Antagonism of the hypermotility response induced by excitatory amino acids in the rat nucleus accumbens

Summary Several compounds have been shown to antagonize the excitation of single neurons produced by excitatory amino acids. This study was designed to determine the effectiveness of these compounds in antagonizing the hypermotility response to excitatory amino acids after intraaccumbens administration. Of the putative antagonists tested, D-aminoadipic acid, diaminopimelic acid and glutamic acid diethyl ester all showed significant inhibitory effects on excitatory amino acid-induced hypermotility while 2-amino-5-phosphonovaleric acid, -D-glutamylglycine, 2-amino-4-phosphonobutyric acid and cis-2,3-piperidine dicarboxylic acid were ineffective. D-Aminoadipic acid decreased N-methyl-aspartic acid-induced hypermotility while having no significant effect on the hypermotility responses induced by kainic or quisqualic acids. Diaminopimelic acid markedly decreased N-methyl-aspartic acid- and kainic acid-induced hypermotility but was totally ineffective on quisqualic acid-induced hypermotility. In contrast to D-aminoadipic acid, glutamic acid diethyl ester antagonized the increase in motility produced by kainic and quisqualic acids but not that produced by N-methyl-aspartic acid. The above data suggests that N-methyl-aspartic acid and quisqualic acid may produce their motor effects through the activation of two different receptors in the nucleus accumbens while kainic acid may mediate its hypermotility response through both N-methyl-aspartic acid and quisqualic acid receptors. However, a third receptor type activated solely by kainic acid cannot be excluded at this time.     

Synaptic potentials mediated by excitatory amino acid receptors in the nucleus accumbens of the rat, in vitro

Focal stimulation of the dorsal regions of the nucleus accumbens of the rat, in vitro, evoked field potentials consisting of two negative waves. The first wave probably reflected electrical activation of the presynaptic fibres. This wave was resistant to excitatory amino acid antagonists and sulpiride. The second negative wave arose from a slower positive going component. These probably evince a population spike and field EPSP, respectively. Intracellular studies also indicated the occurrence of EPSP's and action potentials. The presumed field EPSP and population spike were reduced by kynurenic acid (IC50 approximately 300 microM) but not by AP5 nor sulpiride. When the Mg2+ was removed, the field EPSP was prolonged in a manner fully reversible by the addition of 30 microM D(-)AP5. These results indicate that focal stimulation within the nucleus accumbens may activate excitatory amino acid-ergic fibres that make synaptic contact with an indigenous population of neurones. The postsynaptic receptors were probably of a non-NMDA subtype. However NMDA receptor-mediated responses were evident upon removal of the Mg2+, suggesting that these receptors could also contribute to neuronal excitation under the appropriate physiological conditions.     

Muscarinic receptors discriminated by pirenzepine are involved in the regulation of neurotransmitter release in rat nucleus accumbens.

The effect of pirenzepine, a selective muscarinic antagonist, was tested on the oxotremorine facilitation of the K+-evoked release of [14C]-dopamine from tissue slices of rat nucleus accumbens. The effect of pirenzepine was compared with that of scopolamine and other antagonists which show no heterogeneity in their action on muscarinic receptors in order to determine whether a selective action at a single receptor subtype, M1 or M2, could be distinguished. Pirenzepine and scopolamine both antagonized the oxotremorine-induced (EC50 = 3 X 10(-7) M) facilitation of [14C]-dopamine release with pA2 values of 7.5 and 8.9 respectively. This result indicated that the high affinity pirenzepine receptor (M1) was involved in this response. Low concentrations of 3-quinuclidinyl benzilate (3 X 10(-10) M), N-methylscopolamine (3 X 10(-9) M) and methyl atropine (10(-8) M) also abolished this facilitatory effect of oxotremorine.     

Modulation of extracellular neurotransmitter levels in the nucleus accumbens by a taurine uptake inhibitor

Using in vivo microdialysis, we examined the effect of local perfusion of the taurine uptake inhibitor guanidinoethyl sulfonate on extracellular levels of various neurotransmitters in the rat nucleus accumbens. Guanidinoethyl sulfonate (500 microM-50 mM) produced a concentration-dependent increase in extracellular taurine levels. While 500 microM and 5 mM concentrations of guanidinoethyl sulfonate were largely without effect, 50 mM guanidinoethyl sulfonate produced a significant decrease in extracellular levels of aspartate, glutamate and glycine, with no effect on extracellular dopamine levels. These results indicate that guanidinoethyl sulfonate can modulate extracellular amino acid levels in the nucleus accumbens.     

