Targeting corticostriatal transmission for the treatment of cannabinoid use disorder.

It is generally assumed that the rewarding effects of cannabinoids are mediated by cannabinoid CB1 receptors (CB1Rs) the activation of which disinhibits dopaminergic neurons in the ventral tegmental area (VTA). However, this mechanism cannot fully explain novel results indicating that dopaminergic neurons also mediate the aversive effects of cannabinoids in rodents, and previous results showing that preferentially presynaptic adenosine A2A receptor (A2AR) antagonists counteract self-administration of Δ-9-tetrahydrocannabinol (THC) in nonhuman primates (NHPs). Based on recent experiments in rodents and imaging studies in humans, we propose that the activation of frontal corticostriatal glutamatergic transmission constitutes an additional and necessary mechanism. Here, we review evidence supporting the involvement of cortical astrocytic CB1Rs in the activation of corticostriatal neurons and that A2AR receptor heteromers localized in striatal glutamatergic terminals mediate the counteracting effects of the presynaptic A2AR antagonists, constituting potential targets for the treatment of cannabinoid use disorder (CUD).

Brain Iron Deficiency Changes the Stoichiometry of Adenosine Receptor Subtypes in Cortico-Striatal Terminals: Implications for Restless Legs Syndrome.

Brain iron deficiency (BID) constitutes a primary pathophysiological mechanism in restless legs syndrome (RLS). BID in rodents has been widely used as an animal model of RLS, since it recapitulates key neurochemical changes reported in RLS patients and shows an RLS-like behavioral phenotype. Previous studies with the BID-rodent model of RLS demonstrated increased sensitivity of cortical pyramidal cells to release glutamate from their striatal nerve terminals driving striatal circuits, a correlative finding of the cortical motor hyperexcitability of RLS patients. It was also found that BID in rodents leads to changes in the adenosinergic system, a downregulation of the inhibitory adenosine A1 receptors (A1Rs) and upregulation of the excitatory adenosine A2A receptors (A2ARs). It was then hypothesized, but not proven, that the BID-induced increased sensitivity of cortico-striatal glutamatergic terminals could be induced by a change in A1R/A2AR stoichiometry in favor of A2ARs. Here, we used a newly developed FACS-based synaptometric analysis to compare the relative abundance on A1Rs and A2ARs in cortico-striatal and thalamo-striatal glutamatergic terminals (labeled with vesicular glutamate transporters VGLUT1 and VGLUT2, respectively) of control and BID rats. It could be demonstrated that BID (determined by measuring transferrin receptor density in the brain) is associated with a selective decrease in the A1R/A2AR ratio in VGLUT1 positive-striatal terminals.

Control of glutamate release by complexes of adenosine and cannabinoid receptors.

BACKGROUND: It has been hypothesized that heteromers of adenosine A2A receptors (A2AR) and cannabinoid CB1 receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals. RESULTS: Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure of heteromers of A2AR with adenosine A1 receptors or dopamine D2 receptors, with different heteromeric or homomeric interfaces. The specific quaternary structure of the A2A-CB1R, which depended on intermolecular interactions involving the long C-terminus of the A2AR, determined a significant A2AR and Gs protein-mediated constitutive activation of adenylyl cyclase. Using heteromer-interfering peptides in experiments with striatal glutamatergic terminals, we could then demonstrate the presence of functionally significant A2AR-CB1R heteromers with the same biochemical characteristics of those studied in mammalian transfected cells. First, either an A2AR agonist or an A2AR antagonist allosterically counteracted Gi-mediated CB1R agonist-induced inhibition of depolarization-induced glutamate release. Second, co-application of both an A2AR agonist and an antagonist cancelled each other effects. Finally, a CB1R agonist inhibited glutamate release dependent on a constitutive activation of A2AR by a canonical Gs-Gi antagonistic interaction at the adenylyl cyclase level. CONCLUSIONS: We demonstrate that the well-established cannabinoid-induced inhibition of striatal glutamate release can mostly be explained by a CB1R-mediated counteraction of the A2AR-mediated constitutive activation of adenylyl cyclase in the A2AR-CB1R heteromer.

