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Background: Chronic alcohol intake produces multiple neuroadaptive changes, including up- and down-regulation of neuropeptides and receptors. There are widespread projections of relaxin-3 containing neurons to, and abundant relaxin family peptide 3 receptor (RXFP3) expression within, brain regions involved in modulating alcohol intake. Recently we demonstrated the involvement of relaxin-3/RXFP3 signalling in alcohol-seeking in rats; therefore in this study we examined whether relaxin-3 and/or RXFP3 expression were altered by chronic alcohol intake in alcohol-preferring iP rats., Methods: Expression of relaxin-3 mRNA in the hindbrain nucleus incertus and RXFP3 radioligand binding levels in discrete forebrain regions were investigated following voluntary intake of alcohol or sucrose for 12 weeks, with a 2 day washout, using quantitative in situ hybridisation histochemistry and in vitro receptor autoradiography, respectively, in cohorts of adult, male iP rats., Results: Levels of relaxin-3 mRNA in the hindbrain nucleus incertus were positively correlated with the level of intake of both alcohol (r(12)=0.59, p=0.03) and sucrose (r(7)=0.70, p=0.04) in iP rats. Dense binding of the RXFP3-selective radioligand, [(125)]-R3/I5, was detected in hypothalamic and extrahypothalamic sites, but no significant changes in the density of RXFP3 were observed in the brain regions quantified following chronic sucrose or ethanol intake., Conclusions: Our findings suggest high endogenous relaxin-3 expression may be associated with higher intake of rewarding substances, rather than its expression being regulated in response to their intake, consistent with an active role for the relaxin-3/RXFP3 system in modulating ingestive and alcohol-related behaviours., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

A role for orexin A in mediating the primary and conditioned reinforcing effects of alcohol has been established. It is unclear however whether the contribution of orexins to alcohol reward occurs independently of effects on appetite and feeding, and whether orexins regulate the motivation to consume alcohol compared to other rewards. To examine this further here we investigate the effect of the orexin(1) receptor antagonist, SB-334867, on self-administration of alcohol (10% v/v) under both fixed (FR) and progressive ratio (PR) schedules of reinforcement, and whether this differs from the motivation to administer a natural food reward, sucrose (0.2-0.7% w/v) in alcohol preferring (iP) rats. SB-334867 treatment significantly reduced responding for both alcohol and sucrose under a FR3 schedule; however, at the same dose, reduced responding and break point for ethanol, but not sucrose, under a PR schedule. These findings for the first time implicate a role for orexins in the motivation to self-administer alcohol and suggest that this may occur independent of any generalized effect on appetitive drive., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In the present study, using rats, we have examined acute, contextual, and sensitized patterns of activated or phosphorylated cAMP response element-binding protein (pCREB) expression in parallel, assaying across multiple nuclei that have been implicated in addiction. The paradigm used included a comparison of pretreatment dose of amphetamine upon patterns of cellular activation, following rechallenge. Because efferent orexinergic projections synapse on many targets through the mammalian brain, including mesotelencephalic regions and limbic systems involved in drug reward and reinforcement, we examined for coexpression of pCREB or c-Fos double labeling within orexin A-immunopositive neurons following sensitization. Acute challenge with amphetamine (1.5 mg/kg i.p.) resulted in an increase in the number of pCREB-immunoreactive (-IR) cells within the substantia nigra but a decrease of pCREB-IR cells in the central and medial subnuclei of the amygdala. Contextual re-exposure to the drug treatment environment altered pCREB expression, particularly in the basal ganglia and hypothalamus, although these effects were dictated by pretreatment dose of amphetamine. Sensitization to amphetamine resulted in robust increases in pCREB-IR cell numbers in the basal ganglia and lateral septum of rats that had been pretreated with high-dose (10 mg/kg i.p.) but not low-dose (2 mg/kg i.p.) amphetamine, despite a similar behavioral response. Orexin A-containing cells in the hypothalamus of sensitized rats did not coexpress pCREB; however, these cells double-labeled for c-Fos and orexin A. These data suggest that orexinergic neurons are activated during the expression of behavioral sensitization, although in a heterogenous manner with regard to afferent topologies and functional roles in the nervous system.

