The group II metabotropic glutamate receptor agonist, LY379268, inhibits both cocaine- and food-seeking behavior in rats

Rationale Group II metabotropic glutamate receptor (mGluR2/3) agonists are proposed to serve as potential treatment for addiction.Objectives The present study examined the hypothesis that mGluR2/3 agonists exert inhibitory effects on cocaine-induced reinstatement of cocaine-seeking.Methods Rats were trained to self-administer either cocaine or control reinforcer (food), then responding on the reinforcer-paired lever was extinguished. Reinstatement of responding was induced by a noncontingent presentation of the self-administered reinforcer (10 mg/kg cocaine, i.p. or 765 mg of food). In one experiment, rats were systemically pretreated with vehicle (Veh) or the mGluR2/3 agonist LY379268 (0.3, 1, or 3 mg/kg, i.p.) 30 min before the reinstatement test session. In a second experiment, Veh or LY379268 (0.05, 0.5, or 5 nmol/side) was microinjected into the nucleus accumbens core (NAc core) 5 min before the reinstatement test session. The effects of LY379268 on cocaine- and food-induced reinstatement on reward seeking were assessed.Results Both systemic and intra-NAc core pretreatment with LY379268 inhibited both cocaine- and food-seeking behavior. However, the effect of LY379268 appeared somewhat more effective for cocaine-seeking than food-seeking.Conclusions These results support a potential therapeutic role for mGluR2/3 agonists on relapse of cocaine-seeking. However, doses that inhibited cocaine-seeking were only threefold lower than those inhibiting food-seeking, indicating possible unacceptable nonspecific effects. In addition, the NAc core is one site of action where the mGluR2/3 agonists elicit effects on reward-seeking behavior.     

Anti-epileptic activity of group II metabotropic glutamate receptor agonists (--)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268) and (--)-2-thia-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY389795).

The selective group II metabotropic glutamate receptor (mGlu(2/3)) agonists (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268) and (-)-2-thia-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY389795) have been evaluated as anti-epileptic drugs in dilute brown agouti (DBA/2) mice, lethargic (lh/lh) mice, genetically epilepsy-prone-9 (GEP) rats and amygdala-kindled rats. Sound-induced clonic seizures in DBA/2 mice were transiently inhibited by both agonists intracerebroventricularly (i.c.v.), LY379268 ED(50)=0.08 [0.02-0.33]nmol and LY389795 ED(50)=0.82 [0.27-3.24]nmol or intraperitoneally (i.p.), LY379268 ED(50)=2.9 [0.9-9.6]mg/kg and LY389795 ED(50)=3.4 [1.0-11.7]mg/kg. Both mGlu(2/3) agonists inhibited seizures induced by the group I mGlu receptor agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG), where LY379268, i.c.v. ED(50)=0.3 [0.02-5.0]pmol and LY389795, i.c.v. ED(50)=0.03 [0.05-0.19]nmol. The spike and wave discharge (SWD) duration of absence seizures in lh/lh mice was significantly reduced by both agonists at 1 and 10nmol (i.c.v.) up to 90min following infusion. The electrically induced seizure score and afterdischarge duration of amygdala-kindled rats was partially inhibited by the agonists 30min after i.p. injection of 10mg/kg. The agonists did not inhibit sound-induced seizures in GEP rats (0.1-1mg/kg, 30min 1h, i.p.), but were proconvulsant following sound stimulus (> or =0.1mg/kg). These findings identify a potential role for mGlu(2/3) agonists in the amelioration of generalised and partial epileptic seizures.     

