6-Nitrodopamine potentiates contractions of rat isolated vas deferens induced by noradrenaline, adrenaline, dopamine and electric field stimulation.

6-Nitrodopamine (6-ND) is a novel endogenous catecholamine that is released from the rat isolated vas deferens, and has been characterized as a major modulator of the contractility of rat isolated epididymal vas deferens (RIEVD). Drugs such as tricyclic antidepressants, α1 and ß1ß2 adrenoceptor blockers, act as selective antagonists of the 6-ND receptor in the RIEVD. In the rat isolated atria, 6-ND has a potent positive chronotropic action and causes remarkable potentiation of the positive chronotropic effects induced by dopamine, noradrenaline, and adrenaline. Here, whether 6-ND interacts with the classical catecholamines in the rat isolated vas deferens was investigated. Incubation with 6-ND (0.1 and 1 nM; 30min) caused no contractions in the RIEVD but provoked significant leftward shifts in the concentration-response curves to noradrenaline, adrenaline, and dopamine. Pre-incubation of the RIEVD with 6-ND (1 nM), potentiated the contractions induced by electric-field stimulation (EFS), whereas pre-incubation with 1 nM of dopamine, noradrenaline or adrenaline, did not affect EFS-induced contractions. In tetrodotoxin (1 µM) pre-treated (30 min) RIEVD, pre-incubation with 6-ND (0.1 nM) did not cause leftward shifts in the concentration-dependent contractions induced by noradrenaline, adrenaline, or dopamine. Pre-incubation of the RIEVD with the α2A-adrenoceptor antagonist idazoxan (30 min, 10 nM) did not affect dopamine, noradrenaline, adrenaline, and EFS-induced contractions. However, when idazoxan (10 nM) and 6-ND (0.1 nM) were simultaneously pre-incubated (30 min), a significant potentiation of the EFS-induced contractions of the RIEVD was observed. 6-nitrodopamine causes remarkable potentiation of dopamine, noradrenaline, and adrenaline contractions on the RIEVD, due to activation of adrenergic terminals, possibly via pre-synaptic adrenoceptors.

Alpha-2A but not 2B/C noradrenergic receptors in ventral tegmental area regulate phasic dopamine release in nucleus accumbens core.

Adrenergic receptors (AR) in the ventral tegmental area (VTA) modulate local neuronal activity and, as a consequence, dopamine (DA) release in the mesolimbic forebrain. Such modulation has functional significance: intra-VTA blockade of α1-AR attenuates behavioral responses to salient environmental stimuli in rat models of drug seeking and conditioned fear as well as phasic DA release in the nucleus accumbens (NAc). In contrast, α2-AR in the VTA has been suggested to act primarily as autoreceptors, limiting local noradrenergic input. The regulation of noradrenaline efflux by α2-AR could be of clinical interest, as α2-AR agonists are proposed as promising pharmacological tools in the treatment of PTSD and substance use disorder. Thus, the aim of our study was to determine the subtype-specificity of α2-ARs in the VTA capable of modulating phasic DA release. We used fast scan cyclic voltammetry (FSCV) in anaesthetized male rats to measure DA release in the NAc after combined electrical stimulation and infusion of selected α2-AR antagonists into the VTA. Intra-VTA microinfusion of idazoxan - a non-subtype-specific α2-AR antagonist, as well as BRL-44408 - a selective α2A-AR antagonist, attenuated electrically-evoked DA in the NAc. In contrast, local administration of JP-1302 or imiloxan (α2B- and α2C-AR antagonists, respectively) had no effect. The effect of BRL-44408 on DA release was attenuated by intra-VTA DA D2 antagonist (raclopride) pre-administration. Finally, we confirmed the presence of α2A-AR protein in the VTA using western blotting. In conclusion, these data specify α2A-, but not α2B- or α2C-AR as the receptor subtype controlling NA release in the VTA.

Effects of imidazoline agents in a rat conditioned place preference model of addiction.

