Modulation of feeding behaviour by blocking purinergic receptors in the rat nucleus accumbens: a combined microdialysis, electroencephalographic and behavioural study

The nonspecific P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), the nonspecific P1 receptor antagonist 8-(p-sulphophenyl)-theophylline (8-SPT) and the combination of both were applied by retrograde microdialysis into the nucleus accumbens (NAc) before and during feeding of 18-h food-deprived rats. In addition to the registration of behavioural parameters, such as the amount and duration of food intake, the feeding-induced changes in dopamine (DA) concentration and the concomitant changes of neuronal activity in the NAc and the ventral tegmental area (VTA) were simultaneously determined. The perfusion with PPADS (20 microm) diminished the amount of food intake and the duration of feeding. Furthermore, the P2 receptor antagonist blocked the feeding-induced DA release and prevented the feeding-elicited changes of the electroencephalography (EEG) power distribution which was characterised by an increase in the power of the 8.0-13.0-Hz frequency band in the NAc and the VTA. The effects of PPADS could be completely prevented by the concomitantly perfused adenosine receptor antagonist 8-SPT (100 microm). When given alone, 8-SPT increased the amount of food ingested, the duration of feeding and the EEG power of the higher frequency range, particularly between 19.0 and 30.0 Hz, in both the NAc and the VTA. The feeding-elicited DA release was supplemented to the enhanced DA level caused by the perfusion with 8-SPT in an additive manner. The P2 and P1 receptor antagonists interact antagonistically in the modulation of feeding behaviour and the feeding-induced changes of EEG activity suggesting that both endogenous extracellular ATP and adenosine are involved in the regulation of the feeding-associated mesolimbic neuronal activity in a functionally antagonistic manner.     

Mechanisms of adenosine 5'-triphosphate-induced dopamine release in the rat nucleus accumbens in vivo

The endogenous mechanisms modulating ATP-induced dopamine release in the nucleus accumbens (NAc) were studied by microdialysis in freely moving rats. The ATP analog 2-Methylthio ATP (2-MeSATP) facilitated the release of dopamine in a manner sensitive to pertussis toxin and tetrodotoxin. It is suggested that G-protein-coupled P2Y receptors and voltage-gated sodium channels are involved in this process. N-methyl-D-aspartate (NMDA) applied in a concentration of 100 microM decreased the extracellular dopamine level, whereas 1 and 10 mM NMDA enhanced it. The endogenous agonist glutamate (10 microM) inhibited the basal and facilitated release of dopamine. Infusion with a combination of the ionotropic glutamate receptor antagonists (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), as well as with the metabotropic glutamate receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG) increased the basal level of dopamine and potentiated the 2-MeSATP-facilitated dopamine release, suggesting an ATP-mediated glutamate release. The GABA(A) receptor antagonist bicuculline infused into the NAc also enhanced the basal level of dopamine; however, the application of 2-MeSATP in the presence of bicuculline caused an early decrease and a subsequent increase of dopamine release. The facilitatory phase of the 2-MeSATP effect was comparable with that measured in the absence of bicuculline. By contrast, when bicuculline was infused into the ventral tegmental area (VTA) it elevated the accumbal basal dopamine level and in addition facilitated the 2-MeSATP- and the glutamate-induced dopamine release above that measured in the absence of bicuculline. These results suggest that ATP in the NAc has a physiologically relevant function in modulating dopaminergic transmission depending on the mesolimbic neuronal activity. The first component of the ATP effect involves a direct stimulation of the terminals of VTA neurons, while the second inhibitory component involves a sequential activation of glutamate and, finally, via ionotropic and metabotropic glutamate receptors, of GABA neurons projecting to the VTA.     