Rationale: The development of mouse models of ethanol consumption and ethanol-seeking behavior is of particular importance in understanding the underlying mechanisms of drug abuse because these models can enable an analysis of an effect of specific genotype on drug-seeking behavior and the interaction of potential therapeutics with genotype. However, there are some limitations with present models, notably the inability to examine appetitive and consummatory behavior separately., Materials and Methods: In the present study, C57BL/6 mice were trained to self-administer 10% ethanol in a modified operant protocol that allowed a clear delineation of consummatory and appetitive phases. The utility of this procedure was confirmed with the use of the metabotropic glutamate 5 (mGlu5) receptor antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP)., Results: Limited-access consumption during the dark phase of the light-dark cycle with intermittent access (every second or third day) led to a high level of consumption by the mice. MTEP caused a dose-dependent decrease in both the consumption of ethanol and the appetitive response for ethanol. Furthermore, this effect was unrelated to any effect of MTEP on locomotor activity., Conclusions: The model provides a useful paradigm for examining both the appetitive and consummatory phases of ethanol consumption in mice; furthermore, the data indicate mGlu5 receptors are involved in both phases.

GABA systems have been implicated as targets for ethanol at the cellular, molecular and behavioural level. The present study was designed to further examine the potential of the GABA(B) receptor as a target for regulating operant alcohol responding. Given that the prototypic agonist, baclofen, reduces the self-administration of alcohol, we hypothesized that the GABA(B) receptor allosteric modulator, CGP7930, might have similar actions but a reduced side-effect profile. In this context, inbred alcohol-preferring (iP) rats were trained to respond for 10% v/v ethanol in a fixed ratio paradigm; all drug testing was performed under an FR3 schedule. Both baclofen and CGP7930 independently reduced voluntary responding for 10% ethanol in a dose-related manner. Neither drug impacted upon responding for water. A combination of subthreshold doses of baclofen and CGP7930 was also able to reduce operant responding for ethanol, suggesting that CGP7930 is indeed acting to facilitate GABA(B) receptor-mediated signalling in this paradigm. These data demonstrate the potential of positive allosteric modulators of metabotropic GABA(B) receptors to regulate alcohol responding.

The present study employed standard peroxidase immunohistochemistry to map the distribution of P2Y(1) receptors in the rat brainstem and nodose ganglia and characterised the binding profile of [alpha(33)P]dATP. Binding of [alpha(33)P]dATP was fully displaceable by adenosine 5'-triphosphate (ATP), and was found on both human and rat nodose ganglia, and throughout the rat brainstem, including the nucleus tractus solitarius and ventrolateral medulla. [Alpha(33)P]dATP binding in the human nodose ganglia was significantly displaced by both 2-methylthio ATP and alpha,beta-methylene ATP, but not by uridine 5'-triphosphate, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, 8,8'-(carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3,5-naphtalenetrisulfonic) acid (NF279) or N-ethylcarboxamidoadenosine. [Alpha(33)P]dATP binding in the rat nodose ganglia and brainstem was significantly displaced by only 2-methylthio ATP, suggesting that [alpha(33)P]dATP is binding to P2Y receptors in the rat. Binding of [alpha(33)P]dATP was also significantly displaced by alpha,beta-methylene adenosine 5'-diphosphate, suggesting a component of the binding is to endogenous ecto-5'-nucleotidase, however, almost all binding could be displaced by a combination of receptor agonists (2-methylthio ATP, uridine 5'-triphosphate and alpha,beta-methylene ATP), suggesting preferential binding to receptors. Immunoreactivity to P2Y(1) receptor (P2Y(1)-IR) exhibited similar distribution patterns to [alpha(33)P]dATP binding, with a clear topographic profile. Particularly dense P2Y(1)-IR labeling was evident in cells and fibres of the dorsal vagal complex. Immunolabeling was also present in the dorsal motor nucleus of the vagus and nucleus ambiguus, indicating the possibility of P2Y(1) receptors on vagal efferents. Unilateral vagal ligation was also performed to examine the transport of P2Y(1) receptor, using both immunohistochemistry and [alpha(33)P]dATP autoradiography. Accumulations of both P2Y(1)-IR and [alpha(33)P]dATP binding were apparent adjacent to both ligatures, suggesting bi-directional transport of P2Y(1) receptors along the rat vagus nerve. This current study represents the first description of P2Y(1) receptor distribution within the rodent brainstem and nodose ganglion and also characterises [alpha(33)P]dATP binding to P2Y receptors., (Copyright 2002 IBRO)