Agmatine (AG), idazoxan (IDZ), and efaroxan (EFR) are imidazoline receptor ligands with beneficial effects in central nervous system disorders. The present study aimed to evaluate the interaction between AG, IDZ, and EFR with an opiate, tramadol (TR), in a conditioned place preference (CPP) paradigm. In the experiment, we used five groups with 8 adult male Wistar rats each. During the condition session, on days 2, 4, 6, and 8, the rats received the drugs (saline, or TR, or IDZ and TR, or EFR and TR, or AG and TR) and were placed in their least preferred compartment. On days 1, 3, 5, and 7, the rats received saline in the preferred compartment. In the preconditioning, the preferred compartment was determined. In the postconditioning, the preference for one of the compartments was reevaluated. TR increased the time spent in the non-preferred compartment. AG decreased time spent in the TR-paired compartment. EFR, more than IDZ, reduced the time spent in the TR-paired compartment, but without statistical significance. AG reversed the TR-induced CPP, while EFR and IDZ only decreased the time spent in the TR-paired compartment, without statistical significance.

Idazoxan and Efaroxan Potentiate the Endurance Performances and the Antioxidant Activity of Ephedrine in Rats.

Background and objectives: The connections between the imidazoline system and multiple other neurotransmitter systems in the brain (adrenergic, dopaminergic, serotoninergic, glutamatergic, opioid) indicate the complexity of the mechanisms underlying motor activity and behavior. The aim of the present research was to investigate the effects of the combination of ephedrine (EPD) and imidazoline antagonists idazoxan (IDZ) and efaroxan (EFR) on the endurance performance in the treadmill test in rats. Materials and Methods: We used Wistar rats distributed as follows: Group 1 (Control) receiving distilled water 0.3 mL/100 g body weight; Group 2 (EPD) receiving 20 mg/kg ephedrine; Group 3 (EPD + IDZ) receiving 20 mg/kg ephedrine + 3 mg/kg idazoxan; Group 4 (EPD + EFR) receiving 20 mg/kg ephedrine + 1 mg/kg efaroxan. An additional group (C) of animals receiving 0.3 mL/100 g body weight distilled water (but not subjected) to effort was used. Endurance capacity was evaluated using a treadmill running PanLAB assay. The evaluation of the substances' influence on oxidative stress was performed by spectrophotometric determination of superoxide dismutase (SOD) and glutathione peroxidase (GPX) activity. Results: Treatment with EPD-IDZ and EPD-EFR were correlated with a longer distance traveled on the belt and with a decrease in the necessary electric shocks to motivate the animal to continue running in the forced locomotion test. Additionally, an increase in the activity of antioxidant enzymes was found. Conclusions: Idazoxan and efaroxan potentiated the physical effort-related effects of ephedrine with regard to endurance capacity and antioxidant activity in rats.

Hippocampal monoamine changes in the Flinders sensitive line rat: A case for the possible use of selective α2C-AR-antagonists in stress and anxiety disorders in companion animals.

Non-selective α2-adrenoreceptor (AR) stimulation delivers favourable sedative, analgesic, muscle relaxant and anxiolytic actions in companion animals, but is associated with cardiovascular and respiratory side effects. Anxiety conditions underscore monoamine disturbances amenable to α2-AR modulation. We investigated sub-chronic (14 day s.c.) treatment with the selective α2C-AR antagonist, ORM-10921 (0.03, 0.1, 0.3 mg/kg/d) on hippocampal noradrenaline (NA), dopamine (DA), serotonin (5-HT) and their turnover levels in stress sensitive Flinders Sensitive Line (FSL) rats versus Flinders Resistant Line (FRL) controls, using high performance liquid chromatography. The effects of ORM-10921 were compared to the non-selective α2-AR antagonist, idazoxan (IDAZ; 3 mg/kg/d), and to imipramine (IMI; 15 mg/kg/d), a reference antidepressant in this model. FSL rats displayed significantly reduced 5-HT (p = 0.03) and DA (p = 0.02) levels vs. FRL controls, while NA levels showed a similar trend. ORM-10921 significantly increased NA (all doses p ≤ 0.02), 5-HT (0.1 and 0.3 mg/kg p ≤ 0.03) and DA levels (all doses p ≤ 0.03), which correlated with decreased monoamine turnover. In contrast, IDAZ significantly elevated NA (p < 0.005) and DA (p < 0.004) but not 5-HT levels. IMI also significantly increased 5-HT (p < 0.009), with a tendency to increase NA (p = 0.09) but not DA. ORM-10921 exerts similar albeit broader effects on hippocampal monoamines than IDAZ, explaining earlier established efficacy associated with α2C-AR antagonism in animal models of depression and cognitive dysfunction. These and the current studies encourage application of ORM-10921 in depression in humans, as well as raise the intriguing possibility that selective α2C-AR antagonists may be beneficial in anxiety and stress-related disorders in companion animals. Both warrant further study.