Memory deficits induced by chronic cannabinoid exposure are prevented by adenosine A2AR receptor antagonism.

Patients under cannabis-based therapies are usually chronically exposed to cannabinoids. Chronic treatment with a cannabinoid receptor agonist, WIN 55,212-2, affects brain metabolism and modifies functional connectivity between brain areas responsible for memory and learning. Therefore, it is of uttermost importance to discover strategies to mitigate the negative side-effects of cannabinoid-based therapies. Previously, we showed that a single treatment with the synthetic cannabinoid WIN 55,212-2 disrupts recognition memory, an effect mediated by cannabinoid receptor 1 (CB1R) and cancelled by concomitant administration of adenosine A2A receptor (A2AR) antagonists. We herein evaluate if memory deficits induced by chronic exposure to WIN 55,212-2 can also be reverted by A2AR antagonism, and assessed the synaptic mechanisms that could be involved in that reversal. We show that chronic administration of KW-6002 (istradefylline) (3 mg/kg/28days) reverts memory deficits (evaluated through the Novel Object Recognition Test) induced by chronic cannabinoid exposure (WIN 55,212-2, 1 mg/kg/28 days). Long Term Potentiation (LTP) of synaptic potentials recorded from the CA1 area of the hippocampus was impaired by WIN 55,212-2 (300 nM), an effect partially rescued by the A2AR antagonist, SCH 58261 (100 nM). Chronic administration of KW-6002 or WIN 55,212-2 did not affect A2AR or CB1R binding in the hippocampus and in the prefrontal cortex. These results, showing that A2AR antagonism can still revert memory deficits after chronic administration of a cannabinoid, an effect that involves mitigation of synaptic plasticity impairment, strongly indicate that adenosine A2ARs are appropriate targets to tackle side-effects of putative therapies involving the activation of cannabinoid receptors.

Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation.

Despite the characteristic etiologies and phenotypes, different brain disorders rely on common pathogenic events. Glutamate-induced neurotoxicity is a pathogenic event shared by different brain disorders. Another event occurring in different brain pathological conditions is the increase of the extracellular ATP levels, which is now recognized as a danger and harmful signal in the brain, as heralded by the ability of P2 receptors (P2Rs) to affect a wide range of brain disorders. Yet, how ATP and P2R contribute to neurodegeneration remains poorly defined. For that purpose, we now examined the contribution of extracellular ATP and P2Rs to glutamate-induced neurodegeneration. We found both in vitro and in vivo that ATP/ADP through the activation of P2Y1R contributes to glutamate-induced neuronal death in the rat hippocampus. We found in cultured rat hippocampal neurons that the exposure to glutamate (100 µM) for 30 min triggers a sustained increase of extracellular ATP levels, which contributes to NMDA receptor (NMDAR)-mediated hippocampal neuronal death through the activation of P2Y1R. We also determined that P2Y1R is involved in excitotoxicity in vivo as the blockade of P2Y1R significantly attenuated rat hippocampal neuronal death upon the systemic administration of kainic acid or upon the intrahippocampal injection of quinolinic acid. This contribution of P2Y1R fades with increasing intensity of excitotoxic conditions, which indicates that P2Y1R is not contributing directly to neurodegeneration, rather behaving as a catalyst decreasing the threshold from which glutamate becomes neurotoxic. Moreover, we unraveled that such excitotoxicity process began with an early synaptotoxicity that was also prevented/attenuated by the antagonism of P2Y1R, both in vitro and in vivo. This should rely on the observed glutamate-induced calpain-mediated axonal cytoskeleton damage, most likely favored by a P2Y1R-driven increase of NMDAR-mediated Ca2+ entry selectively in axons. This may constitute a degenerative mechanism shared by different brain diseases, particularly relevant at initial pathogenic stages.

Adenosine A2A Receptors in the Rat Prelimbic Medial Prefrontal Cortex Control Delay-Based Cost-Benefit Decision Making.