Blockade of group II metabotropic glutamate receptors in the nucleus accumbens produces hyperlocomotion in rats previously exposed to amphetamine

The neurotransmitter glutamate is known to participate in both the induction and expression of locomotor sensitization by psychostimulant drugs like amphetamine. Previously, it was reported that subtype nonselective blockade of metabotropic glutamate receptors (mGluRs) in the nucleus accumbens (NAcc) produces hyperlocomotion in rats previously exposed to amphetamine. The present experiments examined whether group II mGluRs may contribute to this effect. Rats in different groups were administered five injections of either saline or amphetamine (1.0 mg/kg, i.p.), one injection given every third day. Two weeks later, they were tested for 2 h following an injection of either saline or the group II mGluR antagonist LY341495. In one experiment, test injections were administered systemically (saline or LY341495, 1.0 mg/kg, i.p.). Rats previously exposed to amphetamine showed a greater locomotor response to LY341495 on the test compared to controls previously exposed to saline. This hyperlocomotor response was absent in rats tested with a combination of LY341495 and the group II mGluR agonist LY379268 (1.0 mg/kg, i.p.). In a second experiment, different rats were tested following microinjections into the NAcc (saline or LY341495, 0.1, 10 or 100 microg/0.5 microl/side). Again, rats previously exposed to amphetamine showed a greater dose-dependent locomotor response to LY341495 on the test relative to saline-exposed controls. Locomotor activity in saline-exposed rats challenged with LY341495 did not differ from that observed in rats previously exposed and tested with saline in either experiment. These results indicate that group II mGluRs, particularly those found in the NAcc, are well positioned to modulate the expression of locomotor sensitization by amphetamine.     

Dopamine D1 receptors and group I metabotropic glutamate receptors contribute to the induction of long-term potentiation in the nucleus accumbens

Long-term changes in the efficacy of glutamatergic synaptic transmission in the striatal complex are proposed to underlie motor learning and neuroadaptations leading to addiction. Dopamine and glutamate play key roles in the induction of long-term potentiation (LTP) and long-term depression (LTD) in the dorsal striatum, but their contribution to synaptic plasticity in the ventral striatum (nucleus accumbens, NAc) has been less extensively studied. We have examined the role of dopamine, glutamate and GABA in the induction of LTP in mouse brain slices containing the NAc. High-frequency stimulation of glutamatergic inputs elicited LTP of field excitatory postsynaptic potentials/population spikes (fEPSP/PSs) in the core region of the NAc. GABA did not seem to participate in LTP induction because LTP was not altered in the presence of either a GABA(A)- (bicuculline) or a GABA(B)- (CGP 55845) receptor antagonist. However, the dopamine D1 receptor antagonist SCH 23390, but not the dopamine D2 receptor antagonist sulpiride, impaired LTP. The dopamine reuptake blocker nomifensine also inhibited LTP induction. We found that group I metabotropic glutamate receptors (mGluRs) contribute to LTP induction because the mGluR1 antagonist LY 367385, or the mGluR5 antagonist MPEP, blocked LTP induction. Furthermore, the glutamate reuptake blocker DL-TBOA also impaired LTP. The present results demonstrate that dopamine and glutamate play critical roles in the mechanisms of induction of LTP in the NAc through the activation of dopamine D1 receptors and group I mGluRs. However, LTP is negatively regulated when endogenous levels of dopamine or glutamate are elevated.     

Dipeptides as effective prodrugs of the unnatural amino acid (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740), a selective group II metabotropic glutamate receptor agonist

(+)-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (1), also known as LY354740, is a highly potent and selective agonist for group II metabotropic glutamate receptors (mGlu receptors 2 and 3) tested in clinical trials. It has been shown to block anxiety in the fear-potentiated startle model. Its relatively low bioavailability in different animal species drove the need for an effective prodrug form that would produce a therapeutic response at lower doses for the treatment of anxiety disorders. We have investigated the increase of intestinal absorption of this compound by targeting the human peptide transporter hPepT1 for active transport of di- and tripeptides derived from 1. We have found that oral administration of an N dipeptide derivative of 1 (12a) in rats shows up to an 8-fold increase in drug absorption and a 300-fold increase in potency in the fear-potentiated startle model in rats when compared with the parent drug 1.     

