Sodium channel blocking activity of AM-36 and sipatrigine (BW619C89): in vitro and in vivo evidence.

Sodium channel blockers are neuroprotective against cerebral ischemia in animal models. A novel neuroprotective compound AM-36, when screened for activity at the most common receptor and ion channel binding sites, revealed activity at site 2 Na+ channels. Studies then investigated this Na+ channel blocking activity in vitro and in vivo relative to other Na+ channel blockers, including the neuroprotective agent sipatrigine (BW619C89). AM-36 inhibited batrachotoxinin (BTX)-sensitive Na+ channel binding in rat brain homogenates with an IC50 of 0.28 microM. Veratridine (100 microM)-induced neurotoxicity in murine cerebellar granule cells was completely inhibited by AM-36 (1.7 microM) compared to only partial inhibition by sipatrigine (26 microM). Veratridine-stimulated glutamate release, as measured through a microdialysis probe in the cortex of anesthetised rats, was inhibited by 90% by superfusion of AM-36 (1000 microM). In the endothelin-1 (ET-1) model of middle cerebral artery occlusion (MCAo) in conscious rats, both AM-36 (6 mg/kg i.p.) and sipatrigine (10 mg/kg i.p.) 30 min post-MCAo significantly reduced cortical, but not striatal infarct volume. As the refractiveness of the striatum is likely to be dependent on the route and time of drug administration, AM-36 (1 mg/kg i.v.) was administered 3 or 5 h after MCAo and significantly reduced both cortical and striatal infarct volumes. The present studies demonstrate Na+ channel blocking activity of AM-36 both in vitro and in vivo, together with significant neuroprotection when administration is delayed up to 5 h following experimental stroke.

Immunoreactive localisation of P2Y1 receptors within the rat and human nodose ganglia and rat brainstem: comparison with [alpha 33P]deoxyadenosine 5'-triphosphate autoradiography.

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.

Functional dopamine D2 receptors on rat vagal afferent neurones.

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 highly selective CRF(2) receptor antagonist K41498 binds to presynaptic CRF(2) receptors in rat brain.

1. Novel analogues of antisauvagine-30 (aSvg-30), a selective antagonist for CRF(2) receptors, have been synthesized and characterized in vitro and in vivo. 2. The analogues were tested for their ability to compete for [(125)I-Tyr(0)]Svg binding and to inhibit Svg-stimulated adenylate cyclase activity in human embryonic kidney (HEK) 293 cells, permanently transfected with cDNA coding for the human CRF(1) (hCRF(1)), hCRF(2alpha) and hCRF(2beta) receptor. One analogue [D-Phe(11), His(12), Nle(17)]Svg(11-40), named K41498, showed high affinity binding to hCRF(2alpha) (K(i)=0.66+/-0.03 nM) and hCRF(2beta) (K(i)=0.62+/-0.01 nM) but not the hCRF(1) receptor (k(i)=425+50 nM) and decreased Svg-stimulated cAMP accumulation in hCRF(2) expressing cells. In conscious Wistar-Kyoto rats, K41498 (1.84 microg, i.v.) antagonized the hypotensive response to systemic urocortin (1.4 microg, i.v.), but did not block the pressor response to centrally administered urocortin (2.35 microg, i.c.v.). 3. K41498 was subsequently radio-iodinated, and in autoradiographic studies, specific (sensitive to rat urocortin, astressin and aSvg30, but insensitive to antalarmin) binding of (125)I-K41498 (100 pM) was detected in the heart and in selected brain regions including the nucleus tractus solitarius (NTS), spinal trigeminal nucleus, lateral septum and around the anterior and middle cerebral arteries. 4. Following unilateral nodose ganglionectomy, binding of (125)I-K41498 was reduced by 65% in the ipsilateral NTS, indicative of presynaptic CRF(2) receptors on vagal afferent terminals. 5. These data demonstrate that K41498 is a useful tool to study native CRF(2) receptors in the brain and periphery.

Functional GABAA receptors on rat vagal afferent neurones.

