Reverse microdialysis of a dopamine D2 receptor antagonist alters extracellular adenosine levels in the rat nucleus accumbens

Recent evidence suggests that modulation of dopaminergic transmission alters striatal levels of extracellular adenosine. The present study used reverse microdialysis of the selective dopamine D₂ receptor antagonist raclopride to investigate whether a blockade of dopamine D₂ receptors modifies extracellular adenosine concentrations in the nucleus accumbens. Results reveal that perfusion of raclopride produced an increase of dialysate adenosine which was significant with a high (10 mM) and intermediate (1 mM) drug concentration, but not with lower drug concentrations (10 and 100 μM). Thus, the present study demonstrates that a selective blockade of dopamine D₂ receptors in the nucleus accumbens produced a pronounced increase of extracellular adenosine. The cellular mechanisms underlying this effect are yet unknown. It is suggested that the increase of extracellular adenosine might be related to a homeostatic modulatory mechanism proposed to be a key function of adenosine in response to neuronal metabolic challenges.     

Serotonin-mediated increases in the extracellular levels of beta-endorphin in the arcuate nucleus and nucleus accumbens: a microdialysis study

Although the involvement of both endogenous opioid and serotonergic systems in modulation of pain and emotion was suggested, the neurochemical interaction between these systems in the brain has not previously been studied directly. Herein, the effects of the local application of serotonin (5-HT) and fluoxetine (a 5-HT reuptake inhibitor) on extracellular levels of beta-endorphin in the arcuate nucleus and nucleus accumbens were assessed in freely moving rats using in vivo microdialysis. The mean basal concentrations of beta-endorphin in dialysates obtained from the arcuate nucleus and nucleus accumbens were 259.9 and 143.3 pM, respectively. Specific lesion of the serotonergic system by 5,7-dihydroxytryptamine (5,7-DHT) caused a significant decrease in these dialysate beta-endorphin levels. When 5-HT (0.25-5 microM) was added to the perfusion solution, the levels of beta-endorphin in the dialysate from the arcuate nucleus increased (186-296% of baseline), in a concentration-dependent manner. In the nucleus accumbens, 0.5 and 2 microM 5-HT in the perfusion fluid did not affect the levels of beta-endorphin in the dialysate, whereas 5 and 10 microM 5-HT caused an increase of approximately 190% of baseline. When fluoxetine (250 microM) was present in the perfusing solution, the levels of beta-endorphin in the dialysates from the arcuate nucleus and nucleus accumbens increased two- to threefold. This effect was not obtained in the 5,7-DHT-lesioned rats. Thus, 5-HT, either endogenously or exogenously delivered, appears to facilitate the release of beta-endorphin in the arcuate nucleus and nucleus accumbens. This indication of an interaction between serotonergic and endorphinic systems may be relevant for assessing pain and mood disorder circuits and the mode of action of antidepressant drugs.     

Long-lasting up-regulation of orexin receptor type 2 protein levels in the rat nucleus accumbens after chronic cocaine administration

Hypothalamic orexin (hypocretin) neurons project to the key structures of the limbic system and orexin receptors, both orexin receptor type 1 (OXR1) and type 2 (OXR2), are expressed in most limbic regions. Emerging evidence suggests that orexin is among important neurotransmitters that regulate addictive properties of drugs of abuse. In this study, we examined the effect of psychostimulant cocaine on orexin receptor protein abundance in the rat limbic system in vivo. Intermittent administration of cocaine (20 mg/kg, i.p., once daily for 5 days) caused a typical behavioral sensitization response to a challenge cocaine injection at a 14-day withdrawal period. Repeated cocaine administration at the same withdrawal time also increased OXR2 protein levels in the nucleus accumbens while repeated cocaine had no effect on OXR1 and orexin neuropeptide (both orexin-A and orexin-B) levels in this region. In contrast to the nucleus accumbens, OXR2 levels in the frontal cortex, the ventral tegmental area, the hippocampus, and the dorsal striatum (caudate putamen) were not altered by cocaine. Remarkably, the up-regulated OXR2 levels in the nucleus accumbens showed a long-lasting nature as it persisted up to 60 days after the discontinuation of repeated cocaine treatments. In contrast to chronic cocaine administration, an acute cocaine injection was insufficient to modify levels of any orexin receptor and peptide. Our data identify the up-regulation of OXR2 in the nucleus accumbens as an enduring molecular event that is correlated well with behavioral plasticity in response to chronic psychostimulant administration. This OXR2 up-regulation may reflect a key adaptation of limbic orexinergic transmission to chronic drug exposure and may thus be critical for the expression of motor plasticity.     