Adenosine A2A receptors (A2ARs) were recently described to control synaptic plasticity and network activity in the prefrontal cortex (PFC). We now probed the role of these PFC A2AR by evaluating the behavioral performance (locomotor activity, anxiety-related behavior, cost-benefit decision making and working memory) of rats upon downregulation of A2AR selectively in the prelimbic medial PFC (PLmPFC) via viral small hairpin RNA targeting the A2AR (shA2AR). The most evident alteration observed in shA2AR-treated rats, when compared to sh-control (shCTRL)-treated rats, was a decrease in the choice of the large reward upon an imposed delay of 15 s assessed in a T-maze-based cost-benefit decision-making paradigm, suggestive of impulsive decision making. Spontaneous locomotion in the open field was not altered, suggesting no changes in exploratory behavior. Furthermore, rats treated with shA2AR in the PLmPFC also displayed a tendency for higher anxiety levels in the elevated plus maze (less entries in the open arms), but not in the open field test (time spent in the center was not affected). Finally, working memory performance was not significantly altered, as revealed by the spontaneous alternation in the Y-maze test and the latency to reach the platform in the repeated trial Morris water maze. These findings constitute the first direct demonstration of a role of PFC A2AR in the control of behavior in physiological conditions, showing their major contribution for the control of delay-based cost-benefit decisions.

Neuronal Adenosine A2A Receptors Are Critical Mediators of Neurodegeneration Triggered by Convulsions.

Neurodegeneration is a process transversal to neuropsychiatric diseases and the understanding of its mechanisms should allow devising strategies to prevent this irreversible step in brain diseases. Neurodegeneration caused by seizures is a critical step in the aggravation of temporal lobe epilepsy, but its mechanisms remain undetermined. Convulsions trigger an elevation of extracellular adenosine and upregulate adenosine A2A receptors (A2AR), which have been associated with the control of neurodegenerative diseases. Using the rat and mouse kainate model of temporal lobe epilepsy, we now tested whether A2AR control convulsions-induced hippocampal neurodegeneration. The pharmacological or genetic blockade of A2AR did not affect kainate-induced convulsions but dampened the subsequent neurotoxicity. This neurotoxicity began with a rapid A2AR upregulation within glutamatergic synapses (within 2 h), through local translation of synaptic A2AR mRNA. This bolstered A2AR-mediated facilitation of glutamate release and of long-term potentiation (LTP) in CA1 synapses (4 h), triggered a subsequent synaptotoxicity, heralded by decreased synaptic plasticity and loss of synaptic markers coupled to calpain activation (12 h), that predated overt neuronal loss (24 h). All modifications were prevented by the deletion of A2AR selectively in forebrain neurons. This shows that synaptic A2AR critically control synaptic excitotoxicity, which underlies the development of convulsions-induced neurodegeneration.

Caffeine Reverts Memory But Not Mood Impairment in a Depression-Prone Mouse Strain with Up-Regulated Adenosine A2A Receptor in Hippocampal Glutamate Synapses.

Caffeine prophylactically prevents mood and memory impairments through adenosine A2A receptor (A2AR) antagonism. A2AR antagonists also therapeutically revert mood and memory impairments, but it is not known if caffeine is also therapeutically or only prophylactically effective. Since depression is accompanied by mood and memory alterations, we now explored if chronic (4 weeks) caffeine consumption (0.3 g/L) reverts mood and memory impairment in helpless mice (HM, 12 weeks old), a bred-based model of depression. HM displayed higher immobility in the tail suspension and forced swimming tests, greater anxiety in the elevated plus maze, and poorer memory performance (modified Y-maze and object recognition). HM also had reduced density of synaptic (synaptophysin, SNAP-25), namely, glutamatergic (vGluT1; -22 ± 7 %) and GABAergic (vGAT; -23 ± 8 %) markers in the hippocampus. HM displayed higher A2AR density (72 ± 6 %) in hippocampal synapses, an enhanced facilitation of hippocampal glutamate release by the A2AR agonist, CGS21680 (30 nM), and a larger LTP amplitude (54 ± 8 % vs. 21 ± 5 % in controls) that was restored to control levels (30 ± 10 %) by the A2AR antagonist, SCH58261 (50 nM). Notably, caffeine intake reverted memory deficits and reverted the loss of hippocampal synaptic markers but did not affect helpless or anxiety behavior. These results reinforce the validity of HM as an animal model of depression by showing that they also display reference memory deficits. Furthermore, caffeine intake selectively reverted memory but not mood deficits displayed by HM, which are associated with an increased density and functional impact of hippocampal A2AR controlling synaptic glutamatergic function.