Deciphering Imidazoline Off-targets by Fishing in the Class A of GPCR field.

Based on the finding that a central antihypertensive agent with high affinity for I1-type imidazoline receptors - rilmenidine, shows cytotoxic effects on cultured cancer cell lines, it has been suggested that imidazoline receptors agonists might have a therapeutic potential in the cancer therapy. Nevertheless, potential rilmenidine side effects caused by activation of α-adrenoceptors, or other associated receptors and enzymes, might hinder its therapeutic benefits. Considering that human α-adrenoceptors belong to the rhodopsin-like class A of G-protein-coupled receptors (GPCRs) it is reasonable to assume that imidazolines might have the affinity for other receptors from the same class. Therefore, to investigate possible off-target effects of imidazoline ligands we have prepared a reverse docking protocol on class A GPCRs, using imidazoline ligands and their decoys. To verify our in silico results, three ligands with high scores and three ligands with low scores were tested for antagonistic activity on α2 - adrenoceptors.

Activation and blockade of α2-adrenoceptors in the prelimbic cortex regulate anxiety-like behaviors in hemiparkinsonian rats.

At present, whether α2-adrenoceptors in the prelimbic cortex (PrL) are involved in Parkinson's disease-related anxiety is unclear. We examined the effects of PrL α2-adrenoceptors on anxiety-like behaviors in rats with unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. Compared to the sham operation, the lesion induced anxiety-like responses as measured by the open field test and elevated plus-maze test. Intra-PrL injection of the α2-adrenoceptor agonist clonidine (1.25, 2.5 or 5 µg/rat) produced anxiolytic effects in sham-operated and lesioned rats. Furthermore, intra-PrL injection of the α2-adrenoceptor antagonist idazoxan (1, 2 or 4 µg/rat) induced anxiogenic effects in two groups of rats. The effective doses produced by clonidine and idazoxan in lesioned rats were higher than those in sham-operated rats. Neurochemical results showed that intra-PrL injection of clonidine (5 µg/rat) or idazoxan (4 µg/rat) decreased or increased dopamine (DA) and noradrenaline (NA) and serotonin (5-HT) levels in the medial prefrontal cortex (mPFC) and amygdala in sham-operated and lesioned rats, respectively. These results suggest that α2-adrenoceptors in the PrL are involved in the regulation of anxiety-like behaviors, which is attributable to changes in DA, NA and 5-HT levels in the mPFC and amygdala after activation and blockade of α2-adrenoceptors.

Analgesic Effect of Melittin on Oxaliplatin-Induced Peripheral Neuropathy in Rats.

Oxaliplatin is a chemotherapeutic agent used for metastatic colon and other advanced cancers. Most common side effect of oxaliplatin is peripheral neuropathy, manifested in mechanical and cold allodynia. Although the analgesic effect of bee venom has been proven to be effective against oxaliplatin-induced peripheral neuropathy, the effect of its major component; melittin has not been studied yet. Thus, in this study, we investigated whether melittin has an analgesic effect on oxaliplatin-induced allodynia. Intraperitoneal single injection of oxaliplatin (6 mg/kg) induced mechanical and cold allodynia, resulting in increased withdrawal behavior in response to von Frey filaments and acetone drop on hind paw. Subcutaneous melittin injection on acupoint ST36 (0.5 mg/kg) alleviated oxaliplatin-induced mechanical and cold allodynia. In electrophysiological study, using spinal in vivo extracellular recording, it was shown that oxaliplatin-induced hyperexcitation of spinal wide dynamic range neurons in response to peripheral stimuli, and melittin administration inhibited this neuronal activity. In behavioral assessment, analgesic effect of melittin was blocked by intrathecal α1- and α2- adrenergic receptor antagonists administration. Based on these results, we suggest that melittin could be used as an analgesic on oxaliplatin-induced peripheral neuropathy, and that its effect is mediated by activating the spinal α1- and α2-adrenergic receptors.

