemide antinociceptive effect. This was confirmed by the coadministration of moclobemide with inactive doses of prototype agonists of the opioid, adrenergic and serotonergic systems. Only clonidine, an alpha2 agonist, significantly shifted (8-fold) the dose response curve of moclobemide. We conclude that there is a selective involvement of the alpha2 adrenergic pathways in the moclobemide-induced antinociceptive effect and a lesser involvement (if any) of the opioid, serotonergic and alpha1 adrenergic mechanisms. More research is needed to establish a possible role for moclobemide in pain management.

In order to delineate the regulatory mechanisms underlying the control of alpha 2 adrenoceptor expression, the sequence of 5' (1145 bp) and 3' (2682 bp) flanking regions of murine alpha 2C2 subtype gene were determined and characterized from a genomic phage clone MA2C2. The 5' flanking region has no TATA box yet with high GC content. The 3' flanking region is marked by the presence of a polyadenylation signal 2.3 kb down stream from the stop codon. The transcription start site was mapped by 5' rapid amplification of cDNA ends (RACE) and primer extension assays at nucleotide A, 415 bp upstream from the first initiation codon and resides in a motif resembling the consensus sequence of initiator found in many TATA-less promoters. An NcoI fragment (4.7 kb) immediately upstream from the translation initiation site was linked to a reporter gene lacZ. Using an in vitro transfection assay, cell lines of renal or neural origin were identified as permissive hosts for alpha 2C2 subtype expression. With its core promoter clearly defined and sequence of the regulatory regions at hand, this in vitro gene transfer system will facilitate the identification of putative cis-elements and transcription factors key to alpha 2C2 adrenoceptor expression.

One of the challenges of restorative neuronal transplantation in the CNS of mammals is the appropriate integration of grafted cells in the host circuitry. One key parameter is the specific influence of grafted cells upon corresponding receptors. In order to test this issue on the lesioned spinal cord of adult rats, two models of spinal cord denervation were used: the first one consisted of a complete transection 1 week prior to an intraspinal transplantation of embryonic locus coeruleus (LC) primordia cell suspension; the second one was a chemical destruction of the spinal noradrenergic (NA) system 1 month prior to a similar transplantation. Five weeks after transplantation, spinal sections were processed for autoradiographic quantification of α2-adrenoceptor binding sites densities. In most regions, α2-adrenoceptor densities remained comparable or higher than before graft; interestingly, in lumbar dorsal horn, lumbar intermediate zone and sacral distal dorsal horn of transected-grafted rats, they returned to control level. Results are discussed in relation to the parallel study performed concerning α1-adrenoceptors [C. Roudet et al., Brain Res., 677 (1995) 1–12.].