Anthranilic sulfonamide CCK1/CCK2 dual receptor antagonists II: Tuning of receptor selectivity and in vivo efficacy

In the previous article we demonstrated how certain CCK2R-selective anthranilic amides could be structurally modified to afford high-affinity, selective CCK1R activity. We now describe our efforts at modulating and optimizing the CCK1R and CCK2R affinities aimed at producing compounds with good pharmacokinetics properties and in vivo efficacy in rat models of gastric acid and pancreatic amylase secretion.     

Neurotensin concentrations in rat striatum and nucleus accumbens: Further studies of their regulation

The possible influence of cholinergic and dopaminergic mechanisms on neurotensin-containing neurones was examined at two different levels; nucleus accumbens and striatum in the rat brain. The acute treatment with the anticholinergic drugs atropine and scopolamine increased neurotensin concentrations in the striatum and, in the former case, also in the nucleus accumbens. Subchronic administration of atropine resulted in tolerance to its neurotensin-elevating action within the accumbens, but not within the striatum. Combined treatment with submaximal doses of haloperidol and atropine resulted in increases in neurotensin content which were greater than those seen with either agent alone. This was true regardless of whether the drugs were administered acutely or subchronically. This observation demonstrated that the tolerance phenomena occurring after subchronic elozapine and fluperlapine were not attributable to their anticholinergic activity. The control of striatal and accumbal neurotensin content by antidopaminergic and anticholinergic drugs seemed to be quite specific: drugs with actions on noradrenergic, serotoninergic, GABA-ergic and opiate systems did not influence the neurotensin content in these two structures. Preliminary studies on the effects of haloperidol on neurotensin release from striatal slices in vitro and that of cycloheximide on haloperidol's effect in vivo, suggest a possible inhibitory action of dopamine receptor blockade on neurotensin release.     

A chronic stress that impairs reactivity in rats also decreases dopaminergic transmission in the nucleus accumbens: a microdialysis study

Chronic stress induces in rats a decreased reactivity toward noxious stimuli (escape deficit), which can be reverted by antidepressant treatments. The present study reports that this condition of behavioral deficit is accompanied by a decreased level of extracellular dopamine in the nucleus accumbens shell. To assess whether this finding was the result of a decreased release or of an enhanced removal of dopamine, we acutely administered cocaine, and 2 h later d-amphetamine, to stressed and control rats. The increases in dopamine output observed in stressed animals after cocaine administration were significantly lower than those observed in control rats; whereas the total amount of dopamine released after d-amphetamine administration was similar in both groups of rats. These data suggest a reduced activity of dopaminergic neurons as the possible mechanism underlying dopamine basal level reduction in stressed animals. It is interesting that the stress group showed a locomotor response to cocaine not different from control rats, thus suggesting a condition of sensitization to dopamine receptor stimulation. Imipramine administered daily concomitantly with stress exposure completely reverted the escape deficit condition of chronically stressed rats. Moreover, stressed rats treated with imipramine showed basal and cocaine stimulated levels of extraneuronal dopamine similar to those observed in control animals.     

Anthranilic sulfonamide CCK1/CCK2 dual receptor antagonists I: Discovery of CCKR1 selectivity in a previously CCKR2-selective lead series

A series of CCK2R-selective anthranilic amides is shown to derive CCK1R affinity via selective substitution of the amide side chain. Thus, extending the length of the original benzamide side chain by a single methylene unit imparts CCK1R affinity to the series, and further fine tuning of the affinity results in CCK1R selectivity of greater than 100-fold.     

Serotonin-2C antagonism augments the effect of citalopram on serotonin and dopamine levels in the ventral tegmental area and nucleus accumbens