Agmatine Inhibits Behavioral Sensitization to Ethanol Through Imidazoline Receptors.

BACKGROUND: Locomotor sensitization to repeated ethanol (EtOH) administration is proposed to play a role in early and recurring steps of addiction. The present study was designed to examine the effect of agmatine on EtOH-induced locomotor sensitization in mice. METHODS: Mice received daily single intraperitoneal injection of EtOH (2.5 g/kg, 20 v/v) for 7 consecutive days. Following a 3-day EtOH-free phase, the mice were challenged with EtOH on day 11 with a single injection of EtOH. Agmatine (10 to 40 µg/mouse), endogenous agmatine enhancers (l-arginine [80 µg/mouse], arcaine [50 µg/mouse], aminoguanidine [25 µg/mouse]), and imidazoline receptor agonist/antagonists were injected (intracerebroventricular [i.c.v.]) either daily before the injection of EtOH during the 7-day development phase or on days 8, 9, and 10 (EtOH-free phase). The horizontal locomotor activity was determined on days 1, 3, 5, 7, and 11. RESULTS: Agmatine (20 to 40 µg/mouse) administration for 7 days (development phase) significantly attenuated the locomotor sensitization response of EtOH challenge on day 11. Further, the agmatine administered only during EtOH-free period (days 8, 9, and 10) also inhibited the enhanced locomotor activity on the 11th day to EtOH challenge as compared to control mice indicating blockade of expression of sensitization. Daily treatment (i.c.v.) with endogenous agmatine enhancers like l-arginine (80 µg/mouse) or arcaine (50 µg/mouse) and aminoguanidine (25 µg/mouse) restrained the development as well as expression of sensitization to EtOH. Imidazoline I1 receptor agonist, moxonidine, and I2 agonist, 2-BFI, not only decreased the development and expression of locomotor sensitization but also potentiated the effect of agmatine when employed in combination. Importantly, I1 receptor antagonist, efaroxan, and I2 antagonist, idazoxan, blocked the effect of agmatine, revealing the involvement of imidazoline receptors in agmatine-mediated inhibition of EtOH sensitization. CONCLUSIONS: Inhibition of EtOH sensitization by agmatine is mediated through imidazoline receptors and project agmatine and imidazoline agents in the pharmacotherapy of alcohol addiction.

Microinjection of 26RFa, an endogenous ligand for the glutamine RF-amide peptide receptor (QRFP receptor), into the rostral ventromedial medulla (RVM), locus coelureus (LC), and periaqueductal grey (PAG) produces an analgesic effect in rats.

26RFa is an endogenous ligand for the QRFP receptor. We previously found that intracerebroventricular injection of 26RFa produces an analgesic effect in a rat formalin test. In the present study, we directly tested the hypothesis that the analgesic effects of 26RFa in the formalin test are mediated in well-recognized regions of the descending inhibitory pain pathways, such as the rostral ventromedial medulla (RVM), locus coeruleus (LC), and periaqueductal grey (PAG) in rats. Injection cannulae were stereotaxically placed in the RVM, LC, or PAG through a burr hole. 26RFa (15 µg) or saline was delivered in a total volume of 0.5 µL. In a formalin test, 50 µL of 5% formalin was injected subcutaneously into the hind paw. In an antagonist study, idazoxan, an α-2 antagonist, or naloxone, an opioid receptor antagonist, was administered. Microinjection of 26RFa into the RVM had no effect compared with that in saline-injected rats. Microinjection of 26RFa into the LC contralateral, but not ipsilateral, to the formalin injection site significantly decreased the number of flinching behaviors compared with that of saline-injected rats. This effect was antagonized by intrathecal injection of idazoxan. Microinjection of 26RFa into the contralateral, but not ipsilateral, PAG produced an analgesic effect, and this effect was partly antagonized by intraperitoneal naloxone. These data suggest that 26RFa microinjected into the contralateral LC induced noradrenaline release in the spinal cord and produced an analgesic effect. In the contralateral PAG, 26RFa activated the opioid system, and some analgesic effects were mediated by opioid system activation.