Purinergic modulation of extracellular glutamate levels in the nucleus accumbens in vivo

In the present study, the P2 receptor-mediated modulation of the extracellular glutamate concentration was investigated by microdialysis in the nucleus accumbens (NAc) of freely moving rats. Because of the known interference of dopaminergic and glutamatergic mechanisms in this area the experiments were performed with animals intra-accumbally treated with 6-hydroxydopamine (6-OHDA) to deplete dopamine pools. Perfusion of the NAc with the prototypic P2 receptor agonist 2-methylthioadenosine 5'-triphosphate (2-MeSATP, 0.1, 1 and 10mM) concentration-dependently increased the extracellular level of glutamate in this area. Pretreatment with the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 0.1mM) decreased the basal extracellular glutamate concentration and inhibited the 2-MeSATP-induced outflow of glutamate. In rats treated with 6-OHDA, 2-MeSATP increased the total extracellular glutamate to an extent about fivefold larger than in sham-lesioned rats. The perfusion of the dopamine-depleted NAc with the D(2)/D(3) dopamine receptor agonist quinpirole (0.1mM) diminished the basal concentration of glutamate and reduced the effect of 2-MeSATP on the extracellular glutamate. These results provide evidence that the stimulation of P2 receptors is involved in the increase of accumbal extracellular glutamate in vivo. This behaviourally relevant mechanism depends on a dopamine D(2) receptor-mediated tone in the nucleus accumbens. Furthermore, the inhibition of P2 receptors may prevent, at least partly, glutamate-mediated neurodegeneration.     

Differential activation of subtype purinergic receptors modulates Ca(2+) mobilization and COX-2 in human microglia

We have studied modulation of purinergic receptors (P(2Y) and P(2X) subtypes) on changes in intracellular Ca(2+) [Ca(2+)](i) and expression and production of COX-2 in human microglia. Measurements using Ca(2+)-sensitive spectrofluorometry showed adenosine triphosphate (ATP) to cause rapid transient increases in [Ca(2+)](i). Application of ATP plus the P(2X) antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), or treatment with adenosine diphosphate-beta-S (ADP-beta-S), a selective P(2Y) agonist, led to a considerable prolongation in [Ca(2+)](i) responses compared with ATP. The prolonged time courses were consistent with sustained activation of store-operated channels (SOC) since SKF96365, an inhibitor of SOC, blocked this component of the response. RT-PCR data showed that microglia expressed no COX-2 either constitutively or following treatment of cells with ATP (100 microM for 8 h). However, treatment using ATP plus PPADS or with ADP-beta-S led to marked expression of COX-2. The enhanced COX-2 with ATP plus PPADS treatment was absent in the presence of SKF96365 or using Ca(2+)-free solution. Immunocytochemistry, using a specific anti-COX-2 antibody, also revealed a pattern of purinergic modulation whereby lack of P(2X) activation enhanced the production of COX-2 protein. These results suggest that modulation of subtypes of purinergic receptors regulates COX-2 in human microglia with a link involving SOC-mediated influx of Ca(2+).     

Facilitation by P(2) receptor activation of acetylcholine release from rat motor nerve terminals: interaction with presynaptic nicotinic receptors

ATP is released from motor nerve endings together with acetylcholine. Released adenine nucleotides can be extracellularly metabolized into adenosine, which is a presynaptic neuromodulator at neuromuscular junctions, but it is not known if P(2) receptor activation also modulates acetylcholine release from mature motor nerve endings. We now tested the effect of a stable ATP analogue, beta,gamma-imido ATP on the nerve-evoked release of acetylcholine from adult rat hemidiaphragm preparations. beta,gamma-Imido ATP (10-100 microM) facilitated in a concentration-dependent manner evoked acetylcholine release, and 30 microM beta,gamma-imido ATP caused a 125% facilitation of evoked acetylcholine release. This facilitatory effect of beta,gamma-imido ATP (30 microM) was abolished by the P(2) receptor antagonists, suramin (100 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 10 microM), but not by the A(1) or A(2A) adenosine receptor antagonists, 1,3-dipropyl-8-cyclopentylxanthine (50 nM) and ZM 241385 (50 nM), respectively. The facilitation of acetylcholine release by beta, gamma-imido ATP (30 microM) was also prevented by the nicotinic acetylcholine receptor antagonist, D-tubocurarine (1 microM) and the facilitatory effect (40%) of the nicotinic acetylcholine receptor agonist, 1,1-dimethyl-4-phenylpiperazinium (1 microM) was abolished by PPADS (10 microM). These results demonstrate a presynaptic facilitatory effect of P(2) receptor activation at the rat phrenic nerve endings, which is tightly coupled with the presynaptic nicotinic autofacilitatory system.     