Many patients with major depression do not respond to selective serotonin reuptake inhibitors (SSRIs). Lack of response could be due to inhibition of dopamine (DA) release by serotonin (5-HT) through 5-HT_2C receptors. Combining an SSRI with a 5-HT_2C antagonist may result in improved efficacy by causing simultaneous increases of 5-HT and DA. In order to test this augmentation strategy, male Wistar rats were treated (s.c.) with an acute dose of the SSRI citalopram (Cit, 5 mg/kg), the 5-HT_2C antagonist SB 242084 (SB, 2 mg/kg), or Cit + SB, and the effect on 5-HT and DA release in the nucleus accumbens (NAcc) was assessed by microdialysis. In a separate experiment, animals were treated with vehicle, Cit (20 mg/kg/d), SB (2 mg/kg/d) or Cit + SB for a period of 2 days (s.c.), and the impact on the release of 5-HT and DA in the ventral tegmental area (VTA) and NAcc was studied. On the day of microdialysis, 5-HT_2C receptor sensitivity was assessed with an SB challenge. Acutely administered Cit + SB increased 5-HT release in the NAcc more than Cit alone. SB alone increased DA release in the NAcc (not in the VTA), but when administered together with Cit, this effect was abolished. A 2-day treatment with Cit or Cit + SB increased 5-HT release in both VTA and NAcc. Combining Cit with SB augmented the effect of Cit in the VTA. DA release in VTA and NAcc was only significantly increased after 2-days of treatment with Cit + SB. In conclusion, Cit + SB had synergistic effects on 5-HT and DA release after 2-days of treatment, probably related to a decreased tonic inhibition of DA release via 5-HT_2C receptors. Regional differences occur and future studies should elucidate if this augmentation strategy is beneficial at the behavioral level.     

Differential effects of galanin and neuropeptide Y on extracellular norepinephrine levels in the paraventricular hypothalamic nucleus of the rat: a microdialysis study.

Evidence suggests that the peptides galanin (GAL) and neuropeptide Y (NPY) interact with the amine norepinephrine (NE) in the hypothalamic paraventricular nucleus (PVN) to stimulate feeding behavior. To directly investigate the nature of these interactions, extracellular levels of PVN NE were monitored in freely-moving rats using the microdialysis/HPLC technique. Following PVN administration of GAL (0.3 nmol), NPY (78 pmol) or Ringer's solution, local NE levels were measured at 20-min intervals for 2 hrs postinjection, under two feeding conditions, namely, in the presence or absence of food. The results demonstrate different effects of these peptides on endogenous NE levels. Following GAL administration, PVN NE levels were enhanced by 80 to 90%, up to 40 min postinjection, independent of food availability. In contrast, following NPY injection, NE levels were significantly reduced 20 min postinjection with food absent, and when food was available, NE levels tended to be enhanced. These results, consistent with pharmacological and biochemical studies, reveal different patterns of peptide-amine interactions in the PVN.     

Okadaic acid: a new probe for the study of cellular regulation

The tumour promoter okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A. Here we review recent studies which demonstrate that this toxin is extremely useful for identifying biological processes that are controlled through the reversible phosphorylation of proteins.     

NMDA-mediated release of glutamate and GABA in the subthalamic nucleus is mediated by dopamine: an in vivo microdialysis study in rats

The present study investigated the effects of N-methyl-D-aspartic acid.H2O (NMDA) on the dopamine, glutamate and GABA release in the subthalamic nucleus (STN) by using in vivo microdialysis in rats. NMDA (100 micromol/L) perfused through the microdialysis probe evoked an increase in extracellular dopamine in the STN of the intact rat of about 170%. This coincided with significant increases in both extracellular glutamate (350%) and GABA (250%). The effect of NMDA perfusion on neurotransmitter release at the level of the STN was completely abolished by co-perfusion of the selective NMDA-receptor antagonist MK-801 (10 micromol/L), whereas subthalamic perfusion of MK-801 alone had no effect on extracellular neurotransmitter concentrations. Furthermore, NMDA induced increases in glutamate were abolished by both SCH23390 (8 micromol/L), a selective D1 antagonist, and remoxipride (4 micromol/L), a selective D2 antagonist. The NMDA induced increase in GABA was abolished by remoxipride but not by SCH23390. Perfusion of the STN with SCH23390 or remoxipride alone had no effect on extracellular neurotransmitter concentrations. The observed effects in intact animals depend on the nigral dopaminergic innervation, as dopamine denervation, by means of 6-hydroxydopamine lesioning of the substantia nigra, clearly abolished the effects of NMDA on neurotransmitter release at the level of the STN. Our work points to a complex interaction between dopamine, glutamate and GABA with a crucial role for dopamine at the level of the STN.     