Hyperthermia during or after stroke is known to worsen neuronal damage. Paradoxically, when hyperthermia precedes stroke, it can protect against a subsequent ischemic insult. Other stressors including restraint also have a similar pre-conditioning effect. In the present study, we report the unanticipated finding that conscious rats, restrained for the purpose of intravenous infusion, had markedly reduced neuronal and functional deficits after middle cerebral artery occlusion compared with unrestrained rats. Restrained rats had significantly higher body temperature prior to stroke than unrestrained rats. The findings suggest restraint leading to mild hyperthermia may be sufficient to induce adaptive processes which protect against subsequent ischemia.

The present study has employed in vitro electrophysiology and radioligand binding assays to determine whether dopamine and adenosine receptors interact with each other on rat vagal afferent neurons. Preincubation of the isolated rat nodose ganglion with the adenosine A2a agonists CGS 21 680 or DPMA (Both 1 microM) resulted in a functional antagonism of the ability of dopamine to depolarise the preparation. Specifically, the concentration-response curve to dopamine was significantly shifted to the right in the presence of CGS 21 680 and DPMA. On the other hand, adenosine itself, A1 and A3 receptor agonists and ATP were all incapable of modulating the electrophysiological response to dopamine. In contrast to the nodose ganglion, CGS 21 680 did not significantly affect the ability of the dopamine D2 ligands quinpirole or raclopride to displace [125I]NCQ298 binding to dopamine D2 receptors in membranes prepared from rat dorsal brain stem. These data indicate the presence of an interaction between high affinity adenosine A2 receptors and dopamine D2 receptors on the soma of rat vagal afferent neurons, whereas the situation in the brain stem remains less clear.

With increased interest in the pharmacology of cholecystokininA (CCKA) receptors, including their trophic and mitogenic effects, the actions of two new non-peptide CCKA receptor antagonists, PD140548 and SR 27897B, were investigated in a convenient model system, the rat isolated nodose ganglion. CCK (1 nM-1 microM) caused concentration-dependent depolarisations when superfused over the nodose ganglion at 37 degrees C as measured by a silicone grease gap technique, and both CCKA antagonists caused significant rightward shifts in the concentration response curve to CCK. SR 27897B (3 and 10 nM) caused 7.9- and 17.9-fold shifts in the CCK concentration-response curve and the apparent-log KB values for each concentration of antagonist were calculated to be 9.36 and 9.23. Further experiments with PD140548 (30 and 100 nM) yielded shifts of 2.9- and 12.5-fold from which -log KB values were determined to be 7.80 and 8.06. Overall SR 27897B was significantly more efficacious than PD140548. Thus, the isolated nodose ganglion preparation allows a functional assessment of CCKA-mediated responses, with the results indicating that both SR 27897B and PD140548 are efficacious CCKA receptor antagonists.

1. In the present study in vitro electrophysiology and receptor autoradiography were used to determine whether rat vagal afferent neurones possess dopamine D2 receptors. 2. Dopamine (10-300 microM) elicited a temperature- and concentration-dependent depolarization of the rat isolated nodose ganglion preparation. When applied to the tissue 15 min prior to agonist, raclopride (10 microM), clozapine (10 microM) or a mixture of raclopride and clozapine (10 microM each) all produced a threefold parallel shift to the right of the dopamine concentration-response curve. In contrast, SCH 23390 (100 nM), phentolamine and propranolol (1 microM each) failed to antagonize the dopamine-mediated depolarization. 3. [125I]-NCQ 298 (0.5 nM), a D2 selective radioligand, bound topographically to sections of rat brainstem. Densitometric quantification of autoradiograms revealed 93.8 +/- 0.5% specific binding of this salicylamide radioligand, as determined by raclopride (10 microM, n = 10 animals). Binding was highest in the nucleus tractus solitarius (NTS), particularly the medial and gelatinous subnuclei. In addition, specific binding was also observed in the interpolar spinal trigeminal nucleus and the inferior olive. 4. Unilateral nodose ganglionectomy caused a 36.6 +/- 3.0% reduction in specific binding in the denervated NTS compared to the contralateral NTS. Furthermore, the loss of binding was confined to the dorsal aspect of the medial subnucleus of the NTS. Sham surgery had no effect on the binding of [125I]-NCQ 298 in rat brainstem. 5. The present data provide evidence for the presence of functionally relevant dopamine D2 receptors on both the soma and central terminals of rat vagal afferent neurones. In addition, the majority of D2 receptors in the rat NTS appear to be located postsynaptically with respect to vagal terminals, and are presumably located either on ascending glossopharyngeal terminals, descending terminals from higher brain regions or on neuronal cell bodies within the NTS.