Laterodorsal tegmental stimulation elicits dopamine efflux in the rat nucleus accumbens by activation of acetylcholine and glutamate receptors in the ventral tegmental area

Cholinergic and glutamatergic neurons in the laterodorsal tegmentum (LDT) and neighbouring mesopontine nuclei are thought to influence mesolimbic dopaminergic neuronal activity involved in goal-directed behaviours. We measured the changes in dopamine oxidation current (corresponding with dopamine efflux) in the nucleus accumbens (NAc) in response to electrical stimulation of the LDT using in vivo chronoamperometry in urethane-anaesthetized rats. LDT stimulation (35 Hz pulse trains for 60 s, 1 s intertrain interval) evoked a three-component change in dopamine efflux in the NAc: (i) an initial stimulation time-locked increase in the dopamine signal above baseline, followed by (ii) an immediate decrease below baseline, and thereafter by (iii) a prolonged increase in the dopamine signal above baseline. Intra-VTA infusion of the nicotinic receptor antagonist mecamylamine (5 microg/0.5 microL) or the ionotropic glutamate receptor antagonist kynurenate (10 microg/microL) attenuated the first LDT-elicited component. The second suppressive component was abolished by intra-LDT infusions of either the nonselective or the M2-selective muscarinic receptor antagonists scopolamine (100 microg/microL) and methoctramine (50 microg/microL), respectively. In contrast, intra-VTA infusions of scopolamine (200 microg/microL) resulted in a selective attenuation of the third facilitatory component, whereas both second and third components were abolished by systemic injections of scopolamine (5 mg/kg). These results suggest that the initial increase, subsequent decrease, and final prolonged increase in extracellular dopamine levels in the NAc are selectively mediated by LDT-elicited activation of (i) nicotinic and glutamatergic receptors in the VTA, (ii) muscarinic M2 autoreceptors on LDT cell bodies, and (iii) muscarinic receptors in the VTA, respectively.     

Accelerated maternal responding following intra-VTA pertussis toxin treatment

Prior studies have supported a role for mesolimbic dopaminergic mechanisms in the regulation of maternal behavior. Accordingly, the ventral tegmental area (VTA) and its dopaminergic projections to the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) have been implicated in both the onset and maintenance of normal maternal behavior. To date, studies of direct manipulation of VTA neurochemistry at the onset of maternal behavior have been limited. The current study was undertaken to directly test the hypothesis that enhancement of dopaminergic transmission in the mesolimbic dopamine system can stimulate maternal activity using a pup-induced virgin model. Nulliparous female rats were stereotaxically infused with pertussis toxin (PTX 0, 0.1, or 0.3 μg/hemisphere) into the VTA to chronically stimulate the activity of dopaminergic projection neurons. After 3 days of recovery, maternal responding to donor pups was tested daily, and latency (in days) to full maternal behavior was recorded. Intra-VTA PTX treatment produced a robust dose-dependent decrease in maternal behavior latency, and a long-lasting increase in locomotor activity. These effects were associated with significantly decreased dopamine D1 receptor mRNA expression in the NAc. No effects of PTX treatment on mesolimbic dopamine utilization or mPFC receptor expression were observed. The findings indicate that chronic neural activation in the VTA accelerates the onset of maternal behavior in virgin female rats via modification of the NAc dopamine D1 receptor.     

ATP-induced in vivo neurotoxicity in the rat striatum via P2 receptors

The present study examined the effects of ATP on the striatum of Sprague-Dawley rats. Intrastriatal administration of ATP produced dose-dependent striatal lesions as confirmed by cresyl violet staining. Additional immunostaining using neuronal nuclear protein (NeuN), OX-42 and GFAP antibodies revealed that ATP caused death of both neurons and glial cells. The nonmetabolizable ATP analogue ATPgammaS and P2X receptor agonist alpha,beta-methylene ATP (alpha,beta-MeATP) mimicked ATP effects, whereas either P2Y receptor agonist ADP or P1 receptor agonist adenosine did not. The P2 receptor antagonist reactive blue 2, but not pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) attenuated ATP-induced striatal injury. These results suggest that intrastriatal administration of ATP causes P2X receptor-mediated cell death in the striatum and support the hypothesis that extracellular ATP can be an important mediator of neuropathological events of brain injuries.     