The metabotropic glutamate receptor 7 (mGluR7) allosteric agonist AMN082 modulates nucleus accumbens GABA and glutamate, but not dopamine, in rats

The group III metabotropic glutamate receptor 7 (mGluR7) has been implicated in many neurological and psychiatric diseases, including drug addiction. However, it is unclear whether and how mGluR7 modulates nucleus accumbens (NAc) dopamine (DA), L-glutamate or gamma-aminobutyric acid (GABA), important neurotransmitters believed to be involved in such neuropsychiatric diseases. In the present study, we found that systemic or intra-NAc administration of the mGluR7 allosteric agonist N,N'-dibenzyhydryl-ethane-1,2-diamine dihydrochloride (AMN082) dose-dependently lowered NAc extracellular GABA and increased extracellular glutamate, but had no effect on extracellular DA levels. Such effects were blocked by (R,S)-alpha-methylserine-O-phosphate (MSOP), a group III mGluR antagonist. Intra-NAc perfusion of tetrodotoxin (TTX) blocked the AMN082-induced increases in glutamate, but failed to block the AMN082-induced reduction in GABA, suggesting vesicular glutamate and non-vesicular GABA origins for these effects. In addition, blockade of NAc GABAB receptors by 2-hydroxy-saclofen itself elevated NAc extracellular glutamate. Intra-NAc perfusion of 2-hydroxy-saclofen not only abolished the enhanced extracellular glutamate normally produced by AMN082, but also decreased extracellular glutamate in a TTX-resistant manner. We interpret these findings to suggest that the increase in glutamate is secondary to the decrease in GABA, which overcomes mGluR7 activation-induced inhibition of non-vesicular glutamate release. In contrast to its modulatory effect on GABA and glutamate, the mGluR7 receptor does not appear to modulate NAc DA release.     

Effects of subchronic methamphetamine exposure on basal dopamine and stress-induced dopamine release in the nucleus accumbens shell of rats

Rationale Subchronic administration of stimulants reduces basal dopamine (DA) concentrations and blocks stress-induced DA release in the nucleus accumbens (NA) of rats during withdrawal. However, no studies have attempted to relate early withdrawal from chronic drug exposure to stress reactivity and changes in DA transmission. Objectives The effects of subchronic low-dose methamphetamine (METH) administration on regional changes in dopamine transporter (DAT) and norepinephrine transporter (NET) immunoreactivity and function during early withdrawal were examined. The effects of subchronic METH on stress responsivity measured by DA release in the nucleus accumbens shell (NA SHELL) and core (NA CORE) during acute restraint stress were also examined. Methods Male rats received single injections of METH (2.0 mg/kg i.p.) or saline (SAL) for 10 days and then were killed 24 h after the last injection. DAT and NET protein in NA, striatum (STR), medial prefrontal cortex (mPFC), and hippocampus were assayed by Western blot analysis. Experiment 2 measured basal extracellular DA concentrations and restraint-stress-induced DA release in vivo in the NA SHELL and CORE of SAL- and METH-pretreated rats after 24-h withdrawal. Experiment 3 examined the in vivo regulation of extracellular DA in the NA SHELL and/or CORE after local administration of GBR12909 (50 μM) or nisoxetine (100 μM; NA SHELL). Results Subchronic METH increased DAT but not NET immunoreactivity in the NA compared to the STR and mPFC. METH reduced basal extracellular DA and blocked restraint-stress-induced DA release in the NA SHELL. DA uptake blockade increased extracellular DA more in the NA SHELL of METH rats, whereas NE uptake blockade increased basal DA concentrations to a similar extent in METH and SAL rats. Conclusions These results suggest that subchronic METH exposure selectively increases NA DAT and consequently reduces basal and stress-induced DA release in the NA SHELL during early withdrawal.     

