Adenosine A2A receptor agonists increase Fos-like immunoreactivity in mesolimbic areas

Expression of the early-gene c-fos is an useful method for studying potential sites of action of drugs active in the CNS. Stimulation of adenosine A_2A receptors by CGS 21680 (5 mg/kg) induced an increase in Fos-like immunoreactivity in the rat nucleus accumbens shell, while in the rostral pole and core CGS 21680 induced Fos-like immunoreactivity only after a high dose. CGS 21680 (5 mg/kg) stimulated c-fos expression also in the lateral septal nucleus and dorso-medial striatum, but not in the dorso-lateral striatum. A similar pattern of Fos-like immunoreactivity was obtained after administration of the A_2A agonist HENECA (5 mg/kg) which displays higher selectivity for A_2A receptors than CGS 21680. Administration of the selective A_2A antagonist SCH 58261 counteracted CGS 21680-induced Fos-like immunoreactivity. Lesions of the dopaminergic mesostriatal projection by 6-hydroxydopamine and stimulation of dopamine D₂/D₃ receptors by quinpirole, prevented CGS 21680-induced Fos-like immunoreactivity in the nucleus accumbens shell. The present results show that stimulation of A_2A receptors induces a profile of c-fos expression similar to that of atypical neuroleptics. A_2A receptor stimulation has been reported to have dopamine antagonistic actions, it is therefore suggested that A_2A agonists might have antipsychotic activity without producing extrapyramidal side effects.     

Neuroprotective effect of L-DOPA co-administered with the adenosine A2A receptor agonist CGS 21680 in an animal model of Parkinson's disease

Adenosine A_2A receptors are a new target for drug development in Parkinson’s disease. Some experimental and clinical data suggest that A_2A receptor antagonists can provide symptomatic improvement by potentiating the effects of -DOPA as well as a decrease in secondary effects such as -DOPA-induced dyskinesia. -DOPA-induced behavioral sensitization in unilateral 6-hydroxydopamine-lesioned rats is frequently used as an experimental model of -DOPA-induced dyskinesia. In the present work this model was used to evaluate the effect of the A_2A receptor agonist CGS 21680 and the A_2A receptor antagonist MSX-3 on -DOPA-induced behavioral sensitization and 6-hydroxydopamine-induced striatal dopamine denervation. -DOPA-induced behavioral sensitization was determined as an increase in -DOPA-induced abnormal involuntary movements and enhancement of apomorphine-induced turning behavior. Striatal dopamine innervation was determined by measuring tyrosine-hydroxylase immunoreactivity. Chronic administration of MSX-3 was not found to be effective at counteracting -DOPA-induced behavioral sensitization. On the other hand, CGS 21680 completely avoided the development of -DOPA-induced behavioral sensitization. The analysis of the striatal dopamine innervation showed that -DOPA-CGS 21680 co-treatment conferred neuroprotection to the toxic effects of 6-hydroxydopamine. This neuroprotective effect was dependent on A_2A and D₂ receptor stimulation, since it was counteracted by MSX-3 and by the D₂ receptor antagonist haloperidol. These results open new therapeutic avenues in early events in Parkinson’s disease.     

Hyperexcitability in CA1 of the rat hippocampal slice following hypoxia or adenosine