1. In the present study, in vitro electrophysiology and receptor autoradiography were used to determine whether rat vagal afferent neurones possess gamma-aminobutyric acid (GABA)A receptors. 2. GABA (1-100 microM) and isoguvacine (3-100 microM) caused a concentration-dependent depolarization of the rat isolated nodose ganglion preparation at room temperature. When applied to the tissue 20 min before the agonist, SR95531 (3 microM) and bicuculline (3 microM) caused a parallel shift to the right of the GABA and isoguvacine concentration-response curves, yielding shifts of 81 fold and 117 fold for SR95531 and 4 fold and 12 fold for bicuculline, respectively. 3. Baclofen (10 nM-100 microM) was unable to elicit a depolarization of the rat isolated nodose ganglion preparation at either room temperature or at 36 degrees C, whilst 5-aminovaleric acid (10 microM), a GABAB receptor antagonist, was unable to antagonize significantly the GABA-induced depolarization at either room temperature or at 36 degrees C. 4. [3H]-SR95531 (7.2 nM), a GABAA receptor-selective antagonist, bound topographically to sections of rat brainstem. Specific binding was highest in the medial nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus nerve (DMVN). Binding was also observed in certain medullary reticular nuclei, in particular the parvocellular reticular nucleus. 5. Unilateral nodose ganglionectomy caused a reduction in GABAA binding site density in the medial NTS from 93 +/- 7 to 68 +/- 6 d.p.m./mm2. This procedure also caused a reduction in GABAA binding site density in the side of the NTS contralateral to the lesion, from 151 +/- 12 to 93 +/- 7 d.p.m./mm2. Sham surgery had no effect on the binding of [3H]-SR95531 in rat brainstem. 6. The present data provide evidence for the presence of GABAA receptors located on the soma and central terminals of rat vagal afferent neurones. Additionally, a population of GABAA receptors is evidenced postsynaptically in the rat NTS with respect to vagal afferent terminals. These data are discussed in relation to the functional pharmacology of GABA in this region of the NTS.

Is Ro 03-7894 an irreversible antagonist at beta-adrenoceptor sites?

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.

Neuroprotective effects of mild hyperthermia prior to focal ischemia in conscious rats.

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.

Autoradiographic visualisation of axonal transport of adenosine A1 receptors along the rat vagus nerve and characterisation of adenosine A1 receptor binding in the dorsal vagal complex of hypertensive and normotensive rats.

The present study had employed in vitro receptor autoradiography with [3H]DPCPX to visualise the presence of adenosine A1 receptors on the rat nodose ganglion, which contains the perikarya of vagal afferent neurons projecting the the nucleus tractus solitarius (NTS). In addition, unilateral vagal ligation resulted in an accumulation of [3H]DPCPX binding adjacent to the ligatures, indication that adenosine A1 receptors are subject to axoplasmic flow along the rat vagus nerve. Radioligand binding assays were utilised to characterise the properties of adenosine A1 receptors in the dorsal vagal complex (NTS, area postrema and dorsal motor nucleus of the vagus) of pup and adult normotensive (Wistar Kyoto, WKY) and hypertensive (spontaneously hypertensive, SHR) rats. Saturation binding indicated that the affinity (KD) of [3H]DPCPX, and the binding site density (Bmax) were not different between the adult WKY and SHR, although the pup SHR had a lower KD value than the pup WKY rat. Competition binding assays revealed complex differences between the two rat strains; however, with respect to hypertension, the affinity of the selective adenosine A1 agonist, cyclohexyladenosine (CHA), was markedly reduced in the membranes from SHR (Ki approximately 93 nM) compared to WKY (approximately 6 nM). Such an observation is consistent with the attenuated responses of SHRs to intra-NTS injections of adenosine.

Presynaptic adenosine A2a receptors on soma and central terminals of rat vagal afferent neurons.

The dorsal vagal complex of the medulla oblongata is a key centre involved in the regulation of numerous autonomic functions, including cardiovascular control. Adenosine has been implicated as a potential neuromodulator of the baroreceptor reflex, and therefore the current study has investigated the presence and characteristics of adenosine receptors on rat vagal afferent neurons. In the nodose-vagal grease gap preparation, the adenosine A2a agonist CGS-21680 evoked a depolarisation only in the presence of the selective adenosine A1 antagonist PACPX. Autoradiography using [3H]NECA (4 nM) with suppression of A1 binding enabled the first visualisation of high affinity adenosine A2 receptors in the nucleus tractus solitarius (NTS). Unilateral nodose ganglionectomy resulted in over 90% reduction in binding in the lesioned (ipsilateral) NTS compared to a sham control. Furthermore, local administration of CGS-21680 increased evoked glutamate release in the NTS, as measured by in vivo microdialysis. These data suggest the presence of presynaptic adenosine A2a receptors on both the soma and central terminals of rat vagal afferent neurons, and thereby support the hypothesis that adenosine may have a modulatory role in the baroreceptor reflex.