Stimulation of brain glucose uptake by cannabinoid CB2 receptors and its therapeutic potential in Alzheimer's disease.

Cannabinoid CB2 receptors (CB2Rs) are emerging as important therapeutic targets in brain disorders that typically involve neurometabolic alterations. We here addressed the possible role of CB2Rs in the regulation of glucose uptake in the mouse brain. To that aim, we have undertaken 1) measurement of (3)H-deoxyglucose uptake in cultured cortical astrocytes and neurons and in acute hippocampal slices; 2) real-time visualization of fluorescently labeled deoxyglucose uptake in superfused hippocampal slices; and 3) in vivo PET imaging of cerebral (18)F-fluorodeoxyglucose uptake. We now show that both selective (JWH133 and GP1a) as well as non-selective (WIN55212-2) CB2R agonists, but not the CB1R-selective agonist, ACEA, stimulate glucose uptake, in a manner that is sensitive to the CB2R-selective antagonist, AM630. Glucose uptake is stimulated in astrocytes and neurons in culture, in acute hippocampal slices, in different brain areas of young adult male C57Bl/6j and CD-1 mice, as well as in middle-aged C57Bl/6j mice. Among the endocannabinoid metabolizing enzymes, the selective inhibition of COX-2, rather than that of FAAH, MAGL or α,ßDH6/12, also stimulates the uptake of glucose in hippocampal slices of middle-aged mice, an effect that was again prevented by AM630. However, we found the levels of the endocannabinoid, anandamide reduced in the hippocampus of TgAPP-2576 mice (a model of ß-amyloidosis), and likely as a consequence, COX-2 inhibition failed to stimulate glucose uptake in these mice. Together, these results reveal a novel general glucoregulatory role for CB2Rs in the brain, raising therapeutic interest in CB2R agonists as nootropic agents.

Adenosine A2B receptor activation stimulates glucose uptake in the mouse forebrain.

ATP consumption during intense neuronal activity leads to peaks of both extracellular adenosine levels and increased glucose uptake in the brain. Here, we investigated the hypothesis that the activation of the low-affinity adenosine receptor, the A2B receptor (A(2B)R), promotes glucose uptake in neurons and astrocytes, thereby linking brain activity with energy metabolism. To this end, we mapped the spatiotemporal accumulation of the fluorescent-labelled deoxyglucose, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), in superfused acute hippocampal slices of C57Bl/6j mice. Bath application of the A(2B)R agonist BAY606583 (300 nM) triggered an immediate and stable (>10 min) increase of the velocity of 2-NBDG accumulation throughout hippocampal slices. This was abolished with the pretreatment with the selective A(2B)R antagonist, MRS1754 (200 nM), and was also absent in A(2B)R null-mutant mice. In mouse primary astrocytic or neuronal cultures, BAY606583 similarly increased (3)H-deoxyglucose uptake in the following 20 min incubation period, which was again abolished by a pretreatment with MRS1754. Finally, incubation of hippocampal, frontocortical, or striatal slices of C57Bl/6j mice at 37 °C, with either MRS1754 (200 nM) or adenosine deaminase (3 U/mL) significantly reduced glucose uptake. Furthermore, A(2B)R blockade diminished newly synthesized glycogen content and at least in the striatum, increased lactate release. In conclusion, we report here that A(2B)R activation is associated with an instant and tonic increase of glucose transport into neurons and astrocytes in the mouse brain. These prompt further investigations to evaluate the clinical potential of this novel glucoregulator mechanism.