Responsiveness of α2-adrenoceptor/I1-imidazoline receptor in the rostral ventrolateral medulla to cardiovascular regulation is enhanced in conscious spontaneously hypertensive rat.

Stimulation of α2-adrenoceptor/I1-imidazoline receptors in the rostral ventrolateral medulla decreases the blood pressure via sympathoinhibition. However, alteration of receptor responses in genetically hypertensive rats remains unclear. We examined cardiovascular responses of α2-adrenoceptor/I1-imidazoline receptor agonist and antagonists microinjected into the rostral ventrolateral medulla of conscious spontaneously hypertensive rats and normotensive Wistar Kyoto rats. Injection of 2-nmol clonidine-an α2-adrenoceptor/I1-imidazoline receptor agonist-unilaterally into the rostral ventrolateral medulla decreased the blood pressure, heart rate, and renal sympathetic nerve activity; the responses were significantly enhanced in spontaneously hypertensive rats than in Wistar Kyoto rats. Co-injection of 2-nmol 2-methoxyidazoxan (a selective α2-adrenoceptor antagonist) or 2-nmol efaroxan (an I1-receptor antagonist) with 2 nmol of clonidine attenuated the hypotensive and bradycardic effects of clonidine-only injection. Injection of 2-methoxyidazoxan alone increased the blood pressure and heart rate in spontaneously hypertensive rats, but not in Wistar Kyoto rats. These results suggest enhanced responsiveness of α2-adrenoceptor/I1-imidazoline receptors in the rostral ventrolateral medulla of spontaneously hypertensive rats.

Stress augments the rewarding memory of cocaine via the activation of brainstem-reward circuitry.

Effects of stress on the reward system are well established in the literature. Although previous studies have revealed that stress can reinstate extinguished addictive behaviors related to cocaine, the effects of stress on the rewarding memory of cocaine are not fully understood. Here, we provide evidence that stress potentiates the expression of rewarding memory of cocaine via the activation of brainstem-reward circuitry using a cocaine-induced conditioned place preference (CPP) paradigm combined with restraint stress in rats. The rats exposed to 30-minute restraint stress immediately before posttest exhibited significantly larger CPP scores compared with non-stressed rats. Intra-laterodorsal tegmental nucleus (LDT) microinjection of a ß or α2 adrenoceptor antagonist attenuated the stress-induced enhancement of cocaine CPP. Consistent with this observation, intra-LDT microinjection of a ß or α2 adrenoceptor agonist before posttest increased cocaine CPP. Additionally, intra-ventral tegmental area (VTA) microinjection of antagonists for the muscarinic acetylcholine, nicotinic acetylcholine or glutamate receptors attenuated the stress-induced enhancement of cocaine CPP. Finally, intra-medial prefrontal cortex (mPFC) microinjection of a D1 receptor antagonist also reduced the stress-induced enhancement of cocaine CPP. These findings suggest a mechanism wherein the LDT is activated by noradrenergic input from the locus coeruleus, leading to the activation of VTA dopamine neurons via both cholinergic and glutamatergic transmission and the subsequent excitation of the mPFC to enhance the memory of cocaine-induced reward value.

Effects of imidazoline I2 receptor agonists on reserpine-induced hyperalgesia and depressive-like behavior in rats.

Pharmacotherapies for fibromyalgia treatment are lacking. This study examined the antinociceptive and antidepressant-like effects of imidazoline I2 receptor (I2R) agonists in a reserpine-induced model of fibromyalgia in rats. Rats were treated for 3 days with vehicle or reserpine. The von Frey filament test was used to assess the antinociceptive effects of I2 receptor agonists, and the forced swim test was used to assess the antidepressant-like effects of these drugs. 2-BFI (3.2-10 mg/kg, intraperitoneally), phenyzoline (17.8-56 mg/kg, intraperitoneally), and CR4056 (3.2-10 mg/kg, intraperitoneally) all dose-dependently produced significant antinociceptive effects, which were attenuated by the I2R antagonist idazoxan. Only CR4056 significantly reduced the immobility time in the forced swim test in both vehicle-treated and reserpine-treated rats. These data suggest that I2R agonists may be useful to treat fibromyalgia-related pain and comorbid depression.