Central integration of cardiovascular and drinking responses elicited by central administration of angiotensin II: divergence of regulation by the ventral tegmental area and nucleus accumbens

Previous studies had implicated the involvement of the ventral tegmental area and its dopamine projections to the nucleus accumbens in goal-directed behavior. This study investigated whether or not the GABAergic inputs to the ventral tegmental area and, in turn, dopaminergic input to the nucleus accumbens from the ventral tegmental area modify drinking and cardiovascular responses elicited by central administration of angiotensin II. Injections of 25 ng of angiotensin II into a lateral cerebral ventricle of the rat elicited water intakes averaging 7-8 mL in 15 min with latencies usually less than 3 min. Pretreatment of the nucleus accumbens with spiperone, a dopamine antagonist, or the ventral tegmental area with gamma-amino butyric acid (GABA) produced dose-dependent reductions in water intake and number of laps taken while increasing the latency to drink. The spiperone injection did not alter the pressor response. On the other hand, the GABA injections attenuated the pressor responses to central angiotensin II administration. These findings suggest that GABA input to the ventral tegmental area modifies both the cardiovascular and drinking responses elicited following central administration of angiotensin II. However, the dopamine projections to the nucleus accumbens appear to be involved only in the drinking responses elicited by central injections of angiotensin II. Divergence for the coordination of the skeletal motor behavioral component and the cardiovascular component elicited by central administration of angiotensin II must occur before the involvement of these dopamine pathways.     

Serotonin 5-HT(2) receptor stimulation of dopamine release in the posterior but not anterior nucleus accumbens of the rat

The objective of the present study was to examine the involvement of serotonin 5-HT(2) receptors within the rat nucleus accumbens (Acc) in the regulation of dopamine (DA) release using in vivo microdialysis. Perfusion with the 5-HT(2) agonist (+)-1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), at concentrations of 25-250 microM, through microdialysis probes located in the posterior Acc increased extracellular DA levels to a maximum of 200% of baseline. DOI-induced increases in the extracellular levels of DA were Ca(2+) dependent and were inhibited by co-perfusion with the 5-HT(2) antagonist LY-53,857. DOI enhancement of the extracellular concentrations of DA was observed when probes were implanted in the Acc core and shell regions posterior to anteroposterior +1.2 mm from bregma, whereas a small reduction in the extracellular levels of DA was observed in the anterior Acc. There were no differences between core and shell subdivisions within either the anterior or the posterior Acc. These results suggest that activation of 5-HT(2) receptors within the posterior, but not anterior, Acc stimulates DA release, indicating rostral-caudal differences in the interactions of 5-HT with DA systems in the Acc.     

Evidence for the dual coupling of the rat neurotensin receptor with pertussis toxin-sensitive and insensitive G-proteins

We previously demonstrated the functional coupling of the rat neurotensin receptor NTS1 with G-proteins on transfected CHO cell homogenates by showing modulation of agonist affinity by guanylyl nucleotides and agonist-mediated stimulation of [(35)S]GTP gamma S binding. In the present study, we observed that G(i/o)-type G-protein inactivation by pertussis toxin (PTx) resulted in a dramatic reduction of the NT-induced [(35)S]GTP gamma S binding whereas the effect of guanylyl nucleotide was almost not affected. As expected, NT-mediated phosphoinositide hydrolysis and intracellular calcium mobilization were not altered after PTx treatment. This suggests the existence of multiple signaling cascades activated by NT. Accordingly, using PTx and the PLC inhibitor U-73122, we showed that both signaling pathways contribute to the NT-mediated production of arachidonic acid. These results support evidence for a dual coupling of the NTS1 with PTx-sensitive and insensitive G-proteins.     

Evidence for alpha-1 adrenergic receptor regulation of atriopeptin release from the isolated rat heart

Atrial myocardium is the source of a recently described peptide hormone termed atriopeptin. Atriopeptin is thought to have a role in the regulation of systemic arterial pressure, fluid balance and plasma electrolyte homeostasis. Isolated rat hearts release atriopeptin into the coronary effluent, and we have found that this release is stimulated by the administration of norepinephrine, a compound with alpha and beta adrenergic properties. Infusion of the pure beta-receptor agonist, isoproterenol, failed to stimulate the release; however, the alpha-1 receptor agonist phenylephrine induced the release in a dose-dependent manner. The stimulation of atriopeptin release by norepinephrine and phenylephrine was inhibited by alpha-blockade with phentolamine. Administration of BHT-920, a selective alpha-2 agonist, had no effect on atriopeptin release. We conclude that atriopeptin secretion by the atrial myocyte is stimulated by activation of the alpha-1 adrenergic receptor. This finding suggests an involvement of the sympathetic nervous system in the physiologic regulation of the secretion of this hormone.     