Adenosine A2b receptors control A1 receptor-mediated inhibition of synaptic transmission in the mouse hippocampus.

Adenosine is a neuromodulator mostly acting through A1 (inhibitory) and A2A (excitatory) receptors in the brain. A2B receptors (A(2B)R) are G(s/q)--protein-coupled receptors with low expression in the brain. As A(2B)R function is largely unknown, we have now explored their role in the mouse hippocampus. We performed electrophysiological extracellular recordings in mouse hippocampal slices, and immunological analysis of nerve terminals and glutamate release in hippocampal slices and synaptosomes. Additionally, A(2B)R-knockout (A(2B)R-KO) and C57/BL6 mice were submitted to a behavioural test battery (open field, elevated plus-maze, Y-maze). The A(2B)R agonist BAY60-6583 (300 nM) decreased the paired-pulse stimulation ratio, an effect prevented by the A(2B)R antagonist MRS 1754 (200 nM) and abrogated in A(2B)R-KO mice. Accordingly, A(2B)R immunoreactivity was present in 73 ± 5% of glutamatergic nerve terminals, i.e. those immunopositive for vesicular glutamate transporters. Furthermore, BAY 60-6583 attenuated the A(1)R control of synaptic transmission, both the A(1)R inhibition caused by 2-chloroadenosine (0.1-1 µM) and the disinhibition caused by the A(1)R antagonist DPCPX (100 nM), both effects prevented by MRS 1754 and abrogated in A(2B)R-KO mice. BAY 60-6583 decreased glutamate release in slices and also attenuated the A(1)R inhibition (CPA 100 nM). A(2B)R-KO mice displayed a modified exploratory behaviour with an increased time in the central areas of the open field, elevated plus-maze and the Y-maze and no alteration of locomotion, anxiety or working memory. We conclude that A(2B)R are present in hippocampal glutamatergic terminals where they counteract the predominant A(1)R-mediated inhibition of synaptic transmission, impacting on exploratory behaviour.

Presynaptic TRPV1 vanilloid receptor function is age- but not CB1 cannabinoid receptor-dependent in the rodent forebrain.

Neocortical and striatal TRPV1 (vanilloid or capsaicin) receptors (TRPV1Rs) are excitatory ligand-gated ion channels, and are implicated in psychiatric disorders. However, the purported presynaptic neuromodulator role of TRPV1Rs in glutamatergic, serotonergic or dopaminergic terminals of the rodent forebrain remains little understood. With the help of patch-clamp electrophysiology and neurochemical approaches, we mapped the age-dependence of presynaptic TRPV1R function, and furthermore, we aimed at exploring whether the presence of CB1 cannabinoid receptors (CB1Rs) influences the function of the TRPV1Rs, as both receptor types share endogenous ligands. We found that the major factor which affects presynaptic TRPV1R function is age: by post-natal day 13, the amplitude of capsaicin-induced release of dopamine and glutamate is halved in the rat striatum, and two weeks later, capsaicin already loses its effect. However, TRPV1R receptor function is not enhanced by chemical or genetic ablation of the CB1Rs in dopaminergic, glutamatergic and serotonergic terminals of the mouse brain. Altogether, our data indicate a possible neurodevelopmental role for presynaptic TRPV1Rs in the rodent brain, but we found no cross-talk between TRPV1Rs and CB1Rs in the same nerve terminal.

Caffeine regulates frontocorticostriatal dopamine transporter density and improves attention and cognitive deficits in an animal model of attention deficit hyperactivity disorder.