Selective inhibition of excitatory amino acid-stimulated phosphoinositide hydrolysis in the rat hippocampus by activation of protein kinase C

The relative roles of protein kinase C in regulating excitatory amino acid-, cholinoceptor-, and adrenoceptor-stimulated phosphoinositide hydrolysis were studied. Slices of rat hippocampus were prelabeled with [3H]-myo-inositol, and agonist-induced [3H]-phosphoinositide hydrolysis was measured by the formation of [3H]-inositol monophosphate ([3H]-IP) in the presence of lithium ion. Activation of protein kinase C with phorbol 12,13-dibutyrate (PDB) (10(-6) M) completely inhibited ibotenate (IBO) (10(-3) M)-induced [3H]-phosphoinositide hydrolysis. Half-maximal inhibition was observed at about 10(-7) M PDB. Higher concentrations of PDB were required to inhibit stimulation of [3H]-IP by either carbachol (CARB) (10(-3) M) or norepinephrine (NE) (10(-4) M, and only partial inhibition could be attained. Preincubation with staurosporine (STAURO) (10(-5) M) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) (10(-4) M), inhibitors of protein kinase C, potentiated IBO- but not CARB- or NE-induced stimulation of [3H]-IP. PDB inhibition of IBO- or NE-stimulated [3H]-phosphoinositide hydrolysis was reversed by co-addition of STAURO or H-7. In the case of IBO + STAURO, this reversal was to the potentiated level observed with STAURO alone. Enhanced agonist stimulation and reversal of PDB inhibition were also produced by STAURO when [3H]-phosphoinositide hydrolysis was stimulated by either L-glutamate or quisqualate. These experiments show that direct activation of protein kinase C by PDB leads to inhibition of phosphoinositide hydrolysis mediated by excitatory amino acid receptors, cholinoceptors, or adrenoceptors. However, the enhanced agonist-stimulated phosphoinositide hydrolysis elicited by inhibitors of protein kinase C suggests that, when protein kinase C is indirectly activated, only excitatory amino acids rapidly inhibit further receptor-coupling.     

Involvement of dopaminergic system in the nucleus accumbens in the discriminative stimulus effects of phencyclidine

The effects of microinjection of phencyclidine (PCP) and dizocilpine, non-competitive NMDA receptor antagonists, and dopamine into the nucleus accumbens were examined in rats trained to discriminate PCP (1.5 mg/kg i.p.) from saline under a two-lever fixed ratio 20 schedule of food reinforcement. Microinjection of PCP (2-40 microg) and dizocilpine (2-12 microg) into the bilateral nucleus accumbens produced a dose-dependent increase in PCP-appropriate responding and fully substituted for systemically administered PCP, whereas microinjection of dopamine (1-4 microg) did not produce PCP-like discriminative stimulus effects. The performance of PCP discrimination was assessed after bilateral destruction of the dopaminergic nerve neurons in the nucleus accumbens with dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA, 4 microg/1 microl/side). The destruction of dopaminergic nerve neurons in the nucleus accumbens failed to prevent the performance of PCP discrimination. There was no difference in the average percentages of PCP-appropriate responding between vehicle and 6-OHDA-treated rats in the dose-response tests. These results suggest that the dopaminergic system in the nucleus accumbens does not play a critical role in the discriminative stimulus effects of PCP.     

Cocaine self-administration reduces excitatory responses in the mouse nucleus accumbens shell.

Drugs of abuse affect behavior by altering neuronal communication within the brain. Previous research examining the effects of intraperitoneally administered cocaine has revealed that cocaine alters excitatory glutamatergic signaling, both directly through regulation of synaptic function, and indirectly through regulation of cellular excitability in areas of the drug reward circuitry such as the nucleus accumbens (NAcc) and ventral tegmental area. We have now extended these findings by testing the hypothesis that self-administration of cocaine might elicit similar alterations in excitatory signaling in the NAcc shell. We observed that cocaine self-administration reduces synaptically evoked excitatory responses recorded extracellularly in the NAcc shell compared to saline self-administration. This alteration was not accompanied by alterations in paired pulse ratio of synaptically evoked responses or in potentiation of these responses by application of the adenylyl cyclase activator forskolin. This reduction in glutamatergic signaling may be one mechanism by which cocaine exerts its long-term behavioral effects.     