Complex interactions between nitric oxide and adenosine receptors in the rat isolated nodose ganglion.

The present study has employed in vitro electrophysiology, utilising the isolated rat nodose ganglion preparation, to determine whether nitric oxide (NO) and adenosine interact with each other in vagal afferent neurons. The nucleophile NO donor, diethylamine-NO, caused reproducible, concentration-related depolarisations of the isolated rat nodose ganglia. Pre-incubation of the isolated rat nodose ganglia with the adenosine A2A receptor agonists CGS 21680 (2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride) and DPMA (N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine) (both 1 microM) resulted in a functional antagonism of the ability of diethylamine-NO to depolarise the preparation. A similar effect was observed with adenosine (10 microM) only in the presence of the adenosine A1 receptor antagonist PACPX (1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine, 100 nM). Conversely, the adenosine A1 receptor agonists ENBA (N6-[2-endo-norbomyl]adenosine, 1 microM) and cyclohexyladenosine (100 nM) potentiated the effect of diethylamine-NO on isolated rat nodose ganglia. Inclusion of either adenosine A3 agonists or ATP had no effect on the diethylamine-NO concentration-response curve. These data suggest an ability of NO to interact, in opposing manner, with adenosine A2A and A1 receptors in rat vagal afferent neurons. On the other hand, neither A3 receptors nor ATP appear capable of interacting with NO.

Actions of nitric oxide and expression of the mRNA encoding nitric oxide synthase in rat vagal afferent neurons.

The present study has investigated whether nitric oxide (NO) is involved in neurotransmission of rat vagal afferent neurons. The diethylamine-NO complex (diethylamine-NO, 10-100 microM) and S-nitroso-N-acetylpenicillamine (3-100 microM) both elicited a concentration-dependent depolarisation of the isolated rat nodose ganglion preparation. Pre-treatment with 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 300 nM), 6-(phenylamino)-5,8-quinolinedione (LY83,583, 30 microM) and Methylene blue (100 microM) all caused a significant shift to the right in the concentration-response curve to diethylamine-NO. Incubation of rat nodose ganglion sections with a 35S-labeled antisense oligonucleotide to neuronal NO synthase resulted in visualisation of the mRNA encoding NO synthase over vagal afferent perikarya. The anatomical findings, therefore, suggest that a number of rat vagal afferent perikarya possess the ability to produce the enzyme required for the biosynthesis of NO. Collectively, these data suggest that NO may be functionally important as a neuromodulator of rat vagal afferent neurons.

Temperature dependence of angiotensin II-mediated depolarisation of the rat isolated nodose ganglion.

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.

Bradykinin B2 receptors in nodose ganglia of rat and human.

The present study has employed in vitro electrophysiology to characterise the ability of bradykinin to depolarise the rat isolated nodose ganglion preparation, containing the perikarya of vagal afferent neurons. Both bradykinin and kallidin elicited a concentration-dependent (1-100 nM) depolarisation when applied to the superfusate bathing the nodose ganglia, whereas the bradykinin B1 receptor agonist, des-Arg9-bradykinin, was only effective in the micromolar range. Furthermore, the electrophysiological response to bradykinin was antagonised by the bradykinin B2 receptor antagonist, D-arginyl-L-arginyl-L-prolyl-trans-4-hydroxy-L-prolylglycyl-3-(2-t hienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl+ ++-L-(2alpha,3beta,7abeta)-octahydro-1H-indole-2-carbonyl-L- arginine (Hoe 140), in a concentration-related manner. To determine the anatomical location of functional bradykinin B2 receptors, in vitro autoradiography with [125I]para-iodophenyl Hoe 140 was performed on sections of rat and human inferior vagal (nodose) ganglia and confirmed the presence of binding over vagal perikarya. Collectively, these data provide evidence for functionally relevant bradykinin B2 receptors on vagal afferent neurons, which are apparently also present on human vagal perikarya.