P2-purinergic receptor antagonists reduce the minimum alveolar concentration of inhaled volatile anesthetics

In the central nervous system (CNS), adenosine triphosphate (ATP) is reported to serve as a fast excitatory neurotransmitter via P2X receptor. To examine possible involvement of inhibition of ATP signal-transmission in anesthetic mechanism, the effect of intracerebroventricular (ICV) administration of P2 receptor antagonists on the minimum alveolar concentration (MAC) of sevoflurane and isoflurane was studied in rat. ICV administration of P2 receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), significantly reduced MAC of both anesthetics. The reduction of the MAC by both suramin and PPADS was dose-dependent and reached plateau at 150 microgram/rat. These results suggest that the inhibition of ATP-signal transmission may be involved in analgesic or anesthetic effect in brain.     

P2X purinergic receptor knockout mice reveal endogenous ATP modulation of both vasopressin and oxytocin release from the intact neurohypophysis

Bursts of action potentials are crucial for neuropeptide release from the hypothalamic neurohypophysial system (HNS). The biophysical properties of the ion channels involved in the release of these neuropeptides, however, cannot explain the efficacy of such bursting patterns on secretion. We have previously shown that ATP, acting via P2X receptors, potentiates only vasopressin (AVP) release from HNS terminals, whereas its metabolite adenosine, via A1 receptors acting on transient Ca(2+) currents, inhibits both AVP and oxytocin (OT) secretion. Thus, purinergic feedback-mechanisms have been proposed to explain bursting efficacy at HNS terminals. Therefore, in the present study, we have used specific P2X receptor knockout (rKO) mice and purportedly selective P2X receptor antagonists to determine the P2X receptor subtype responsible for endogenous ATP induced potentiation of electrically-stimulated neuropeptide release. Intact neurohypophyses (NH) from wild-type (WT), P2X3 rKO, P2X2/3 rKO and P2X7 rKO mice were electrically stimulated with four 25-s bursts (3 V at 39 Hz) separated by 21-s interburst intervals with or without the P2X2 and P2X3 receptor antagonists, suramin or pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). These frequencies, number of bursts, and voltages were determined to maximise both AVP and OT release by electrical stimulations. Treatment of WT mouse NH with suramin/PPADS significantly reduced electrically-stimulated AVP release. A similar inhibition by suramin was observed in electrically-stimulated NH from P2X3 and P2X7 rKO mice but not P2X2/3 rKO mice, indicating that endogenous ATP facilitation of electrically-stimulated AVP release is mediated primarily by the activation of the P2X2 receptor. Unexpectedly, electrically-stimulated OT release from WT, P2X3, P2X2/3 and P2X7 rKO mice was potentiated by suramin, indicating nonpurinergic effects by this 'selective' antagonist. Nevertheless, these results show that sufficient endogenous ATP is released by bursts of action potentials to act at P2X2 receptors in a positive-feedback mechanism to 'differentially' modulate neuropeptide release from central nervous system terminals.     

ATP induces long-term potentiation of C-fiber-evoked field potentials in spinal dorsal horn: the roles of P2X4 receptors and p38 MAPK in microglia

Many studies have shown that adenosine triphosphate (ATP), as a neurotransmitter, is involved in plastic changes of synaptic transmission in central nervous system. In the present study, we tested whether extracellular ATP can induce long-term potentiation (LTP) of C-fiber-evoked field potentials in spinal dorsal horn. The results showed the following: (1) ATP at a concentration of 0.3 mM induced spinal LTP of C-fiber-evoked field potentials, lasting for at least 5 h; (2) spinal application of 2',3'-O-(2,4,6-trinitrophenyl)adenosine-5-triphosphate (TNP-ATP; an antagonist of P2X(1-4) receptors), but not pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; an antagonist of P2X(1,2,3,5,7) receptors), 30 min before ATP blocked ATP-induced LTP, indicating that ATP may induce spinal LTP by activation of P2X(4) receptors; (3) at 60 min after LTP induction the level of phospho-p38 mitogen-activated protein kinase (p-p38 MAPK) was significantly elevated and at 180 min after LTP the number of P2X(4) receptors increased significantly; both p-p38 and P2X(4) receptors were exclusively co-located with the microglia marker, but not with neuronal or astrocyte marker; (4) spinal application of TNP-ATP but not PPADS prevented p38 activation; (5) spinal application of SB203580, a p38 MAPK inhibitor, prevented both spinal LTP and the upregulation of P2X(4) receptors. The results suggested that ATP may activate p38 MAPK by binding to intrinsic P2X(4) receptors in microglia, and subsequently enhance the expression of P2X(4) receptors, contributing to spinal LTP.     