Participation of adenosine receptors in the depression of synaptic transmission during hypoxia, and the production of multiple populations spikes in the pyramidal neurons following hypoxia, has been investigated in the CA₁ area of the rat hippocampal slice. A method is presented for analysing such hyperexcitability, using input/output curves of the second population spike. This method provides evidence that rebound hyperexcitability following hypoxia or prolonged adenosine-mediated inhibition results from an increase in excitability of the CA₁ pyramidal neurons rather than from an increase in excitatory neurotransmitter release. Hypoxia-induced depression of the synaptic components of evoked field potentials was blocked in a concentration dependent manner by the selective A₁ receptor antagonist 8-cyclopenthyltheophylline (8-CPT), demonstrating extracellular accumulation of adenosine during hypoxia. Upon reoxygenation of slices following 30 min hypoxia, multiple population spikes were evoked by a single orthodromic stimulus in slices that exhibited only a single population spike prior to hypoxia. Such post-hypoxic hyperexcitability was not prevented by superfusion of slices with 8-CPT during hypoxia. Depression of synaptic transmission by 30 min superfusion of slices with 50 μM adenosine was also followed, upon washout, by the appearance of multiple population spikes. However, such hyperexcitability could not be produced by superfusion with adenosine analogues selective for A₁ receptors, cyclopentyladenosine, selective for A_2a receptors, 2-p-(2-carboxyethyl)phenetheylamino-5′-ethylcarboxamidoadenosine (CGS 21680), or active at A_2a and A_2b receptors,N⁶-[2-(3,5-dimethyoxyphenyl)-2-(2-methyl-phenyl)ethyl]adenosine, suggesting that adenosine receptors other than the A₁, A_2a or A_2b subtypes are involved in its generation.     

A single (−)-nicotine injection causes change with a time delay in the affinity of striatal D2 receptors for antagonist, but not for agonist, nor in the D2 receptor mRNA levels in the rat substantia nigra

The in vitro and in vivo effects of (−)-nicotine on dopamine D₂ receptors in the rat neostriatum have been studied using biochemical binding, in situ hybridization and immunocytochemistry. A single i.p. injection (1 mg/kg) of (−)-nicotine resulted in a reduction of theK_D value of the D₂ antagonist [³H]raclopride binding sites in rat neostriatal membrane preparations at 12 h without any significant change in theB_max value. This action of (−)-nicotine was counteracted by pretreatment 15 min earlier with the nicotine antagonist mecamylamine (1 mg/kg, i.p.). However, theK_D and theB_max values of the D₂ agonist [³H]NPA binding sites in the rat neostriatal membrane preparations were not significantly affected 0.5–48 h after a single i.p. injection with 1 mg/kg of (−)-nicotine. No significant change in neostriatal D₂ receptor mRNA levels was observed at any time interval after the (−)-nicotine injection. No significant change was observed in tyrosine hydroxylase (TH) immunoreactivity in either the substantia nigra or the neostriatum, nor in nigral TH mRNA levels during the time interval studied (4–24 h posttreatment). Furthermore, addition of low (10 nM) or high (1 μM) concentrations of (−)-nicotine in vitro to rat neostriatal membranes did not alter the characteristics of [³H]raclopride or [³H]NPA binding. These results indicate that a single (−)-nicotine injection can produce a selective and delayed increase in the affinity of D₂ receptors for the antagonist, but not for the agonist without modifying the levels of D₂ receptor mRNA, probably via the activation of central nicotinic receptors.     

5-HT2 receptor regulation of acetylcholine release induced by dopaminergic stimulation in rat striatal slices

The role of 5-hydroxytryptamine (5-HT) receptor subtypes in acetylcholine (ACh) release induced by dopamine or neurokinin receptor stimulation was studied in rat striatal slices. The dopamine D₁ receptor agonist SKF 38393 potentiated in a tetrodotoxin-sensitive manner the K⁺-evoked [³H]ACh release while SCH 23390, a dopamine D₁ receptor antagonist, had no effect. [³H]ACh release was decreased by the dopamine D₂ receptor agonist LY 171555 (quinpirole) and slightly potentiated by the dopamine D₂ receptor antagonist haloperidol. The selective neurokinin NK₁ receptor agonist [Sar⁹, met(O₂)¹¹]SP also potentiated K⁺-evoked release of [³H]ACh. GR 82334, a NK₁ receptor antagonist, blocked not only the effect of [Sar⁹, met(O₂)¹¹]SP but also the release of ACh induced by the D₁ receptor agonist SKF 38393. Among the 5-HT agents studied, only the 5-HT_2A receptor antagonists ketanserin and ritanserin were able to reduce the ACh release induced by dopamine D₁ receptor stimulation. Mesulergine, a more selective 5-HT_2C antagonist, showed an intrinsic releasing effect but did not affect K⁺-evoked ACh release induced by SKF 38393. Methysergide and methiothepin, mixed 5-HT_1/2 antagonists, as well as ondansetron, a 5-HT₃ receptor antagonist, showed an intrinsic effect on ACh release, their effects being additive to that of SKF 38393. 5-HT₂ receptor agonists were ineffective. However, the 5-HT₂ agonist DOI was able to prevent the antagonism by ketanserin of the increased [³H]ACh efflux elicited by SKF 38393, suggesting a permissive role of 5-HT_2A receptors. None of the above indicated 5-HT agents was able to reduce the ACh release induced by the selective NK₁ agonist. The results suggest that 5-HT₂ receptors, probably of the 5-HT_2A subtype, modulate the release of ACh observed in slices from the rat striatum after stimulation of dopamine D₁ receptors. It seems that this serotonergic control is exerted on the interposed collaterals of substance P-containing neurons which promote ACh efflux through activation of NK₁ receptors located on cholinergic interneurons.     