Sex differences and effects of cocaine on excitatory synapses in the nucleus accumbens

Human and animal studies indicate that drugs of abuse affect males and females differently, but the mechanism(s) underlying sex differences are unknown. The nucleus accumbens (NAc) is central in the neural circuitry of addiction and medium spiny neurons (MSNs) in the NAc show drug-induced changes in morphology and physiology including increased dendritic spine density. We previously showed in drug-na?ve rats that MSN dendritic spine density is higher in females than males. In this study, we investigated sex differences in the effects of cocaine on locomotor activity as well as MSN dendritic spine density and excitatory synaptic physiology in rats treated for 5 weeks followed by 17-21 days of abstinence. Females showed a greater locomotor response to cocaine and more robust behavioral sensitization than males. Spine density was also higher in females and, particularly in the core of the NAc, the magnitude of the cocaine-induced increase in spine density was greater in females. Interestingly, in cocaine-treated females but not males, cocaine-induced behavioral activation during treatment was correlated with spine density measured after treatment. Miniature EPSC (mEPSC) frequency in core MSNs also was higher in females, and increased with cocaine in both the core and shell of females more than males. We found no differences in mEPSC amplitude or paired-pulse ratio of evoked EPSCs, suggesting that sex differences and cocaine effects on mEPSC frequency reflect differences in excitatory synapse number per neuron rather than presynaptic release probability. These studies are the first to demonstrate structural and electrophysiological differences between males and females that may drive sex differences in addictive behavior.     

Interactions between CB1 cannabinoid and mu 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.     

Regulation of serotonin release by GABA and excitatory amino acids

Regulation of serotonin release by gamma-aminobutyric acid (GABA) and glutamate was examined by microdialysis in unanaesthetized rats. The GABA(A) receptor agonist muscimol, or the glutamate receptor agonists kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolaproprionate or N-methyl-D-aspartate were infused into the dorsal raphe nucleus (DRN) while extracellular serotonin was measured in the DRN and nucleus accumbens. Muscimol produced decreases, and the glutamate receptor agonists produced increases in serotonin. To determine if these receptors have a tonic influence on serotonergic neurons, glutamate or GABA(A) receptor antagonists were infused into the DRN. Kynurenate, a nonselective glutamate receptor blocker, produced a small, 30% decrease in serotonin. A similar decrease was obtained with combined infusion of AP-5 and DNQX into the DRN. The GABAA receptor blocker bicuculline produced an approximately three-fold increase in DRN serotonin. In conclusion, glutamate neurotransmitters have a weak tonic excitatory influence on serotonergic neurons in the rat DRN. However, the predominate influence is mediated by GABA(A) receptors.     

Inhibition of basal and stress-induced dopamine release in the cerebral cortex and nucleus accumbens of freely moving rats by the neurosteroid allopregnanolone

The neurosteroid allopregnanolone is a potent and efficacious modulator of γ-aminobutyric acid (GABA) type A receptors. The effects of intracerebroventricular injection of allopregnanolone (5 to 15 μg in 5 μl) on basal and stress-induced changes in the extracellular concentrations of dopamine were investigated by microdialysis in various brain areas of freely moving rats and compared with those of the benzodiazepine midazolam (1 to 10 μg in 5 μl). Allopregnanolone reduced (by a maximum of 65 to 75%) basal dopamine content in the prefrontal cortex and nucleus accumbens in a dose-dependent manner, but had no effect on dopamine output in the striatum. Allopregnanolone (10 to 15 μg) also completely prevented the increase in extracellular dopamine concentrations in the nucleus accumbens and cerebral cortex induced by foot-shock stress. Midazolam reduced basal dopamine content in all three brain regions studied as well as the stress- induced increase in dopamine content in the nucleus accumbens and cerebral cortex with a greater potency than allopregnanolone. These results suggest that endogenous neurosteroids may participate in the GABAergic modulation of dopaminergic transmission in the rat cerebral cortex and nucleus accumbens, two brain areas which are important in the regulation of emotional processes. These agents do not appear to affect striatal dopaminergic transmission which modulates motor function.     