Cytoprotection against oxidative stress-induced damage of astrocytes by extracellular ATP via P2Y1 receptors

Oxidative stress is the main cause of neuronal damage in traumatic brain injury, hypoxia/reperfusion injury, and neurodegenerative disorders. Although extracellular nucleosides, especially adenosine, are well known to protect against neuronal damage in such pathological conditions, the effects of these nucleosides or nucleotides on glial cell damage remain largely unknown. We report that ATP but not adenosine protects against the cell death of cultured astrocytes induced by hydrogen peroxide (H2O2). ATP ameliorated the H2O2-induced decrease in cell viability of astrocytes in an incubation time- and concentration-dependent fashion. Protection by ATP was inhibited by P2 receptor antagonists and was mimicked by P2Y1 receptor agonists but not by adenosine. The expressions of P2Y1 mRNAs and functional P2Y1 receptors in astrocytes were confirmed. Thus, ATP, acting on P2Y1 receptors in astrocytes, showed a protective action against H2O2. The astrocytic protection by the P2Y1 receptor agonist 2-methylthio-ADP was inhibited by an intracellular Ca2+ chelator and a blocker of phospholipase C, indicating the involvement of intracellular signals mediated by Gq/11-coupled P2Y1 receptors. The ATP-induced protection was inhibited by cycloheximide, a protein synthesis inhibitor, and it took more than 12 h for the onset of the protective action. In the DNA microarray analysis, ATP induced a dramatic upregulation of various oxidoreductase genes. Taken together, ATP acts on P2Y1 receptors coupled to Gq/11, resulting in the upregulation of oxidoreductase genes, leading to the protection of astrocytes against H2O2.     

P2Y1 receptor inhibits GABA transport through a calcium signalling‐dependent mechanism in rat cortical astrocytes

Astrocytes express a variety of purinergic (P2) receptors, involved in astrocytic communication through fast increases in [Ca²⁺]_i. Of these, the metabotropic ATP receptors (P2Y) regulate cytoplasmic Ca²⁺ levels through the PLC‐PKC pathway. GABA transporters are a substrate for a number of Ca²⁺‐related kinases, raising the possibility that calcium signalling in astrocytes impact the control of extracellular levels of the major inhibitory transmitter in the brain. To access this possibility we tested the influence of P2Y receptors upon GABA transport into astrocytes. Mature primary cortical astroglial‐enriched cultures expressed functional P2Y receptors, as evaluated through Ca²⁺ imaging, being P2Y₁ the predominant P2Y receptor subtype. ATP (100 μM, for 1 min) caused an inhibition of GABA transport through either GAT‐1 or GAT‐3 transporters, decreasing the V_max kinetic constant. ATP‐induced inhibition of GATs activity was still evident in the presence of adenosine deaminase, precluding an adenosine‐mediated effect. This, was mimicked by a specific agonist for the P2Y_1,12,13 receptor (2‐MeSADP). The effect of 2‐MeSADP on GABA transport was blocked by the P2 (PPADS) and P2Y₁ selective (MRS2179) receptor antagonists, as well as by the PLC inhibitor (U73122). 2‐MeSADP failed to inhibit GABA transport in astrocytes where intracellular calcium had been chelated (BAPTA‐AM) or where calcium stores were depleted (α‐cyclopiazonic acid, CPA). In conclusion, P2Y₁ receptors in astrocytes inhibit GABA transport through a mechanism dependent of P2Y₁‐mediated calcium signalling, suggesting that astrocytic calcium signalling, which occurs as a consequence of neuronal firing, may operate a negative feedback loop to enhance extracellular levels of GABA. GLIA 2014;62:1211–1226     

Differential alterations in sympathetic neurotransmission in mesenteric arteries and veins in DOCA-salt hypertensive rats