Agonists of A1 and A2A adenosine receptors attenuate methamphetamine-induced overflow of dopamine in rat striatum

The effect of adenosine receptor agonists on the release of striatal dopamine (DA), induced by repeated doses of methamphetamine (MTH), was evaluated. Rats received three injections of MTH (5 mg/kg i.p.) at 2-h intervals. The release of DA in the striatum was measured by a microdialysis in freely moving animals. The agonist of adenosine A₁ receptor, N⁶-cyclopentyladenosine (CPA) and the agonist of adenosine A_2A receptor, 2-[p-(carboxy-ethyl)phenylethylamino]-5′-N-ethylcarboxyamidoadenosine (CGS 21680), either of them being infused locally into the striatum at concentrations of 50 and 100 μM, produced decreases in the extracellular DA level during exposure to MTH, and a weaker effect on the levels of DOPAC and HVA. The above effects were reversed by the specific antagonists of adenosine A₁ and A_2A receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 3,7-dimethyl-1-propargylxanthine (DMPX), respectively. Our results indicate that both the A₁ and A_2A adenosine receptors appear to be involved in reducing the excessive release of DA in the striatum; furthermore, they suggest a neuroprotective role of adenosine in MTH neurotoxicity.     

Identification of extrastriatal dopamine D2 receptors in post mortem human brain with [I]epidepride

The regional distribution of striatal and extrastriatal dopamine D₂ receptors in human brain was studied in vitro with(S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-[¹²⁵I]iodo-2,3-dimethoxybenzamide, [¹²⁵I]epidepride, using post mortem brain specimens from six subjects. Scatchard analysis of the saturation equilibrium binding in twenty-three regions of post mortem brain revealed highest levels of binding in the caudate (16.5 pmol/g tissue) and putamen (16.6 pmol/g tissue) with lower levels seen in the globus pallidus (7.0 pmol/g tissue), nucleus accumbens (7.2 pmol/g tissue), hypothalamus (1.8 pmol/g tissue), pituitary (1.3 pmol/g tissue), substantia innominata (1.0 pmol/g tissue), and amygdala (0.87 pmol/g tissue). Of note was the presence of dopamine D₂ receptors in the four thalamic nuclei studied, i.e. anterior nucleus (1.0 pmol/g tissue), dorsomedial nucleus (0.96 pmol/g tissue), ventral nuclei (0.72 pmol/g tissue), and pulvinar (0.86 pmol/g tissue), at levels comparable to the amygdala (0.87 pmol/g tissue) and considerably higher than levels seen in anterior cingulate (0.26 pmol/g tissue) or anterior hippocampus (0.36 pmol/g tissue). The frontal cortex had very low levels of dopamine D₂ receptors (0.17–0.20 pmol/g tissue) while the inferior and medial temporal cortex had relatively higher levels (0.31–0.46 pmol/g tissue). Inhibition of [¹²⁵I]epidepride binding by a variety of neurotransmitter ligands to striatal, ventral thalamic and inferior temporal cortical homogenates demonstrated that [¹²⁵I]epidepride binding was potently inhibited only by dopamine D₂ ligands. The present study demonstrates that dopamine D₂ receptors are present in basal ganglia, many limbic regions, cortex and in the thalamus. The density of thalamic D₂ receptors is comparable to many limbic regions and is considerably higher than in cortex. Very few frontal lobe D₂ receptors are present in man.     

