A.N.M. Schoffelmeer

Interactions between CB1 cannabinoid and μ opioid receptors mediating inhibition of neurotransmitter release in rat nucleus accumbens core

We examined the occurrence of functional interactions between CB1 cannabinoid and mu opioid receptors in the core of rat nucleus accumbens (NAc core). To that end, receptor-mediated inhibition of depolarization (4-aminopyridine)-induced [3H]glutamate release and glutamate (NMDA) receptor-stimulated [14C]acetylcholine (ACh) and [3H]GABA release was studied in superfused NAc core slices. The inhibitory effects of the mu receptor agonist morphine and the CB1 receptor agonist HU210 on the release of these neurotransmitters were selectively antagonized by the mu receptor antagonist naloxone and the CB1 receptor antagonist SR141716A, respectively. Surprisingly, naloxone prevented the antagonistic action of SR141716A at CB1 receptors and SR141716A abolished that of naloxone at mu receptors mediating inhibition of [3H]glutamate and [3H]GABA release. Therefore, these antagonists seem to allosterically interact, indicating the involvement of physically associated mu opioid and CB1 cannabinoid receptors. Such an interaction between antagonists was not observed at the receptors mediating inhibition of [14C]ACh release. Moreover, dose-response curves of the agonists showed that mu and CB1 receptors mediating inhibition of [3H]glutamate release display a non-additive interaction, whereas these receptors synergistically interact regarding their inhibitory control of [3H]GABA release. Finally, the apparent allosteric interaction between antagonists was also observed regarding the effects of other receptor-selective agonists and antagonists at mu opioid and CB1 cannabinoid receptors (mediating inhibition of NMDA-induced [3H]GABA release) and must therefore be a unique property of the receptors involved. These data suggest the existence of physically associated mu opioid and CB1 cannabinoid receptors, whereby activation of these receptors results in either a non-additive (glutamate release) or a synergistic (GABA release) effect. It is proposed that these allosterically interacting mu and CB1 receptors in the NAc core may represent G-protein coupled heterodimeric receptor complexes.

Ontogeny of μ-, δ- and κ-opioid receptors mediating inhibition of neurotransmitter release and adenylate cyclase activity in rat brain

The ontogeny was examined of functional opioid receptors mediating presynaptic inhibition of neurotransmitter release and inhibition of dopamine (DA)-sensitive adenylate cyclase in the rat brain, using highly selective agonists for μ-, δ- and κ-receptors. On gestational day 17 (E17) strong inhibitory effects of the selective μ-agonist DAGO on the electrically evoked release of [3H]noradrenaline from cortical slices and of the selective κ-agonist U-50,488 on the electrically evoked release of [3H]DA from striatal slices were found. Electrically evoked release of [3H]acetylcholine from striatal slices was not detectable before postnatal day 7 (P7), but on that day it was already strongly inhibited by the selective δ-agonist DPDPE. Although μ- and δ-opioid receptors coupled to DA-sensitive adenylate cyclase in the striatum are likely to be physically associated in an opioid receptor complex in the adult, they were found to develop asynchronously. Whereas selective activation of μ-receptors with DAGO resulted in an inhibition of D1 dopamine receptor-stimulated adenylate cyclase activity on E17, activation of δ-receptors with DPDPE was not effective until P14. This study confirms the early appearance of μ- and κ-opioid receptors and the relatively late development of δ-opioid receptors in the rat brain. Most importantly, it shows that in an early stage of development opioids are already able to mediate modulation of noradrenergic (via activation of μ-receptors) and dopaminergic (via activation of μ- and κ-receptors) neurotransmission processes. Therefore, these opioid receptor types could play a role in brain development and/or developmental disturbances.

Stimulation of D-2 dopamine receptors decreases the evoked in vitro release of [3H]acetylcholine from rat neostriatum: role of K+ and Ca2+

Abstract: Reportedly, stimulation of D‐2 dopamine receptors inhibits the depolarization‐induced release of acetylcholine from the neostriatum in a cyclic AMP‐independent manner. In the present study, we investigated the role of K+ and Ca2+ in the D‐2 receptor‐mediated inhibition of evoked [3H]acetylcholine release from rat striatal tissue slices. It is shown that the D‐2 receptor‐mediated decrease of K+‐evoked [3H]acetylcholine release is not influenced by the extracellular Ca2+ concentration. However, increasing extracellular K+, in the presence and absence of Ca2+, markedly attenuates the effect of D‐2 stimulation on the K+‐evoked [3H]acetylcholine release. Furthermore, it is shown that activation of D‐2 receptors in the absence of Ca2+ also inhibits the veratrine‐evoked release of [3H]acetylcholine from rat striatum. These results suggest that the D‐2 dopamine receptor mediates the decrease of depolarization‐induced [3H]acetylcholine release from rat striatum primarily by stimulation of K+ efflux (opening of K+ channels) and inhibition of intracellular Ca2+ mobilization.