Lack of tolerance to nicotine-induced dopamine release in the nucleus accumbens

The extent to which repeated administration produces tolerance to nicotine-induced increases in dopamine transmission in the nucleus accumbens was investigated in rats. In vivo microdialysis was used to sample extracellular dopamine and metabolites after a nicotine challenge (0.35 mg/kg) in (1) naive rats, (2) acutely pretreated rats (1 prior nicotine injection), and (3) chronically pretreated rats (12-15 prior daily nicotine injections, 0.35 mg/kg per injection). Nicotine increased extracellular DA and its metabolites, and these increases were not significantly altered by either acute or chronic prior exposure to the drug. The failure to find evidence of tolerance is compatible with the hypothesis that the mesolimbic dopaminergic system is a substrate for the reinforcing properties of chronically administered nicotine.     

Pentylenetetrazol-kindling modulates stimulated dopamine release in the nucleus accumbens of rats

Kindling-induced activation of dopaminergic neurones in the nucleus accumbens in pentylenetetrazol (PTZ)-kindled rats was studied using microdialysis. Dopamine (DA) release after PTZ challenge was measured: (1) two weeks and (2) ten weeks after kindling completion and (3) two weeks after a kindling procedure with diazepam (DZP) treatment. In (1) a significant increase in DA concentration was found after PTZ challenge and this increase was still evident 10 weeks after kindling completion (2). Coadministration of DZP in the course of kindling development inhibited the increase in sensitivity of the accumbens dopaminergic system (3).     

Opposite effects of inhibitory and excitatory neurosteroids on [3H]dopamine release from rat nucleus accumbens.

Neurosteroids with GABAA receptor antagonistic properties increase K(+)-evoked [3H]dopamine release from rat nucleus accumbens slices, whereas neurosteroid positive modulators of GABAA exert an opposite effect.     

The release and uptake of excitatory amino acids

In this article, David Nicholls and David Attwell describe recent advances in our understanding of the mechanisms by which excitatory amino acids are released from cells, and of the way in which a low extracellular glutamate concentration is maintained. Glutamate can be released from cells by two mechanism: either by Ca2(+)-dependent vesicular release or, in pathological conditions, by reversal of the plasma membrane uptake carrier. The contrasting pharmacology and ionic dependence of the glutamate uptake carriers in the vesicle membrane and in the plasma membrane explain how glutamate (but probably not aspartate) can function as a neurotransmitter, and why the extracellular glutamate concentration rises to neurotoxic levels in brain anoxia.     

Basal and feeding-evoked dopamine release in the rat nucleus accumbens is depressed by leptin

The involvement of the satiety-controlling hormone leptin in the modulation of the reward-associated dopamine release was investigated by monitoring the extracellular dopamine concentration in microdialysates from the nucleus accumbens of rats during feeding after infusion of leptin or artificial cerebrospinal fluid into the lateral ventricle of rats. Leptin suppressed the basal as well as the feeding-evoked extracellular dopamine concentration and reduced the amount and duration of food intake compared to the pair-feed vehicle-treated controls. These results suggest that leptin is involved in the dopaminergic modulation of feeding-induced rewarding functions.     

Inhibition of calcium channels by opioid- and adenosine-receptor agonists in neurons of the nucleus accumbens