Synergistic interaction between an adenosine antagonist and a D1 dopamine agonist on rotational behavior and striatal c-Fos induction in 6-hydroxydopamine-lesioned rats

The interaction between adenosine and D₁ dopamine systems in regulating motor behavior and striatal c-Fos expression was examined in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. These results were compared to the synergistic interaction between D₁ and D₂ dopamine systems in 6-OHDA rats. Coadministration of the adenosine antagonist 3,7-dimethyl-1-propargylxanthine (DMPX: 10 mg/kg) and the D₁ dopamine agonist SKF38393 (0.5 mg/kg) to 6-OHDA-lesioned rats produced significant contralateral rotation and c-Fos expression in the ipsilateral striatum compared to 6-OHDA rats treated with either drug alone. However, the regional pattern of striatal c-Fos activation following treatment of 6-OHDA rats with SKF38393 and DMPX was different from the dorsolateral pattern of striatal c-Fos induction observed after coadministration of D₁ and D₂ dopamine agonists (SKF38393: 0.5 mg/kg+quinpirole: 0.05 mg/kg). These data are consistent with a functional interaction between D₁ dopamine and adenosine systems in the striatum, but suggest that activation of different subsets of striatal neurons underlie the behavioral synergy observed following combined adenosine antagonist-D₁ dopamine agonist and combined D₁ dopamine agonist–D₂ dopamine agonist treatment.     

The role of dopamine in the maintenance and breakdown of D1/D2 synergism

The neurochemical factors involved in the maintenance and breakdown of dopamine D₁/D₂ receptor synergism were investigated by giving rats various pharmacological treatments that diminish the ability of dopamine to interact with its D₁ and/or D₂ receptors. Following these treatments, rats were observed for the expression of stereotyped motor behavior in response to independent stimulation of D₁ or D₂ receptors. Independent D₂-mediated responses were observed: (a) 2 h after the last of three daily reserpine (1 mg/kg) injections, (b) 48 h after bilateral 6-hydroxydopamine (6-OHDA) lesions of the mesostriatal pathways, (c) 24 h after a concentrated 48-h regimen (one injection/6 h) of eticlopride (0.5 mg/kg) or eticlopride + SCH 23390 (0.5 mg each), and (d) 2 h after a concentrated 48-h regimen (one injection/6 h) of α-methyl-p-tyrosine (αMPT; 100 mg/kg), but not after control treatments or a concentrated regimen of SCH 23390 alone. By contrast, independent D₁-mediated responses were observed only after three daily reserpine injections or 48 h after bilateral 6-OHDA lesions. Independent D₁-mediated stereotypy was not observed under control conditions or following a concentrated 48-h regimen of (a) SCH 23390 or eticlopride (0.5 mg/kg each) alone or in combination, (b) a high dose of SCH 23390 (1.0 mg/kg), (c) αMPT (100 mg/kg), or (d) αMPT (100 mg/kg)+SCH 23390 (1.0 mg/kg). Reserpine, bilateral 6-OHDA, and αMPT treatments produced striatal dopamine depletions of 96%, 92%, and 71%, respectively. These data indicate that the breakdown in D₁/D₂ synergism consists of two components: (a) D₁ independence from the controlling influence of D₂ receptors, and (b) D₂ independence from the controlling influence of D₁ receptors. The interaction of synaptic DA with its D₂ receptors plays a major role in determining whether these receptors can function independently of D₁ receptors, whereas reduced DA-D₁ activity alone appears insufficient to elicit D₁ independence.     