Attention deficit hyperactivity disorder (ADHD) likely involves dopaminergic dysfunction in the frontal cortex and striatum, resulting in cognitive and motor abnormalities. Since both adenosine and dopamine modulation systems are tightly intertwined, we tested if caffeine (a non-selective adenosine receptor antagonist) attenuated the behavioral and neurochemical changes in adolescent spontaneously hypertensive rats (SHR, a validated ADHD animal model) compared to their control strain (Wistar Kyoto rats, WKY). SHR were hyperactive and had poorer performance in the attentional set-shifting and Y-maze paradigms and also displayed increased dopamine transporter (DAT) density and increased dopamine uptake in frontocortical and striatal terminals compared with WKY rats. Chronic caffeine treatment was devoid of effects in WKY rats while it improved memory and attention deficits and also normalized dopaminergic function in SHR. Additionally, we provide the first direct demonstration for the presence of adenosine A2A receptors (A2AR) in frontocortical nerve terminals, whose density was increased in SHR. These findings underscore the potential for caffeine treatment to normalize frontocortical dopaminergic function and to abrogate attention and cognitive changes characteristic of ADHD.

Cannabinoids inhibit the synaptic uptake of adenosine and dopamine in the rat and mouse striatum.

Adenosine, dopamine and endocannabinoids strictly modulate the release of one another in the dorsolateral striatum thereby controlling synaptic plasticity. As a second level of interaction, they regulate the action of one another via receptor heteromer formation. Here we investigated a putative third level of interaction, i.e. the possible control by cannabinoids of synaptic dopamine and adenosine reuptake. We found that a large number of endo- and exogenous cannabinoid ligands inhibit the uptake of [(3)H]adenosine and [(3)H]dopamine in rat sriatal nerve terminals. Maximal effects were often comparable to those of the dopamine transporter inhibitor, GBR12783 and the equilibrative nucleoside transporter inhibitor, dipyridamole. Cannabinoid ligands were generally more potent to inhibit the uptake of adenosine than that of dopamine. The inhibitory effect was: (1) unrelated to the pharmacological profile(s) of the ligands at the cannabinoid CB(1), CB(2), GPR55 and at the vanilloid TRPV(1) receptors; (2) not prevented by the cannabinoid CB(1) receptor antagonist/inverse agonist, LY320135; and (3) maintained in the cannabinoid CB(1) receptor knockout mice. In the same experiments, only O-2050, cannabidiol, and WIN55212-3 inhibited the simultaneously measured DL-TBOA-sensitive uptake of [(14)C]glutamate. In summary, many cannabinoid ligands are able to inhibit the synaptic uptake of adenosine and dopamine. These effects are not mediated by cannabinoid CB(1) receptors, and should be an additional mechanism to consider when interpreting synaptic effects of cannabinoids.

Pre-synaptic adenosine A2A receptors control cannabinoid CB1 receptor-mediated inhibition of striatal glutamatergic neurotransmission.

An interaction between adenosine A(2A) receptors (A(2A) Rs) and cannabinoid CB(1) receptors (CB(1) Rs) has been consistently reported to occur in the striatum, although the precise mechanisms are not completely understood. As both receptors control striatal glutamatergic transmission, we now probed the putative interaction between pre-synaptic CB(1) R and A(2A) R in the striatum. In extracellular field potentials recordings in corticostriatal slices from Wistar rats, A(2A) R activation by CGS21680 inhibited CB(1) R-mediated effects (depression of synaptic response and increase in paired-pulse facilitation). Moreover, in superfused rat striatal nerve terminals, A(2A) R activation prevented, while A(2A) R inhibition facilitated, the CB(1) R-mediated inhibition of 4-aminopyridine-evoked glutamate release. In summary, the present study provides converging neurochemical and electrophysiological support for the occurrence of a tight control of CB(1) R function by A(2A) Rs in glutamatergic terminals of the striatum. In view of the key role of glutamate to trigger the recruitment of striatal circuits, this pre-synaptic interaction between CB(1) R and A(2A) R may be of relevance for the pathogenesis and the treatment of neuropsychiatric disorders affecting the basal ganglia.

Modification upon aging of the density of presynaptic modulation systems in the hippocampus.