Blockade of the locomotor stimulant effects of amphetamine by group I,group II,and group III metabotropic glutamate receptor ligands in the rat nucleus accumbens: possible interactions with dopamine receptors

Previous investigations have shown that mGlu receptors would be involved in the amphetamine-induced motor response. However, data are somewhat controversial across studies where methodological protocols vary. The aim of the present study was to determine the involvement of mGlu receptors in the NAcc in the locomotor-activating properties of amphetamine in rats well habituated to their experimental environment, a condition known to modulate the motor response to amphetamine. Focal infusion of the group I mGlu receptor antagonist S-4-CPG, which has no effect on basal motor activity, virtually suppressed the locomotor response to amphetamine, while infusion of the group II mGlu receptor antagonist LY 341495 or the group III mGlu receptor agonist AP4, at the minimal dose that produces locomotor activation, reduced it by approximately a half. These effects were blocked by the group I mGlu receptor agonist DHPG, the group II mGlu receptor agonist APDC, and the group III mGlu receptor antagonist MPPG, respectively. These data confirm that mGlu receptors in the NAcc contribute to the psychostimulant motor effect of amphetamine. Results are discussed from the view of recent neuropharmacological studies that have defined the effects of these mGlu receptor ligands on basal motor activity and DA receptor agonists-induced locomotor responses in rats exposed to similar experimental procedures (Eur J Neuroscience 13 (2001) 2157; Neuropharmacology 41 (2001) 454; Eur J Neuroscience 13 (2001) 869). It is suggested that the contribution of mGlu receptors to the amphetamine-induced motor response may result mainly from their functional, either direct or indirect, interactions with D1-like receptors in the NAcc.     

Role of muscarinic receptors in the activation of the ventral subiculum and the consequences for dopamine release in the nucleus accumbens

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active herbal component traditionally used in China for treating various ailments. Emodin exerts antiproliferative effects in many cancer cell lines and the actual molecular mechanism of which is still not clear. Since apoptosis could be a potential mechanism to explain these effects, we tested whether emodin induces cell death in human cervical cancer cells. Our results suggest that emodin exerts antiproliferative effects in human cervical cancer cells. Emodin inhibited DNA synthesis and induced apoptosis as demonstrated by increased nuclear condensation, annexin binding and DNA fragmentation in Bu 25TK cells in the presence of emodin. Moreover, we demonstrate for the first time in human cervical cancer cells that the apoptotic pathway involved in emodin-induced apoptosis is caspase-dependent and presumably through the mitochondrial pathway, as shown by the activation of caspases-3, -9 and cleavage of poly(ADP-ribose) polymerase.     

Ethanol induces stronger dopamine release in nucleus accumbens (shell) of alcohol-preferring (bibulous) than in alcohol-avoiding (abstainer) rats

Several studies on the differences between ethanol-preferring versus non-preferring rat lines suggest an innate deficit in the mesolimbic dopaminergic system as an underlying factor for ethanol volition. Rats would try to overcome such deficit by engaging in a drug-seeking behaviour, when available, to drink an ethanol solution over water. Thus, in the present study we compared the effect of a single dose of ethanol (1 g/kg, i.p.) on the extracellular levels of monoamines measured by microdialysis in the shell of nucleus accumbens of University of Chile bibulous (UChB) and University of Chile Abstainer (UChA) rats, bred for 79 and 88 generations to prefer or reject ethanol, respectively. It is reported that under basal conditions extracellular dopamine levels are lower in the bibulous than in the abstainer rats, while ethanol induced a 2-fold greater increase of dopamine release in bibulous than in abstainer rats. The greater effect of ethanol in bibulous rats was not associated to differences in blood ethanol levels, since the concentration and elimination of ethanol were virtually identical in both rat lines, indicating that bibulous rats are more sensitive to the stimulation of dopamine release by ethanol than abstainer rats. No differences were observed in 5-hydroxytryptamine or metabolites measured simultaneously under basal or ethanol-stimulating conditions in bibulous and abstainer rats. Overall, the present results suggest that a low dopaminergic tone and a strong mesolimbic dopamine response to ethanol are concerted neurochemical features associated to an ethanol-seeking behaviour in rats.     