Selective unilateral inactivation of striatal D1 and D2 dopamine receptor subtypes by EEDQ: turning behavior elicited by D2 dopamine receptor agonists

The unilateral intrastriatal injection of the irreversible dopamine (DA) receptor blockerN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) induces a marked decrease in the density of D₁ (-48%) and D₂ (-51%) DA receptors available for binding to [³H]SCH 23390 and [³H]raclopride, respectively. A challenge dose of the D₂ agonist LY 171555 (1 mg/kg, i.p., 24 h after EEDQ) causes intensive ipsiversive circling behavior, whereas the selective D₁ agonist SKF 38393 (20 mg/kg, i.p., 24 h after EEDQ) is unable to induce rotations. The density of D₁ and D₂ DA receptors returns to basal levels by 7 days after the intrastriatal infusion of EEDQ. This biochemical recovery is associated with a progressive decrease in the number of rotations elicited by a challenge dose of LY 171555, suggesting the EEDQ does not cause any relevant neuronal damage. A selective inactivation of striatal D₁ or D₂ DA receptors can be obtained by injecting EEDQ 30 min after the administration of the D₂ antagonist raclopride (20 mg/kg, i.p.) or of the D₁ antagonist SCH 23390 (2 mg/kg, s.c.), respectively. The intensity of the circling behavior induced by LY 171555 24 h after EEDQ in animals with a selective inactivation of D₂ DA receptors is similar to that found in rats in which both D₁ and D₂ DA receptors have been inactivated. In contrast, LY 171555 does not cause rotations when the density of D₁ DA receptors is selectively decreased by EEDQ in rats pretreated with raclopride. These results indicate that the imbalance in striatal D₂ receptors, but not in D₁ receptors, is a critical factor for the expression of the motor effects elicited by LY 171555 in EEDQ-treated rats.     

Decreased striatal D2 dopamine receptors in obese Zucker rats: changes during aging

The specific binding of [³H]YM-09151-2 was used to investigate the possible differences in age-associated changes in striatal D₂ dopamine (DA) receptor properties in genetically obese (fa/fa) Zucker rats and their lean3(Fa/?) littermates. The maximal binding sites (B_max) of D₂ DA receptors was found to decline with age in both obese and lean rats: the rate of decline in receptor B_max was slightly higher in lean than obese rats. However. the B_max of D₂ DA receptor in 6-, 12- and 18-month-old obese rats was significantly lower compared to the age-matched lean rats. These data indicate that obesity decreases the number of striatal D₂ DA receptors without affecting the rate at which receptor number decreases with age.     

Opposing actions of adenosine A2a and dopamine D2 receptor activation on GABA release in the basal ganglia: Evidence for an A2a/D2 receptor interaction in globus pallidus

There is increasing evidence that adenosine (ADO) and dopamine (DA) interact directly in the basal ganglia via actions at ADO A_2a and DA D₂ receptors, respectively. The purpose of this study was to determine 1) the extent to which these receptors modulate endogenous GABA release in discrete regions of the rat basal ganglia and 2) whether GABA release is modulated by a direct and opposing interaction between ADO A_2a and DA D₂ receptors. Tissue slices of striatum (STR) containing globus pallidus (GP; STR/GP) and micropunches of STR, GP, and substantia nigra pars reticulata (SNr) were studied. Radioligand binding demonstrated that ADO A₁, ADO A_2a, and DA D₂ receptors were present in each of the tissue preparations with the exception of SNr, in which ADO A_2a receptors were not detected. Stimulation of ADO A_2a receptors with CGS 21680 (1–10 nM) increased electrically stimulated GABA release in STR/GP slices and GP micropunches. Consistent with the lack of A_2a receptors in SNr, CGS 21680 had no effect on GABA release from this region. In contrast, stimulation of DA D₂ receptors with N-0437 (1–100 nM) inhibited evoked GABA release from STR/GP slices and both GP and SNr micropunches. The D₂-mediated inhibition of GABA release in GP was abolished in the presence of CGS 21680 (10 nM). These experiments demonstrate that stimulation of ADO A_2a and DA D₂ receptors has opposing effects on endogenous GABA release in STR and GP. These opposing actions may explain the antagonistic interactions between ADO and DA that have been observed in behavioral studies and support the hypothesis that the striatopallidal efferent system is an important anatomical substrate for the A_2a/D₂ receptor interaction. © 1996 Wiley-Liss, Inc.     