The sulphated octapeptide, cholecystokinin (CCK-8S), is believed to be a neurotransmitter of vagal sensory neurones, and here the presence of functional receptors for CCK-8S in the rat vagus nerve has been investigated by electrophysiological and autoradiographic techniques. CCK-8S caused concentration-dependent depolarizations when superfused over the rat isolated nodose ganglion at 37 degrees C as measured by a silicone grease gap technique. Concentration-response curves to CCK-8S were shifted to the right by low concentrations of the CCKA receptor antagonist, Devazepide, but not by the CCKB receptor antagonist, L-365,260, data which indicate that receptors were of the CCKA subtype. Consistent with this notion, the CCKB agonist, unsulphated CCK-8, was without effect until high concentrations (> 1 microM) were used. A synthetic analogue of CCK-8S, D-Tyr25(Nle28,31)-CCK 25-33S, which has been reported to be more stable and peptidase-resistant than CCK-8S, was equipotent with CCK-8S in depolarizing the nodose ganglion. When D-Tyr25(Nle28,31)-CCK 25-33S was labelled with 125I, it bound to tissue sections of nodose ganglion. By light microscopic autoradiography, silver grains were found to be highly localized over cell bodies of vagal sensory neurones. An excess of CCK-8S inhibited binding as did Devazepide, but not L-365,260, confirming that binding sites were CCKA subtype receptors. These results indicate the existence of functional CCKA receptors in the nodose ganglion and strengthen the case for the involvement of vagal sensory neurones in gastric emptying and satiety.

Previous autoradiographic studies have identified angiotensin II (AII) binding sites over the nodose ganglion and along the vagal afferent neurons. In the present study, we examined whether these binding sites are functional receptors by measuring d.c. potential changes by extracellular recording techniques in the rat isolated nodose ganglion preparation in response to superfusion of angiotensin peptides. It was found that AII, as well as AI and AIII elicited concentration-dependent depolarisation of the nodose ganglion. However, the amino terminal angiotensin heptapeptide, A(1-7), failed to evoke any significant response. The AII receptor antagonist, saralasin had no intrinsic activity, but caused a concentration-dependent blockade of AII-induced depolarisation. This study provides evidence for direct neuronal effects of angiotensin peptides on rat vagal afferent neurons. Moreover, this preparation is a relatively convenient one in which to study functional neuronal AII receptor mechanisms on central or peripheral terminals of vagal sensory neurons.

The ability of angiotensin II (A II) and 5-hydroxytryptamine (5-HT) to depolarise the rat isolated nodose ganglion preparation was examined. 5-HT depolarised the nodose ganglion, both at room temperature (20-24 degrees C) and at 35-37 degrees C. However, A II depolarised the nodose ganglion only under the latter condition, and these responses were blocked by the A II receptor antagonist saralasin. This study extends previous findings which have demonstrated A II binding sites on the nodose ganglion and axon, and identifies the rat nodose ganglion as a sensitive preparation in which to study the interactions between neuronal A II receptor activation and its blockade by A II receptor antagonists.