Different presynaptic neuromodulation systems have been explored as possible targets to manage neurodegenerative diseases. However, most studies used young adult animals whereas neurodegenerative diseases are prevalent in the elderly. Thus, we now explored by Western blot analysis how the density of different presynaptic markers and receptors changes with aging in rat hippocampal synaptosomes (purified nerve terminals). Compared to synaptosomal membranes from 2-month-old rats, the density of presynaptic proteins (synaptophysin or SNAP-25) decreased at 18-24 months. In parallel, markers of glutamatergic terminals (vGluT1 or vGluT2) and cholinergic terminal markers (vAChT) constantly decreased with aging from 12 to 18 months onwards, whereas the densities of GABAergic (vGAT) only decreased after 24 months. Inhibitory A(1) and CB(1) receptor density tended to decrease with aging, whereas facilitatory mGluR5 and P2Y1 receptor density was roughly constant and facilitatory A(2A) receptor density increased at 18-24 months. Thus aging causes an imbalance of excitatory versus inhibitory nerve terminal markers and causes a predominant decrease of inhibitory rather than facilitatory presynaptic modulation systems.

Differential glutamate-dependent and glutamate-independent adenosine A1 receptor-mediated modulation of dopamine release in different striatal compartments.

Adenosine and dopamine are two important modulators of glutamatergic neurotransmission in the striatum. However, conflicting reports exist about the role of adenosine and adenosine receptors in the modulation of striatal dopamine release. It has been previously suggested that adenosine A(1) receptors localized in glutamatergic nerve terminals indirectly modulate dopamine release, by their ability to modulate glutamate release. In the present study, using in vivo microdialysis, we provide evidence for the existence of a significant glutamate-independent tonic modulation of dopamine release in most of the analyzed striatal compartments. In the dorsal, but not in the ventral, part of the shell of the nucleus accumbens (NAc), blockade of A(1) receptors by local perfusion with the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine or by systemic administration of the non-selective adenosine antagonist caffeine induced a glutamate-dependent release of dopamine. On the contrary, A(1) receptor blockade induced a glutamate-independent dopamine release in the core of the NAc and the nucleus caudate-putamen. Furthermore, using immunocytochemical and functional studies in rat striatal synaptosomes, we demonstrate that a fraction of striatal dopaminergic terminals contains adenosine A(1) receptors, which directly inhibit dopamine release independently of glutamatergic transmission.

Supersensitivity of P2X receptors in cerebrocortical cell cultures after in vitro ischemia.

Neuronally enriched primary cerebrocortical cultures were exposed to glucose-free medium saturated with argon (in vitro ischemia) instead of oxygen (normoxia). Ischemia did not alter P2X7 receptor mRNA, although serum deprivation clearly increased it. Accordingly, P2X7 receptor immunoreactivity (IR) of microtubuline-associated protein 2 (MAP2)-IR neurons or of glial fibrillary acidic protein (GFAP)-IR astrocytes was not affected; serum deprivation augmented the P2X7 receptor IR only in the astrocytic, but not the neuronal cell population. However, ischemia markedly increased the ATP- and 2'-3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP)-induced release of previously incorporated [3H]GABA. Both Brilliant Blue G and oxidized ATP inhibited the release of [3H]GABA caused by ATP application; the Brilliant Blue G-sensitive, P2X7 receptor-mediated fraction, was much larger after ischemia than after normoxia. Whereas ischemic stimulation failed to alter the amplitude of ATP- and BzATP-induced small inward currents recorded from a subset of non-pyramidal neurons, BzATP caused a more pronounced increase in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) after ischemia than after normoxia. Brilliant Blue G almost abolished the effect of BzATP in normoxic neurons. Since neither the amplitude of mIPSCs nor that of the muscimol-induced inward currents was affected by BzATP, it is assumed that BzATP acts at presynaptic P2X7 receptors. Finally, P2X7 receptors did not enhance the intracellular free Ca2+ concentration either in proximal dendrites or in astrocytes, irrespective of the normoxic or ischemic pre-incubation conditions. Hence, facilitatory P2X7 receptors may be situated at the axon terminals of GABAergic non-pyramidal neurons. When compared with normoxia, ischemia appears to markedly increase P2X7 receptor-mediated GABA release, which may limit the severity of the ischemic damage. At the same time we did not find an accompanying enhancement of P2X7 mRNA or protein expression, suggesting that receptors may become hypersensitive because of an increased efficiency of their transduction pathways.