Adenosine-dopamine receptor interactions in the isolated rat nodose ganglion but not in membranes of dorsal vagal complex.

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.

Electrophysiological studies of the cholecystokininA receptor antagonists SR27897B and PD140548 in the rat isolated nodose ganglion.

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.

Relaxin-3 mRNA levels in nucleus incertus correlate with alcohol and sucrose intake in rats.

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.

Discrete cue-conditioned alcohol-seeking after protracted abstinence: pattern of neural activation and involvement of orexin1 receptors.

BACKGROUND AND PURPOSE: The enduring propensity for alcoholics to relapse even following years of abstinence presents a major hurdle for treatment. Here we report a model of relapse following protracted abstinence and investigate the pattern of neuronal activation following cue-induced reinstatement and administration of the orexin1 receptor antagonist SB-334867 in inbred alcohol-preferring rats. EXPERIMENTAL APPROACH: Rats were trained to self-administer alcohol under operant conditions and divided into two groups: immediate (reinstated immediately following extinction) and delayed (extinguished and then housed for 5 months before reinstatement). Prior to reinstatement, animals were treated with vehicle (immediate n= 11, delayed n= 11) or SB-334867 (20 mg·kg⁻¹ i.p.; immediate n= 6, delayed n= 11). Fos expression was compared between each group and to animals that underwent extinction only. KEY RESULTS: SB-334867 significantly attenuated cue-induced reinstatement in both groups. Immediate reinstatement increased Fos expression in the nucleus accumbens (NAc), infra-limbic (IL), pre-limbic (PrL), orbitofrontal (OFC) and piriform cortices, the lateral and dorsomedial hypothalamus, central amygdala and basolateral amygdala (BLA), and the bed nucleus of the stria terminalis. Following delayed reinstatement, Fos expression was further elevated in cortical structures. Concurrent with preventing reinstatement, SB-334867 decreased Fos in NAc core, PrL and OFC following immediate reinstatement. Following protracted abstinence, SB-334867 treatment decreased reinstatement-induced Fos in the PrL, OFC and piriform cortices. CONCLUSIONS AND IMPLICATIONS: Cue-induced alcohol seeking can be triggered following protracted abstinence in rats. The effects of SB-334867 on both behaviour and Fos expression suggest that the orexin system is implicated in cue-induced reinstatement, although some loci may shift following protracted abstinence.

Chronic corticotropin-releasing factor type 1 receptor antagonism with antalarmin regulates the dopaminergic system of Fawn-Hooded rats.

Corticotropin-releasing factor is a neuropeptide associated with the integration of physiological and behavioural responses to stress and also in the modulation of affective state and drug reward. The selective, centrally acting corticotropin-releasing factor type 1 receptor antagonist, antalarmin, is a potent anxiolytic and reduces volitional ethanol consumption in Fawn-Hooded rats. The efficacy of antalarmin to reduce ethanol consumption increased with time, suggestive of adaptation to reinforcement processes and goal-directed behaviour. The aim of the present study was to examine the effects of chronic antalarmin treatment on reward-related regions of Fawn-Hooded rat brain. Bi-daily antalarmin treatment (20 mg/kg, i.p.) for 10 days increased tyrosine hydroxylase messenger RNA expression throughout the ventral mesencephalon. Following chronic antalarmin the density of dopaminergic terminals within the basal ganglia and amygdaloid complex were reduced, as was dopamine transporter binding within the striatum. Receptor autoradiography indicated an up-regulation of dopamine D2, but no change in D1, binding in striatum, and Golgi-Cox analysis of striatal medium spiny neurones indicated that chronic antalarmin treatment increased spine density. Thus, chronic antalarmin treatment modulates dopaminergic pathways and implies that chronic treatment with drugs of this class may ultimately alter postsynaptic signaling mechanisms within the basal ganglia.

Comparison of guinea pig uterine and rat vas deferens preparations for assessment of beta 2-adrenoceptor-mediated activity.

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.