Noradrenaline-induced contractions of the rabbit and rat isolated aorta and guinea-pig spleen strips were inhibited by concentrations of phenoxybenzamine which did not affect responses to calcium. This may suggest a specific action on alpha-adrenoceptors. However, analysis of noradrenaline concentration-effect curves in guinea-pig spleen indicated that 1 mumol/l phenoxybenzamine should have reduced the available receptor population to 6% of control, but data from radioligand binding experiments on the same tissues using [3H]-prazosin indicated a reduction of the receptor population to only 82% of control. The reduced responsiveness observed in the organ bath study after phenoxybenzamine pretreatment, whilst not apparently related to effects on voltage-dependent calcium channels, could be due to the actions of phenoxybenzamine on other (non-receptor) processes such as receptor-operated calcium channels. Maximal contractile responses to histamine in rabbit isolated aorta but not those in guinea-pig isolated ileal preparations, were depressed by concentrations of phenoxybenzamine which depressed responses to calcium. Phenoxybenzamine produced parallel rightward shifts of curves to carbachol in guinea-pig ileal preparations but only depressed maximal responses to the agonist in higher concentrations which reduced responses to calcium. On the basis of the results obtained with calcium it is possible that the effects of phenoxybenzamine on receptor-mediated responses could be produced through the actions of this antagonist at less specific sites such as voltage-dependent calcium channels for histamine in rabbit aorta and carbachol in guinea-pig ileum. For alpha-receptor mediated responses in aortic and splenic strip preparations and for histamine-mediated responses in guinea-pig ileum, the actions of phenoxybenzamine may reflect an interaction of the antagonist with receptor-operated calcium channels.

In the present study, investigations were performed to determine the subtype(s) of beta-receptor-mediating relaxation in K+-depolarized guinea pig uterine preparations, and inhibition of twitches in field-stimulated rat vas deferens preparations. The relative activities of (-)-isoprenaline, salbutamol, terbutaline, noradrenaline, and RO363, and the affinity constants determined for the beta 1-receptor antagonist atenolol, and the beta 2-receptor antagonist lCl 118551, indicated that all the inhibitory responses in both preparations were mediated solely through beta 2-adrenoceptor stimulation. In the uterine preparations there was a small population of alpha-receptors which were of little consequence when assessing beta 2-receptor-mediated activity. During K+-induced contractures, both neuronal and extraneuronal uptake systems were inactivated. In rat vas deferens preparations, it was necessary to pretreat the tissues with phenoxybenzamine in order to prevent alpha-receptor actions and neuronal and extraneuronal uptake systems interfering with beta-receptor-mediated activity. In both tissues, 6-8 highly reproducible concentration-effect curves to (-)-isoprenaline could be established. However, the uterine preparation responded more robustly to repeated washings, required less sensitive recording apparatus, and showed responses to individual concentrations of drugs that were more readily quantified. However, in general, both preparations appear to be suitable as tissues for assessing the beta 2-adrenoceptor actions of drugs.

Ro 03-7894 (0.6 mM) produced a non-parallel shift to the right of dose-response curves to (-)-isoprenaline in K+ depolarized uterine preparations from the guinea-pig. The displacement of the curves was readily reversed by washing. A rightward shift of similar magnitude was also produced by Ro 03-7894 in transmurally stimulated ileal preparations. The relaxant effects of fenoterol in carbachol-contracted guinea-pig tracheal preparations (in the presence of 2 microM atenolol) were not altered by 0.6 mM Ro 03-7894. In the three tissues there was no evidence of a reduction in the maximal inhibitory response to the agonists. In uterine and tracheal preparations, Ro 03-7894 (0.6 mM) depressed contractile responses to exogenous calcium. The depression of responses was enhanced after washout of Ro 03-7894 for 80 min. Contractile responses of ileal preparations to transmural stimulation were also depressed by Ro 03-7894. Concentration-effect curves for the positive inotropic effects of (-)-isoprenaline in guinea-pig left atrial preparations were markedly shifted to the right and the maximum response depressed by 0.6 mM Ro 03-7894. Although the rightward shift of the curves was fully reversed during the 120 min washout period, the maximal responses remained depressed. In similar experiments, Ro 03-7894 produced a washout-resistant depression of inotropic responses to histamine and calcium. The results of radioligand binding studies in left atria using (-)-[125I]-iodocyanopindolol indicated that, when compared to the untreated atria, there was no reduction in the maximal density of binding sites 120 min after washout of 0.6 mM Ro 03-7894. 5 On the basis of the present results it is concluded that Ro 03-7894 induces a non-specific depressant effect on smooth and cardiac muscle preparations during exposure to the drug. This depressant effect persists following washout of the drug. There is no evidence for an irreversible effect of Ro 03-7894 at beta-adrenoceptor sites.