Noradrenergic modulation determines respiratory network activity during temperature changes in the in vitro brainstem of bullfrogs.

Among vertebrate ectotherms, air breathing frequency is generally constrained across warmer temperatures, but decreases during cooling. The brainstem mechanisms that give rise to this ventilatory strategy are unclear. Neuromodulation has recently been shown to stabilize motor circuit output across temperatures. Therefore, we tested the hypothesis that an important neuromodulatory system in respiratory control network, norepinephrine, produces this pattern of respiratory motor activity across temperatures. To this end, we used in vitro brainstem-spinal cord preparations from adult bullfrogs, Lithobates catesbeianus, to assess the role of noradrenergic signaling in shaping the frequency response of the respiratory network during temperature changes. We identified that noradrenergic signaling through the α1 adrenergic receptor constrains motor output from the respiratory network across warm temperatures. In contrast, the α2 adrenergic receptor actively inhibits respiratory motor output during cooling. These results indicate that noradrenergic tuning, rather than passive thermal responses, produces temperature responses of the respiratory circuits.

Agmatine modulates spontaneous activity in neurons of the rat medial habenular complex-a relevant mechanism in the pathophysiology and treatment of depression?

The dorsal diencephalic conduction system connects limbic forebrain structures to monaminergic mesencephalic nuclei via a distinct relay station, the habenular complexes. Both habenular nuclei, the lateral as well as the medial nucleus, are considered to play a prominent role in mental disorders like major depression. Herein, we investigate the effect of the polyamine agmatine on the electrical activity of neurons within the medial habenula in rat. We present evidence that agmatine strongly decreases spontaneous action potential firing of medial habenular neurons by activating I1-type imidazoline receptors. Additionally, we compare the expression patterns of agmatinase, an enzyme capable of inactivating agmatine, in rat and human habenula. In the medial habenula of both species, agmatinase is similarly distributed and observed in neurons and, in particular, in distinct neuropil areas. The putative relevance of these findings in the context of depression is discussed. It is concluded that increased activity of the agmatinergic system in the medial habenula may strengthen midbrain dopaminergic activity. Consequently, the habenular-interpeduncular axis may be dysregulated in patients with major depression.

Blockade of α2-adrenergic or metabotropic glutamate receptors induces glutamate release in the locus coeruleus to activate descending inhibition in rats with chronic neuropathic hypersensitivity.

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.

Imidazolines increase the levels of the autophagosomal marker LC3-II in macrophage-like RAW264.7 cells.

This study evaluated whether imidazolines can induce autophagy in the murine macrophage-like cell line RAW264.7. Idazoxan increased the content of LC3-II, an autophagosomal marker, in RAW264.7 cells. To determine whether this effect was due to the induction of its synthesis or inhibition of its degradation, idazoxan treatment was performed in the presence of bafilomycin A1, which blocks autophagosome-lysosome fusion, as well as Pepstatin A and E-64d, both of which block protein degradation in autolysosomes. An increased content of LC3-II was observed in the presence of bafilomycin A1 as well as the protease inhibitors. Furthermore, an increased number of autophagosomes was observed following idazoxan treatment using an autophagosome-specific dye. This indicated that idazoxan induced autophagy. Other imidazolines, such as efaroxan, clonidine, and 2-(2-benzofuranyl)-2-imidazoline, also increased the LC3-II content in RAW264.7 cells in the presence of bafilomycin A1. Taken together, these results indicate that some imidazolines, including idazoxan, can induce autophagy in RAW264.7 cells.

On the central noradrenergic mechanism underlying the social play-suppressant effect of methylphenidate in rats.