Activation of presynaptic α2-adrenoceptors attenuates the inhibitory effect of μ-opioid receptor agonists on noradrenaline release from brain slices

Summary3H-noradrenaline release from rat neocortical slices induced by 15 mM K+ was concentration-dependently inhibited by morphine, [D-Ala2-D-Leu5] enkephalin (DADLE) and the calcium entry blocker Cd2+. Blockade of presynaptic α2-adrenoceptors with phentolamine, almost doubling K+-induced 3H-noradrenaline release, slightly enhanced the relative inhibitory effects of morphine and DADLE, whereas that of Cd2+ remained unaffected. In contrast, activation of presynaptic α2-adrenoceptors with clonidine (1 μM) or TL-99 (1 μM), inhibiting release by about 50%, completely abolished the inhibitory effects of morphine and DADLE without affecting that of Cd2+. When in the presence of 1 μM clonidine adenylate cyclase was activated with forskolin (10 μM), which restored release to the drug-free control level, the opioids still did not display their inhibitory effects. Therefore, μ-opioid receptor efficacy appears to be dependent on the degree of activation of α2-adrenoceptors in central noradrenergic nerve terminals, probably through a local receptor interaction within the nerve terminal membrane.

mu-Opioid Receptors in the Nucleus Accumbens Shell Region Mediate the Effects of Amphetamine on Inhibitory Control But Not Impulsive Choice

Acute challenges with psychostimulants such as amphetamine affect impulsive behavior in both animals and humans. With regard to amphetamine, it is important to unravel how this drug affects impulsivity since it is not only a widely abused recreational drug but also regularly prescribed to ameliorate maladaptive impulsivity. Therefore, we studied the effects of amphetamine in two rat models of impulsivity, the five-choice serial reaction time task and the delayed-reward task, providing measures of inhibitory control and impulsive choice, respectively. We focused on the role of opioid receptor activation in amphetamine-induced impulsivity as there is ample evidence indicating an important role for endogenous opioids in several behavioral and neurochemical effects of amphetamine. Results showed that amphetamine-induced inhibitory control deficits were dose-dependently attenuated by the preferential μ-opioid receptor antagonist naloxone, but not by the selective δ-opioid receptor antagonist naltrindole or κ-opioid receptor antagonist nor-BNI (nor-binaltorphimine dihydrochloride). In contrast, naloxone did not affect amphetamine-induced improvements in impulsive decision making. Naloxone also completely prevented inhibitory control deficits induced by GBR 12909 [1-(2-[bis(4-fluorophenyl)methoxy] ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride], a selective dopamine transporter inhibitor. Intracranial infusions of naloxone, the selective μ-opioid receptor antagonist CTAP (H-d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2), morphine, and the selective μ-opioid receptor agonist DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin acetate salt) revealed that μ-opioid receptor activation in the shell rather than the core subregion of the nucleus accumbens (NAc) modulates inhibitory control and subserves the effect of amphetamine thereon. Together, these results indicate an important role for NAc shell μ-opioid receptors in the regulation of inhibitory control, probably via an interaction between these receptors and the mesolimbic dopamine system.

Insulin Modulates Cocaine-Sensitive Monoamine Transporter Function and Impulsive Behavior