Blockade of NMDA receptors in the prefrontal cortex increases dopamine and acetylcholine release in the nucleus accumbens and motor activity

Objectives  The present study investigates the effects of injections of a specific N-methyl-d-aspartic acid (NMDA) antagonist 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phophonic acid (CPP) into the prefrontal cortex (PFC) on the extracellular concentrations of dopamine and acetylcholine in the nucleus accumbens (NAc) and on motor activity in the freely moving rat. Materials and methods  Sprague–Dawley male rats were implanted with guide cannulas into the medial PFC and NAc to perform bilateral microinjections and microdialysis experiments. Spontaneous motor activity was monitored in the open field. Results  Injections of CPP (1 μg/0.5 μL) into the PFC produced a significant increase of the baseline extracellular concentrations of dopamine (up to 130%), dihydroxyphenylacetic acid (DOPAC; up to 120%), homovanillic acid (HVA; up to 130%), and acetylcholine (up to 190%) in the NAc as well as motor hyperactivity. In the NAc, perfusion of the NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate antagonists CPP (50 μM)+6,7-dinitroquinoxaline-2,3-dione (DNQX; 50 μM) through the microdialysis probe blocked acetylcholine release, but not DOPAC and HVA increases produced by CPP injections into the PFC. Also, increases in motor activity produced by prefrontal injections of CPP were significantly reduced by bilateral injections into the NAc of a mixed D1/D2 antagonist, flupenthixol (5 and 25 μg/0.5 μL). Injections into the NAc of the muscarinic antagonist scopolamine (1 and 10 μg/0.5 μL) further increased, and of the nicotinic antagonist mecamylamine (1 and 10 μg/0.5 μL) did not change, the increases in motor activity produced by prefrontal CPP injections. Conclusions  These results suggest that the dysfunction of NMDA receptors in the PFC could be a key factor in the neurochemical and motor effects associated with corticolimbic hyperactivity.     

Effects of dorsal and ventral hippocampal NMDA stimulation on nucleus accumbens core and shell dopamine release

This study has analysed the effects of infusing N-methyl-D-aspartate (NMDA) into either the ventral or dorsal hippocampus on dopamine (DA) transmission in the nucleus accumbens (NAC) core or shell for the first time. Dopamine was measured using in vivo microdialysis with high performance liquid chromatography with electrochemical detection (HPLC-EC). Unilateral NMDA infusion (0.5 microg) into the ventral hippocampus (VH) increased extracellular DA levels in NAC shell during the first 30 min following infusion compared to saline (SAL) infused animals. In contrast, NAC core DA levels were unaffected. NMDA infusion into the dorsal hippocampus (DH) led to a decrease in NAC core DA levels; this effect was not observed in the SAL-infused group. DA levels in NAC shell remained unaltered. At the end of the experiments, we examined the response to a systemic amphetamine (AMPH) injection of 1mg/kg on extracellular DA levels of the NAC core and shell. Interestingly, on2ly animals previously infused with NMDA into the VH exhibited a sensitized DA response in the NAC shell in response to the AMPH injection. We can conclude that VH activation has an acute stimulatory effect on DA release in the shell and that DH activation has a suppressive effect on extracellular DA levels in the core.     

Role of GABA B receptors in the endomorphin-1-, but not endomorphin-2-, induced dopamine efflux in the nucleus accumbens of freely moving rats

In vivo microdialysis was used to study the effects of the locally applied GABA B receptor antagonist 2-hydroxysaclofen and GABA B receptor agonist baclofen on the basal dopamine efflux as well as on the endomorphin-1- and endomorphin-2-induced dopamine efflux in the nucleus accumbens of freely moving rats. 2-Hydroxysaclofen (100 and 500 nmol) increased basal dopamine efflux. Baclofen (2.5 and 5 nmol) failed to affect basal dopamine efflux. 2-Hydroxysaclofen (1 and 10 nmol) which did not alter the basal dopamine efflux, enhanced the endomorphin-1 (25 nmol)-induced dopamine efflux. Baclofen (2.5 and 5 nmol) failed to affect endomorphin-1 (25 nmol)-induced dopamine efflux, but it counteracted the 2-hydroxysaclofen-induced increase of the endomorphin-1-elicited dopamine efflux. Neither 2-hydroxysaclofen (10 nmol) nor baclofen (5 nmol) affected the endomorphin-2 (25 nmol)-induced dopamine efflux. The doses mentioned are the total amount of drug over the infusion period that varied across the drugs (25 or 50 min). These results suggest that accumbal GABA B receptor plays an inhibitory role on the basal as well as the endomorphin-1-elicited accumbal dopamine efflux. The present results support our earlier reported notion that endomorphin-1 and endomorphin-2 increase accumbal dopamine efflux by different mechanisms. Finally, it is suggested that a decrease of endogenous accumbal GABA reduces the accumbal GABA B receptor-mediated GABA-ergic inhibition, enhancing thereby the accumbal dopamine efflux.     