Central adenosine A2A receptors: an overview

Recent advances in molecular biology, biochemistry, cell biology and behavioral pharmacology together with the development of more selective ligands to the various adenosine receptors have increased our understanding of the functioning of central adenosine A_2A receptors. The A_2A receptor is one of four adenosine receptors found in the brain. Its expression is highest in striatum, nucleus accumbens and olfactory tubercles, although it also occurs in neurons and microglia in most other brain regions. The receptor has seven transmembrane domains and couples via Gs to adenyl cyclase stimulation. Antagonistic interactions between A_2A receptors and dopamine D₂ receptors have been described, as stimulation of the A_2A receptor leads to a reduction in the affinity of D₂ receptors for D₂ receptor agonists. The A_2A receptor is thought to play a role in a number of physiological responses and pathological conditions. Indeed, A_2A receptor antagonists may be useful for the treatment of acute and chronic neurodegenerative disorders such as cerebral ischemia or Parkinson's disease. A_2A receptor agonists may treat certain types of seizures or sleep disorders. This review discusses the characteristics, distribution, pharmacochemical properties and regulation of central A_2A receptors, as well as A_2A receptor-mediated behavioural responses and their potential role in various neuropsychiatric disorders.     

Single and repeated episodes of ethanol withdrawal increase adenosine A1, but not A2A, receptor density in mouse brain

A history of multiple ethanol withdrawal experiences has been shown to exacerbate the severity of future withdrawal episodes, and this sensitization of the withdrawal response has been hypothesized to represent a ‘kindling' phenomenon. Since adenosine functions as an inhibitory modulator of seizure activity and may interact with ethanol to influence neuronal excitability, the present study was conducted to examine the effects of single and repeated episodes of ethanol withdrawal on adenosine A₁ and A_2A receptors in adult C3H/He mice. Mice were chronically exposed to ethanol vapor in inhalation chambers and tested for withdrawal seizures following multiple withdrawal (MW) experience (four cycles of 16 h ethanol intoxication interrupted by 8 h periods of abstinence), single withdrawal experience following 16 h (SW) or 64 h (CE) continuous ethanol intoxication, or no ethanol exposure (controls). Separate groups of mice from each withdrawal condition were used to generate pooled cortical and striatal tissue for ligand saturation experiments using [³H]cyclohexyladenosine to label A₁ receptors and [³H]CGS 21680 to label A_2A receptors. Results indicated that withdrawal seizures were significantly more severe in mice with multiple withdrawal experience in comparison to animals that experienced only a single withdrawal episode, even when total amount of ethanol exposure was equated among groups. The density of A₁ receptors in cerebral cortex was significantly increased over controls 8 h following final ethanol withdrawal by approximately 35% in SW and CE groups, with the largest increase observed in the MW group (56%). Withdrawal treatment groups did not differ in cortical A₁ binding sites immediately upon withdrawal from ethanol, and no significant differences in binding of [³H]CGS 21680 to striatal A_2A receptors were observed following ethanol withdrawal. Ethanol exposure and withdrawal did not significantly alter ligand affinity for either adenosine receptor. These results indicate that adenosine A₁ receptors are selectively upregulated during ethanol withdrawal and that the degree of upregulation may be enhanced following multiple withdrawal episodes. Further, these observations suggest that the upregulation of brain A₁ receptors during ethanol withdrawal may represent a compensatory inhibitory response to increased seizure severity associated with repeated episodes of ethanol withdrawal.     