Because insulin acutely enhances the function of dopamine transporters, the tyrosine kinase receptors activated by this hormone may modulate transporter-dependent neurochemical and behavioral effects of psychoactive drugs. In this respect, we examined the effects of insulin on exocytotic monoamine release and the efficacy of the monoamine transporter blocker cocaine in rat nucleus accumbens. Whereas insulin reduced electrically evoked exocytotic [3H]dopamine release in nucleus accumbens slices, the hormone potentiated the release-enhancing effect of cocaine thereon. The phosphatidylinositol 3-kinase inhibitor LY294002 abolished these effects, indicating the involvement of insulin receptors. Similar insulin effects were observed on the release of [3H]norepinephrine in nucleus accumbens slices, but not on that of [3H]serotonin, and were also apparent in medial prefrontal cortex slices. As might then be expected, insulin also potentiated the dopamine and norepinephrine release-enhancing effects of the selective monoamine uptake inhibitors GBR12909 and desmethylimipramine, respectively. In subsequent behavioral experiments, we investigated the role of insulin in motor impulsivity that depends on monoamine neurotransmission in the nucleus accumbens. Intracranial administration of insulin in the nucleus accumbens alone reduced premature responses in the five-choice serial reaction time task and enhanced the stimulatory effect of peripheral cocaine administration on impulsivity, resembling the observed neurochemical effects of the hormone. In contrast, cocaine-induced locomotor activity remained unchanged by intra-accumbal insulin application. These data reveal that insulin presynaptically regulates cocaine-sensitive monoamine transporter function in the nucleus accumbens and, as a consequence, impulsivity. Therefore, insulin signaling proteins may represent targets for the treatment of inhibitory control deficits such as addictive behaviors.

μ‐Opioid Receptors Mediate the Inhibitory Effect of Opioids on Dopamine‐Sensitive Adenylate Cyclase in Primary Cultures of Rat Neostriatal Neurons

Abstract: The receptors mediating the inhibition of D1 dopamine receptor‐stimulated adenylate cyclase by opioids were examined in primary cultures of rat neostriatal neurons. Adenylate cyclase activity was dose‐dependently increased by the selective D1 dopamine receptor agonist SKF 38393 (EC50= 0.05 μM). This stimulation was fully antagonized by the selective D1 dopamine receptor antagonist SCH 23390 (1 μM). SKF 38393 (1 μM)‐stimulated adenylate cyclase activity was strongly reduced (by almost 60%) by the highly selective μ‐agonist [D‐Ala2,MePhe4,Gly‐ol5]‐enkephalin (DAGO; EC50= 0.006μM) and high concentrations of the selective δ‐agonist [d‐Ser2(O‐tert‐butyl), Leu5]‐enkephaIyl‐Thr6 (DSTBU‐LET; EC50= 0.13 μM) but not by the selective δ‐agonist [d‐penicillamine2, d‐penicillamine5]enkephalin (DPDPE). D1 dopamine receptor‐stimulated adenylate cyclase activity was also slightly reduced (by ∼20%) by high concentrations of the k‐agonist U50,488 (EC50= 0.63 μM). The inhibitory effects of submaximally effective concentrations of DAGO, DSTBULET, and U50,488 were equally well antagonized by the μ‐opioid receptor‐selective antagonist naloxone (EC50 of ∼0.1 μM). Neither the irreversible δ‐ligand fentanyl isothiocyanate (1 μM) nor the reversible δ‐antagonist ICI 174864 (1 μM) reversed the inhibitory effects of DSTBULET. The inhibitory effects of DAGO and U50,488 were equally well reversed by high concentrations (>0.1 μM) of the k‐opioid receptor‐selective antagonist norbinaltorphimine. The effect of DAGO (1 μM) was already detectable after 1 day in culture, whereas DPDPE (1 μM) had no effect even after 28 days in culture. These data indicate that an homogeneous population of μ‐opioid receptors coupled as inhibitors to D1 dopamine receptor‐stimulated adenylate cyclase is expressed in rat neostriatal neurons in primary culture.

Lack of Cross-Sensitization of the Locomotor Effects of Morphine in Amphetamine-Treated Rats

Repeated exposure to morphine and amphetamine induces long-lasting sensitization of their psychomotor stimulant properties, whereas pretreatment with morphine causes cross-sensitization of the locomotor effects of amphetamine. Here, we investigated whether pre-exposure to amphetamine also results in cross-sensitization to morphine. Rats pretreated with amphetamine (5 × 2.5 mg/kg, i.p.) displayed neither short-term (3 days post-treatment) nor long-term (3 weeks post-treatment) cross-sensitization of the locomotor effects of morphine (2 or 5 mg/kg, s.c.). Two other amphetamine pretreatment protocols (1 × 5 mg/kg, i.p. and 14 × 2.5 mg/kg, i.p.) also failed to induce cross-sensitization to morphine. In contrast, all amphetamine pretreatment regimens induced sensitization of the locomotor effects of amphetamine (1 mg/kg, i.p.) and pretreatment with morphine (14 × 10 mg/kg, s.c.) induced both short- and long-term sensitization of the locomotor effects of both morphine and amphetamine. These data suggest that the expression of sensitization of the locomotor effects of morphine and amphetamine, at least partially, involves distinct neuroadaptive phenomena.