Social play behaviour is a vigorous, highly rewarding social activity abundant in the young of most mammalian species, including humans. Social play is thought to be important for social, emotional and cognitive development, yet its neural underpinnings are incompletely understood. We have previously shown that low doses of methylphenidate suppress social play behaviour through a noradrenergic mechanism of action, and that methylphenidate exerts its effect within the prefrontal cortex, amygdala and habenula. In the present study, we sought to reveal whether these regions work in parallel or in series to mediate the play-suppressant effect of methylphenidate. To that aim, we tested whether infusion of the α2-adrenoceptor antagonist RX821002 into the anterior cingulate cortex, infralimbic cortex, basolateral amygdala or habenula prevents the effect of methylphenidate on social play behaviour, or the psychomotor stimulant effect of methylphenidate. We found that the social play-suppressant effect of methylphenidate was not prevented by infusion of the α2-adrenoceptor antagonist into either region, or by infusion of RX821002 into both the anterior cingulate and infralimbic cortex. By contrast, RX821002 infusion into the anterior cingulate modestly enhanced social play, and infusion of the antagonist into the infralimbic cortex attenuated the psychomotor stimulant effect of methylphenidate. We conclude that there is redundancy in the neural circuitry that mediates the play-suppressant effect of methylphenidate, whereby prefrontal cortical and subcortical limbic mechanisms act in parallel. Moreover, our data support the notion that prefrontal noradrenergic mechanisms contribute to the locomotor enhancing effect of psychostimulant drugs.

α2A- and α2C-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands.

The poor norepinephrine innervation and high density of Gi/o-coupled α2A- and α2C-adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D2-like receptor ligands, such as the D3 receptor agonist 7-OH-PIPAT and the D4 receptor agonist RO-105824, to α2-adrenoceptors in cortical and striatal tissue, which express α2A-adrenoceptors and both α2A- and α2C-adrenoceptors, respectively. The affinity of dopamine for α2-adrenoceptors was found to be similar to that for D1-like and D2-like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α2A- and α2C-adrenoceptors. Their ability to activate Gi/o proteins through α2A- and α2C-adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α2-adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity, and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α2A- and α2C-adrenoceptors was nearly identical to its binding to the crystallized D3 receptor. Therefore, we provide conclusive evidence that α2A- and α2C-adrenoceptors are functional receptors for norepinephrine, dopamine, and other previously assumed selective D2-like receptor ligands, which calls for revisiting previous studies with those ligands.

Opioid and noradrenergic contributions of tapentadol to the inhibition of locus coeruleus neurons in the streptozotocin rat model of polyneuropathic pain.

Tapentadol is an analgesic that acts as an agonist of µ opioid receptors (MOR) and that inhibits noradrenaline reuptake. Data from healthy rats show that tapentadol inhibits neuronal activity in the locus coeruleus (LC), a nucleus regulated by both the noradrenergic and opioid systems. Thus, we set out to investigate the effect of tapentadol on LC activity in streptozotocin (STZ)-induced diabetic rats, a model of diabetic polyneuropathy, by analyzing single-unit extracellular recordings of LC neurons. Four weeks after inducing diabetes, tapentadol dose-response curves were obtained from animals pre-treated with RX821002 or naloxone (alpha2-adrenoceptors and opioid receptors antagonists, respectively). In STZ rats, the spontaneous activity of LC neurons (0.9 ±â€¯0.1 Hz) was lower than in naïve animals (1.5 ±â€¯0.1 Hz), and tapentadol's inhibitory effect was also weaker. Alpha2-adrenoceptors blockade by RX821002 (100 µg/kg i.v.) in STZ animals significantly increased the spontaneous activity (from 0.8 ±â€¯0.1 to 1.4 ±â€¯0.2 Hz) and it dampened the inhibition of LC neurons produced by tapentadol. However, opioid receptors blockade following naloxone pre-treatment (5 mg/kg i.v.) did not alter the spontaneous firing rate (0.9 ±â€¯0.2 vs 0.9 ±â€¯0.2 Hz) or the inhibitory effect of tapentadol on LC neurons in STZ animals. Thus, diabetic polyneuropathy appears to exert neuroplastic changes in LC neurotransmission, enhancing the sensitivity of alpha2-adrenoceptors and dampening opioid receptors expression. Tapentadol's activity seems to be predominantly mediated through its noradrenergic effects rather than its influence on opioid receptors in the STZ model of diabetic polyneuropathy.