Endogenous adenosine facilitates neurotransmission via A2A adenosine receptors in the rat superior colliculus in vivo

The concentration of endogenous adenosine in the cerebrospinal fluid increased 2–3-fold of the original level in the area of rat superior colliculus after the intraperitoneal administration of an adenosine deaminase inhibitor, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenosine, 10 mg/kg). Potentials evoked in the superior colliculus by optic tract stimulation were also facilitated by 120–160% of their initial amplitudes. A selective A₁ adenosine receptor antagonist, DPCPX (8-cyclopentyl-1,3-dipropylxanthine), failed to reduce such EHNA-induced facilitation. However, a selective A_2A adenosine receptor antagonist, KF17837 (8(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine) completely eliminated the facilitatory effects of EHNA. Northern blot analysis demonstrated abundant expression of A₁ adenosine receptor mRNA in the superior colliculus. RT-PCR analysis was able to detect the concomitant expression of A_2A adenosine receptor mRNA, but at levels lower than one-tenth of the striatal expression. In the superior colliculus, A_2A adenosine receptors function predominantly on the facilitatory effects of adenosine, irrespective of the ubiquitous expression of A₁ adenosine receptors.     

Evidence for the behavioral supersensitivity of dopamine D2 receptors without receptor up-regulation in morphine-abstinent rats

The effect of morphine tolerance-dependence and abstinence on the characteristics of dopamine D₂ receptors in brain regions and spinal cord was determined in the rat. Male Sprague-Dawley rats were implanted s.c. under light ether anesthesia with 6 morphine pellets for a 7-day period, each containing 75 mg of morphine free base. Rats implanted with placebo served as controls. This procedure resulted in the development of tolerance to morphine as evidenced by decreased analgesic response to a challenge dose of morphine. Similarly, the development of physical dependence was evidenced by a decreased in body weight and colonic temperature after morphine pellet removal (withdrawal). The binding characteristics (B_max andK_d values) of [³H]spiroperidol to dopamine D₂ receptors were determined in the tissues of morphine-tolerant and morphine-abstinent rats. In the tolerant rats, the pellets were left intact at the time of sacrificing, whereas, in the abstinent rats the pellets were removed 18 h prior to sacrificing. The binding of [³H]spiroperidol was determined in membranes prepared from brain regions (hypothalamus, hippocampus, cortex, pons and medulla, midbrain, corpus striatum and amygdala) and spinal cord of rats from various treatment groups. [³H]Spiroperidol bound to brain regions and spinal cord at a single high affinity site. TheB_max or theK_d values in brain regions and spinal cord of morphine-tolerant and -abstinent rats did not differ from their respective placebo controls. The behavioral responses to a selective dopamine D₂ receptor agonist, 2-bromo-α-ergocryptine were also determined in the morphine-abstinent rats. In morphine-abstinent rats, increased behavioral activity, such as total distance travelled, number of movements, and the number of stereotypic movements was seen as compared to placebo controls. The dose of 2-bromo-α-ergocryptine which by itself had no effect on any type of behavioral activity in placebo-treated rats, increased the total distance travelled, horizontal activity, number of movements, and movement time in morphine-abstinent rats. Although in morphine-tolerant or morphine-abstinent rats, the characteristics of [³H]spiroperidol binding to dopamine D₂ receptors in brain regions and spinal cord were unchanged, the supersensitivity was observed to behavioral responses of 2-bromo-α-ergocryptine, a selective dopamine D₂ receptor agonist. These results provide an evidence for behavioral responses of 2-bromo-α-ergocryptine, a selective up-regulation in morphine abstinent rats. Previously, we have show that dopamine D₁ receptors are unaffected in morphine tolerant rats but are modified in morphine-abstinent rats. Thus, in the morphine abstinent process a significant difference was noted in the biochemical characteristics of dopamine D₁ and D₂ receptors.     