The pharmacological effects of opioid- and adenosine-receptor agonists on neural signalling were investigated by measuring drug actions on barium current flowing through calcium channels in acutely-dissociated neurons of the rat nucleus accumbens (NAc). Under whole-cell voltage clamp, opioids acted via mu, but not delta or kappa, receptors to partially inhibit barium current. Mean inhibition was 35+/-2% (+/-s.e.mean, n = 33) for methionine-enkephalin and 37+/-1% (n = 65) for the selective mu receptor agonist DAMGO, both measured at saturating agonist concentrations in neurons with diameter > or = 20 microm. EC(50) for DAMGO was 100 nM. Perfusion of naloxone reversed the current inhibition by DAMGO. Adenosine also partially inhibited barium current in these neurons. Mean inhibition was 28+/-2% (n = 29) for adenosine and 33+/-3% (n = 27) for the selective A1 receptor agonist N(6)CPA, both at saturating concentrations in neurons with diameter > or = 20 microm. EC(50) for N(6)CPA was 34 nM. Adenosine inhibition was reversed by perfusion of an A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, while the selective A2A receptor agonist, CGS 21680, had no effect. Inhibition by opioids and adenosine was mutually occlusive, suggesting a converging pathway onto calcium channels. These actions involved a G-protein-coupled mechanism, as demonstrated by the partial relief of inhibition by strong depolarization and by the application of N-ethylmaleimide or GTP-gamma-S. Inhibition of barium current by opioids had their greatest effect in large neurons, that is, in presumed interneurons. In contrast, opioid inhibition in neurons with diameter < or = 15 microm was 11+/-2% (n = 26) for methionine-enkephalin and 11+/-4% (n = 17) for DAMGO, both measured at saturating agonist concentrations. Adenosine inhibition in neurons with diameter < or = 15 microm was 22+/-5% (n = 9). These results implicate the interneurons as a locus for the modulation of the excitability of projection neurons in the NAc during the processes of addiction and withdrawal.     

Dopamine and serotonin release in the nucleus accumbens during starvation-induced hyperactivity

Activity-based anorexia (ABA) is considered an animal model for anorexia nervosa (AN). By scheduled feeding and voluntary wheel running, it mimics severe body weight loss and increased physical activity in AN. Pharmacological, genetic and imaging studies implicate dopamine and serotonin in the regulation of feeding behavior, food-anticipatory activity, and food reward. Previous studies propose that the nucleus accumbens (NAc) plays an important role in these food-related processes. Here we determined dopamine and serotonin levels in the NAc upon exposure to the ABA model. Surprisingly, the release of dopamine and serotonin in the NAc were not increased during the initiation of food-anticipatory behavior in ABA rats. Dopamine release in the NAc was increased during feeding behavior in ABA rats. During ABA, levels of serotonin were low and circadian activity is blunted. We conclude that during the early stages of development of food-anticipatory activity, increased dopamine does not trigger hyperactivity.     

Effects of excitatory amino acids on locomotor activity after bilateral microinjection into the rat nucleus accumbens: Possible dependence on dopaminergic mechanisms

In order to study the role of excitatory amino acids on motor function, the effects of kainic, quisqualic, and $\text{N-}\text{methyl}\text{-}\text{dl}\text{-}\text{aspartic}$ acids on locomotor activity were determined after their direct injection into the nucleus accumbens. These three amino acids have been used in previous studies to classify receptors for excitatory amino acids in the mammalian spinal cord. After injection into the nucleus accumbens all three amino acids stimulated locomotor activity, with kainic acid being the most potent and $\text{N-}\text{methyl}\text{-}\text{dl}\text{-}\text{aspartic}$ acid the least potent. The maximum intensity of the stimulation produced by kainic and quisqualic acids was greater than that produced by $\text{N-}\text{methyl}\text{-}\text{dl}\text{-}\text{aspartic}$ acid. These results suggest that receptors in the nucleus accumbens, sensitive to kainic and quisqualic acids, play a more important role in the stimulation of locomotor activity than those sensitive to $\text{N-}\text{methyl}\text{-}\text{dl}\text{-}\text{aspartic}$ acid. In addition to the above amino acids, the administration of large doses of l-aspartic and d-glutamic acids also produced hyperactivity, while l-glutamic acid had no effect. To determine whether endogenous dopamine mediates the hypermotility produced by the excitatory amino acids, the response to these amino acids was studied after treatment with reserpine or dopamine receptor blocking agents. Reserpine (5 mg/kg, i.p.), haloperidol (0.8 mg/kg, i.p.) or fluphenazine [5.0 μg (total dose) injected into the nucleus accumbens] markedly attenuated the hypermotility induced by excitatory amino acids. These results suggest that the hypermotility produced by excitatory amino acids is mediated through release of dopamine and the subsequent stimulation of dopamine receptors in the nucleus accumbens.