Sympathetic control of arteries and veins may be altered in hypertension. To test this hypothesis, constrictions of mesenteric arteries and veins caused by nerve stimulation and by norepinephrine (NE) and ATP were studied in vitro in tissues from deoxycorticosterone acetate (DOCA)-salt hypertensive and sham normotensive rats. In DOCA-salt arteries, the maximum neurogenic response was greater than that in sham arteries. The P2 receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 10 microM), greatly reduced neurogenic responses in sham but not DOCA-salt arteries. The alpha1-adrenergic receptor antagonist, prazosin (0.1 microM), inhibited responses in DOCA-salt but not sham arteries. Concentration-response curves for norepinephrine and ATP were similar in sham and DOCA-salt arteries, indicating that reactivity to sympathetic vasoconstrictor transmitters was not changed in DOCA-salt arteries. Neurogenic constrictions in sham and DOCA-salt veins were similar in amplitude, and they were completely blocked by prazosin. However, concentration-response curves for norepinephrine in DOCA-salt veins were right-shifted compared to those in sham veins. Cocaine (10 microM) and corticosterone (10 microM) caused a leftward shift in norepinephrine concentration-response curves in DOCA-salt but not sham veins. Norepinephrine content was decreased in DOCA-salt arteries and veins, and there was an increased norepinephrine transporter (NET) level in DOCA-salt veins. These data indicate that, in DOCA-salt hypertension, there is an increased norepinephrine release from sympathetic nerves associated with mesenteric arteries and veins. In arteries, this results in an increase in the amplitude of neurogenic constrictions. In veins, increased norepinephrine release maintains neurogenic constrictions in the presence of increased NET levels.     

Involvement of 5-HT1B receptors within the ventral tegmental area in regulation of mesolimbic dopaminergic neuronal activity via GABA mechanisms: a study with dual-probe microdialysis

This study was designed to assess the involvement of 5-HT1B receptors within the ventral tegmental area (VTA) in the regulation of mesolimbic dopaminergic transmission. Dual-probe microdialysis was performed in freely moving adult Sprague-Dawley rats with one probe within the VTA and the other within the ipsilateral nucleus accumbens (NACC). Drugs were administered into the VTA via retrograde dialysis. Dialysates from both the VTA and the NAC were collected for determination of dopamine (DA) and gamma-aminobutyric acid (GABA) by high-performance liquid chromatography with electrochemical detection. Intra-tegmental infusion of CP 93129 (20, 40, and 80 microM), a 5-HT1B receptor agonist, increased extracellular DA concentrations in a concentration-dependent manner not only in the NACC but also in the VTA, indicating increased mesolimbic DA neuron activity. Administration of CP 93129 at 80 microM into the VTA also significantly decreased extracellular GABA concentrations in this region. Co-infusion of the 5-HT1B receptor antagonist SB 216641 (10 microM), but not the 5-HT1A receptor antagonist WAY 100635 (10 microM) or the 5-HT1D/1A receptor antagonist BRL 15572 (10 microM), antagonized not only the effects of intra-tegmental CP 93129 (80 microM) on VTA DA and NAC DA but also on VTA GABA. The results suggest that activation of VTA 5-HT1B receptors increases mesolimbic DA neuron activities. The increased DA neuron activity may be associated, at least in part, with the 5-HT1B receptor-mediated inhibition of VTA GABA release.     

Effects of ATP and derivatives on neuropile glial cells of the leech central nervous system