Synthesis, pharmacological characterization, and molecular modeling of heterobicyclic amino acids related to (+)-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylic acid (LY354740): identification of two new potent, selective, and systemically active agonists for group II metabotropic glutamate receptors

As part of our ongoing research program aimed at the identification of highly potent, selective, and systemically active agonists for group II metabotropic glutamate (mGlu) receptors, we have prepared novel heterobicyclic amino acids (-)-2-oxa-4-aminobicyclo[3.1. 0]hexane-4,6-dicarboxylate (LY379268, (-)-9) and (-)-2-thia-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY389795, (-)-10). Compounds (-)-9 and (-)-10 are structurally related to our previously described nanomolar potency group II mGlu receptor agonist, (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate (LY354740 monohydrate, 5), with the C4-methylene unit of 5 being replaced with either an oxygen atom (as in (-)-9) or a sulfur atom (as in (-)-10). Compounds (-)-9 and (-)-10 potently and stereospecifically displaced specific binding of the mGlu2/3 receptor antagonist ([3H]LY341495) in rat cerebral cortical homogenates, displaying IC50 values of 15 +/- 4 and 8.4 +/- 0.8 nM, respectively, while having no effect up to 100 000 nM on radioligand binding to the glutamate recognition site on NMDA, AMPA, or kainate receptors. Compounds (-)-9 and (-)-10 also potently displaced [3H]LY341495 binding from membranes expressing recombinant human group II mGlu receptor subtypes: (-)-9, Ki = 14.1 +/- 1.4 nM at mGlu2 and 5.8 +/- 0.64 nM at mGlu3; (-)-10, Ki = 40.6 +/- 3.7 nM at mGlu2 and 4.7 +/- 1.2 nM at mGlu3. Evaluation of the functional effects of (-)-9 and (-)-10 on second-messenger responses in nonneuronal cells expressing human mGlu receptor subtypes demonstrated each to be a highly potent agonist for group II mGlu receptors: (-)-9, EC50 = 2.69 +/- 0.26 nM at mGlu2 and 4.58 +/- 0.04 nM at mGlu3; (-)-10, EC50 = 3.91 +/- 0.81 nM at mGlu2 and 7.63 +/- 2. 08 nM at mGlu3. In contrast, neither compound (up to 10 000 nM) displayed either agonist or antagonist activity in cells expressing recombinant human mGlu1a, mGlu5a, mGlu4a, or mGlu7a receptors. The agonist effects of (-)-9 and (-)-10 at group II mGlu receptors were not totally specific, however, as mGlu6 agonist activity was observed at high nanomolar concentrations for (-)-9 (EC50 = 401 +/- 46 nM) and at micromolar concentrations (EC50 = 2 430 +/- 600 nM) for (-)-10; furthermore, each activated mGlu8 receptors at micromolar concentrations (EC50 = 1 690 +/- 130 and 7 340 +/- 2 720 nM, respectively). Intraperitoneal administration of either (-)-9 or (-)-10 in the mouse resulted in a dose-related blockade of limbic seizure activity produced by the nonselective group I/group II mGluR agonist (1S,3R)-ACPD ((-)-9 ED50 = 19 mg/kg, (-)-10 ED50 = 14 mg/kg), indicating that these molecules effectively cross the blood-brain barrier following systemic administration and suppress group I mGluR-mediated limbic excitation. Thus, heterobicyclic amino acids (-)-9 and (-)-10 are novel pharmacological tools useful for exploring the functions of mGlu receptors in vitro and in vivo.