Involvement of dopamine D1 and D2 receptors in the ethanol-associated place preference in rats exposed to conditioned fear stress

The present study was designed to investigate: (1) the involvement of dopamine D₁ and D₂ receptors, and (2) the roles of these receptors and endogenous opioid systems (endorphinergic and enkephalinergic systems) in the ethanol-induced place preference in rats exposed to conditioned fear stress using the conditioned place preference paradigm. The administration of ethanol (300 mg/kg, i.p.) induced a significant place preference. The selective D₁ receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H3-benzazepine)hydrochloride (SCH23390; 0.01 and 0.03 mg/kg, s.c.) and the selective D₂ receptor antagonist S(−)-5-(aminosulfonyl)-N-[(1-ethyl-2-pyrrolidinyl)-methyl]-2-methoxybenzamide (sulpiride; 20 and 40 mg/kg, s.c.) significantly attenuated the ethanol-induced place preference. The administration of ethanol (75 mg/kg, i.p.) tended to produce a place preference, but this effect was not significant. SCH23390 (0.03 mg/kg, s.c.) and sulpiride (40 mg/kg, s.c.) significantly attenuated the enhancement of the ethanol (75 mg/kg, i.p.)-induced place preference produced by the μ-opioid receptor agonist morphine (0.1 mg/kg, s.c.). In addition, SCH23390 (0.03 mg/kg, s.c.) also significantly attenuated the enhancement of the ethanol (75 mg/kg, i.p.)-induced place preference produced by the selective δ-opioid receptor agonist 2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aα-octahydroquinolino[2,3,3,-g]isoquinoline (TAN-67; 20 mg/kg, s.c.). On the other hand, sulpiride (40 mg/kg) had no significant effect on the enhancement of the ethanol (75 mg/kg, i.p.)-induced place preference produced by TAN-67. These results suggest that D₁ and D₂ receptors may be involved in the rewarding mechanism of ethanol under psychological stress. In addition, D₁ receptors may participate in the rewarding effect of ethanol modulated by the activation of μ- and δ-opioid receptors, whereas D₂ receptors may participate in the rewarding effect of ethanol modulated by the activation of μ-opioid receptors, but not in that modulated by the activation of δ-opioid receptors.     

Adenosine A2 receptor-mediated excitation of a subset of AH/Type 2 neurons and elevation of cAMP levels in myenteric ganglia of guinea-pig ileum

Abstract The aim of the study was to test the hypothesis that excitatory A₂ and inhibitory A₁ receptors coexist on myenteric AHIType 2 neurons, and are positively coupled to adenylate cyclase to stimulate cAMP formation. The A₂ agonists NECA and CGS 21680 increased excitability and depolarized the membrane in 40% of 71 AH/Type 2 neurons. In the remainder, the agonists depressed excitability and hyperpolarized the neurons. In 13% of neurons, A₂ agonists caused a concentration-dependent depolarization at nanomolar concentrations, followed by hyperpolarization at higher concentrations. CGS 21680 (EC₅₀=0.15 nM) was 133-fold more potent than NECA (EC₅₀= 20 nM) in depolarizing AH/Type 2 neurons. The A₁ agonist, CCPA, caused hyperpolarization and depressed excitability in more than 90% of neurons. The potency profile of agonists for depolarization was CGS 21680 ≫ NECA ≫> CCPA. NECA augmented at nanomolar and inhibited at micromolar concentrations, excitatory depolarizing responses to forskolin in AH/Type 2 neurons; whereas, CCPA only inhibited the action of forskolin. In parallel studies on enzymatically dissociated myenteric ganglia, when the ganglia were exposed to priming concentrations of forskolin (5 μM) in the presence of Ro-20 1724, NECA enhanced the stimulatory action of forskolin on CAMP formation. This effect was abolished by the adenosine receptor antagonist DPSPX. The potency of NECA for stimulation of adenylate cyclase equalled that for depolarization of the AH/Type 2 neurons. The results suggest that high affinity excitutory A₂ receptors are coupled to adenylate cyclase in a minority subset of AH/Type 2 myenteric neurons, and that inhibitory A₁ and excitatory A₂ receptors are co-localized on some AH/Type 2 neurons.