We investigated the effects of ATP (adenosine 5'-triphosphate) and derivatives on leech neuropile glial cells, focusing on exposed glial cells. ATP dose-dependently depolarized or hyperpolarized neuropile glial cells in situ as well as exposed neuropile glial cells. These potential shifts varied among cells and repetitive ATP application did not change their amplitude, duration or direction. In exposed neuropile glial cells, ATP most frequently induced a Na(+)-dependent depolarization and decreased the input resistance. The agonist potency ATP > ADP (adenosine 5'-diphosphate) > AMP (adenosine 5'-monophosphate) > adenosine indicates that P2 purinoceptors mediate this depolarization. The P2Y agonist 2-methylthio-ATP mimicked the ATP-induced depolarization, whereas the P2Y antagonist PPADS (pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid) reduced it. P2X agonists were without effect. Because the P1 antagonist 8-SPT (8-(p-sulphophenyl)-theophylline) also depressed ATP-induced depolarizations and some ATP-insensitive glial cells responded to adenosine, we suggest coexpression of metabotropic P2Y and P1 purinoceptors. The ATP-induced depolarization requires activation of Na(+) channels or nonselective cation channels, whereas the ATP-induced hyperpolarization indicates activation of K(+) channels. ATP also increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), that is independent of Ca(2+) influx but reflects intracellular Ca(2+) release possibly triggered by IP(3) formation. ADP and AMP also increased [Ca(2+)](i), but were less efficient than ATP; adenosine and 2-methylthio-ATP did not affect [Ca(2+)](i). In view of the mobilization of intracellular Ca(2+), ATP is clearly different from other leech neurotransmitters, because it enables intracellular Ca(2+) signaling without causing prominent changes in glial membrane potential. Thus disturbance of the extracellular microenvironment and the demand for metabolic energy are minimized.     

Adenosine triphosphate accelerates recovery from hypoxic/hypoglycemic perturbation of guinea pig hippocampal neurotransmission via a P(2) receptor

The present study was designed to assess the effects of adenosine triphosphate (ATP) on hippocampal neurotransmissions under the normal and hypoxic/hypoglycemic conditions. ATP reversely depressed population spikes (PSs), which were monitored in the dentate gyrus of guinea pig hippocampal slices, in a dose-dependent manner at concentrations ranged from 0.1 micro M to 1 mM. A similar depression was obtained with the P(2) receptor agonist, alpha,beta-methylene ATP (alpha,beta-MeATP), and the effect was inhibited by the P(2) receptor antagonists, suramin and PPADS. The inhibitory action of ATP or alpha,beta-MeATP was inhibited by the gamma-aminobutyric acid(A) (GABA(A)) receptor antagonist, bicuculline, but it was not affected by theophylline, a broad inhibitor of adenosine (P(1)) receptors, tetraethylammonium, a broad inhibitor of K(+) channels, or ecto-protein kinase inhibitors. ATP or alpha,beta-MeATP enhanced GABA release from guinea pig hippocampal slices, that was inhibited by deleting extracellular Ca(2+) or in the presence of tetrodotoxin, while ATP had no effect on GABA release from cultured rat hippocampal astrocytes or postsynaptic GABA-gated channel currents in cultured rat hippocampal neurons. Twenty-minutes deprivation of glucose and oxygen from extracellular solution abolished PSs, the amplitude recovering to about 30% of basal levels 50 min after returning to normal conditions. ATP or alpha,beta-MeATP accelerated the recovery after hypoxic/hypoglycemic insult (approximately 80% of basal levels). Adenosine diphosphate and adenosine monophosphate accelerated the recovery, but to a much lesser extent, and adenosine had no effect. The results of the present study thus suggest that ATP inhibits neuronal activity by enhancing neuronal GABA release via a P(2) receptor, perhaps a P2X receptor, thereby protecting against hypoxic/hypoglycemic perturbation of hippocampal neurotransmission.     

Insulin in the ventral tegmental area reduces cocaine‐evoked dopamine in the nucleus accumbens in vivo

Mesolimbic dopamine circuits, implicated in incentive motivation, are sensitive to changes in metabolic state such as weight loss and diet‐induced obesity. These neurons are important targets for metabolic hormones such as leptin, glucagon‐like peptide‐1, ghrelin and insulin. Insulin receptors are located on dopamine neurons in the ventral tegmental area (VTA) and we have previously demonstrated that insulin induces long‐term depression of excitatory synapses onto VTA dopamine neurons. While insulin can decrease dopamine concentration in somatodendritic regions, it can increase dopamine in striatal slices. Whether insulin directly targets the VTA to alter dopamine release in projection areas, such as the nucleus accumbens (NAc), remains unknown. The main goal of the present experiments was to examine NAc dopamine concentration following VTA administration of insulin. Using in vivo FSCV to detect rapid fluctuations in dopamine concentration, we showed that intra‐VTA insulin via action at insulin receptors reduced pedunculopontine nucleus‐evoked dopamine release in the NAc. Furthermore, intra‐VTA insulin reduced cocaine‐potentiated NAc dopamine. Finally, intra‐VTA or intranasal insulin decreased locomotor responses to cocaine, an effect blocked by an intra‐VTA administered insulin receptor antagonist. Together, these data demonstrate that mesolimbic dopaminergic projections are important targets of the metabolic hormone, insulin.     