High temporal resolution of amino acid levels in rat nucleus accumbens during operant ethanol self-administration: involvement of elevated glycine in anticipation

Capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF) provides 15-s temporal resolution of amino acid levels in microdialysate, which, for the first time, allows almost real time measurement of changes during episodes of behavior. We trained Sprague-Dawley rats to self-administer either 10% ethanol-containing gelatin or non-alcoholic gelatin in a typical operant chamber. After rats reached stable daily levels of responding, microdialysis probes were inserted into nucleus accumbens and samples were collected before, during and after operant sessions with on-line analysis via CE-LIF. During the first 15 min of the operant session, there was a significant increase in taurine that correlated with the amount of ethanol consumed ( R ² = 0.81) but no change in rats responding for plain gel. There were large, consistent increases in glycine in both the ethanol and plain gel groups which correlated with the amount of gel consumed. A smaller increase was observed in rats with free non-operant access to plain gel compared to the increase seen with the same amount of gel consumed under operant conditions. When rats were given a time out after each delivery of gel in the operant protocol, the greatest increase of glycine was obtained with the longest time out period. Thus, increases in glycine in nucleus accumbens appear to be related to anticipation of reinforcement.     

In vivo characterization of basal amino acid levels in subregions of the rat nucleus accumbens: effect of a dopamine D(3)/D(2) agonist.

Recent evidence demonstrates that two subdivisions of the nucleus accumbens, the dorsolateral core and the ventromedial shell can be distinguished by morphological, immunohistochemical and chemoarchitectural differences. In the present study, we measured basal levels of amino acids in microdialysates from both the shell and core subterritories of the nucleus accumbens in freely moving rats using HPLC with fluorescence detection. The effect of the dopamine D(3)/D(2) receptor agonist quinelorane (30 microg/kg s.c.) was then investigated in both subregions. With the exception of glutamate, histidine, and serine, which showed similar levels in both subterritories, alanine, arginine, aspartate, gamma-aminobutyric acid, glutamine, and tyrosine were significantly higher in the shell compared with the core. In contrast, taurine levels were significantly lower in the shell than in the core. A particularly striking difference across subregions of the nucleus accumbens was observed for basal GABA levels with a shell/core ratio of 18.5. Among all the amino acids investigated in the present study, quinelorane selectively decreased dialysate GABA levels in the core subregion of the nucleus accumbens. The results of the present study point to specific profiles of both shell and core in terms of: (1) basal chemical neuroanatomical markers for amino acids; and (2) GABAergic response to the DA D(3)/D(2) agonist quinelorane.     

Dopamine D1-D2 receptor heteromer in dual phenotype GABA/glutamate-coexpressing striatal medium spiny neurons: regulation of BDNF, GAD67 and VGLUT1/2

In basal ganglia a significant subset of GABAergic medium spiny neurons (MSNs) coexpress D1 and D2 receptors (D1R and D2R) along with the neuropeptides dynorphin (DYN) and enkephalin (ENK). These coexpressing neurons have been recently shown to have a region-specific distribution throughout the mesolimbic and basal ganglia circuits. While the functional relevance of these MSNs remains relatively unexplored, they have been shown to exhibit the unique property of expressing the dopamine D1-D2 receptor heteromer, a novel receptor complex with distinct pharmacology and cell signaling properties. Here we showed that MSNs coexpressing the D1R and D2R also exhibited a dual GABA/glutamate phenotype. Activation of the D1R-D2R heteromer in these neurons resulted in the simultaneous, but differential regulation of proteins involved in GABA and glutamate production or vesicular uptake in the nucleus accumbens (NAc), ventral tegmental area (VTA), caudate putamen and substantia nigra (SN). Additionally, activation of the D1R-D2R heteromer in NAc shell, but not NAc core, differentially altered protein expression in VTA and SN, regions rich in dopamine cell bodies. The identification of a MSN with dual inhibitory and excitatory intrinsic functions provides new insights into the neuroanatomy of the basal ganglia and demonstrates a novel source of glutamate in this circuit. Furthermore, the demonstration of a dopamine receptor complex with the potential to differentially regulate the expression of proteins directly involved in GABAergic inhibitory or glutamatergic excitatory activation in VTA and SN may potentially provide new insights into the regulation of dopamine neuron activity. This could have broad implications in understanding how dysregulation of neurotransmission within basal ganglia contributes to dopamine neuronal dysfunction.