Dopamine and adenosine mediate substance P-induced depression of evoked IPSCs in the rat nucleus accumbens in vitro

The major projection cells of the nucleus accumbens (NAc) are under a strong inhibitory influence from GABAergic afferents and depend on afferent excitation to produce their output. We have earlier reported that substance P (SP), a peptide which is colocalized with GABA in these neurons, depresses excitatory synaptic transmission in this nucleus (Kombian, S.B., Ananthalakshmi, K.V.V., Parvathy, S.S. & Matowe, W.C. (2003) J. Neurophysiol., 89, 728-738). In order to better understand the role of this peptide in the synaptic physiology of the NAc, it is important to determine its effects on inhibitory synaptic responses. Using whole-cell recording in rat forebrain slices, we show here that SP also depresses evoked inhibitory postsynaptic currents (IPSCs) in the NAc via intermediate neuromodulators. SP caused a partially reversible, dose-dependent decrease in evoked IPSC amplitude. This effect was present without measurable changes in the holding current, input resistance of recorded cells or decay rate (tau) of IPSCs. It was mimicked by a neurokinin-1 (NK1) receptor-selective agonist, [Sar9, Met (O2)11]-SP, and blocked by an NK1 receptor-selective antagonist, L 732 138. The SP-induced IPSC depression was prevented by SCH23390, a dopamine D1-like receptor antagonist and by 8-cyclopentyltheophylline, an adenosine A1 receptor blocker. Furthermore, the SP effect was also markedly attenuated by exogenous adenosine, dipyridamole, rolipram and barium. These data show that SP, acting on NK1 receptors, depresses inhibitory synaptic transmission indirectly by enhancing extracellular dopamine and adenosine levels. SP therefore acts in the NAc to modulate both excitatory and inhibitory afferent inputs using the same mechanism(s).     

Muscarinic receptor blockade in the ventral tegmental area attenuates cocaine enhancement of laterodorsal tegmentum stimulation‐evoked accumbens dopamine efflux in the mouse

The reinforcing properties of cocaine have been related to increased extracellular concentrations of dopamine in the nucleus accumbens (NAc). M5 muscarinic acetylcholine receptors (mAChRs) on dopamine cells in the ventral tegmental area (VTA) facilitate mesoaccumbens dopamine transmission and are critically involved in mediating natural and drug reinforcement. We investigated the effects of pharmacological blockade of mAChRs in the VTA on cocaine's ability to enhance electrically evoked NAc dopamine efflux. Using fixed potential amperometry together with carbon fiber recording microelectrodes positioned in the NAc core, we quantified dopamine oxidation currents (dopamine efflux) evoked by brief stimulation (15 monophasic pulses at 50 Hz every 30 s) of the laterodorsal tegmentum (LDT) in urethane (1.5 g/kg, i.p.) anesthetized mice. Compared to predrug baseline responses, cocaine (5 or 10 mg/kg, i.p.) dose‐dependently enhanced LDT stimulation‐evoked NAc dopamine efflux, whereas the nonsubtype selective mAChR antagonist scopolamine (10 μg/0.5 μl) microinfused into the VTA diminished LDT‐evoked NAc dopamine efflux. Preinfusion of scopolamine into the VTA diminished the facilitatory actions of cocaine on LDT stimulation‐evoked NAc dopamine efflux, and when infused at the peak effect of cocaine attenuated LDT‐evoked dopamine efflux to below predrug baseline levels. These findings suggest that LDT cholinergic inputs to dopamine neurons in the VTA, via activation of mAChRs (probably of the M5 subtype), are involved in modulating the facilitatory effects of cocaine on NAc dopamine neurotransmission. They also suggest that the development of antagonists aimed at selectively disrupting M5 receptor function may be valuable in reducing abuse liability of psychostimulants. Synapse 64:216–223, 2010. © 2009 Wiley‐Liss, Inc.