P2X receptor activation elicits transporter-mediated noradrenaline release from rat hippocampal slices.

This study was designed to test the hypothesis of whether activation of presynaptic P2X receptor-gated ion channels elicits noradrenaline release from central catecholaminergic terminals. ATP, alpha,beta-methylene-adenosine 5'-triphosphate (alpha,beta-methyleneATP), and ADP elicited concentration-dependent [3H]noradrenaline outflow from superfused rat hippocampal slices with the following rank order of agonist potency: alpha,beta-methyleneATP > ATP > ADP. Among P2 receptor antagonists, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (30 microM), 4,4',4",4"'-[carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino))]tetrakis-1,3-benzenedisulfonic acid (100 nM), and 8,8'-[carbonybis(imino-3,1-phenylenecarbonylimino)]bis1,3,5-naphthalenetrisulphonic acid (10 microM) significantly inhibited the outflow of [3H]noradrenaline, evoked by ATP, whereas Brilliant Blue G (100 nM), 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate tetraammonium (10 microM), the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (250 nM), and the A2A receptor antagonist 3,7-dimethyl-1-propargylxanthine (250 nM) were ineffective. Pretreatment with the Gi protein inhibitor pertussis toxin (2.5 microg/ml) did not change the effect of ATP on [3H]noradrenaline outflow. In contrast, a decrease in extracellular pH from 7.4 to 6.6 significantly attenuated the response by ATP. When extracellular Na+ was replaced by choline chloride and in the presence of the noradrenaline uptake inhibitor desipramine (10 microM), the ATP-evoked [3H]noradrenaline outflow was almost completely abolished, indicating that its underlying mechanism is the sodium-dependent reversal of the noradrenaline transporter. Reverse transcription-polymerase chain reaction analysis revealed that mRNA encoding P2X1, P2X2, P2X3, P2X4, P2X6, P2X7 and P2Y1 receptor subunits were expressed in the brainstem containing catecholaminergic nuclei projecting to the hippocampus, whereas mRNA encoding P2X5, P2Y2, P2Y4, and P2Y6 receptors were absent. Taken together, these results indicate that noradrenergic terminals of the rat hippocampus are equipped with presynaptic facilitatory P2X receptors, displaying a pharmacological profile similar to homomeric P2X1 and P2X3 receptors.

Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus.

Although originally cloned from rat brain, the P2X7 receptor has only recently been localized in neurones, and functional responses mediated by these neuronal P2X7 receptors (P2X7 R) are largely unknown. Here we studied the effect of P2X7 R activation on the release of neurotransmitters from superfused rat hippocampal slices. ATP (1-30 mm) and other ATP analogues elicited concentration-dependent [3 H]GABA outflow, with the following rank order of potency: benzoylbenzoylATP (BzATP) > ATP > ADP. PPADS, the non-selective P2-receptor antagonist (3-30 microm), Brilliant blue G (1-100 nm) the P2X7 -selective antagonist and Zn2+ (0.1-30 microm) inhibited, whereas lack of Mg2+ potentiated the response by ATP. In situ hybridization revealed that P2X7 R mRNA is expressed in the neurones of the cell body layers in the hippocampus. P2X7 R immunoreactivity was found in excitatory synaptic terminals in CA1 and CA3 region targeting the dendrites of pyramidal cells and parvalbumin labelled structures. ATP (3-30 microm) and BzATP (0.6-6 microm) elicited concentration-dependent [14 C]glutamate efflux, and blockade of the kainate receptor-mediated transmission by CNQX (10-100 microm) and gadolinium (100 microm), decreased ATP evoked [3 H]GABA efflux. The Na+ channel blocker TTX (1 microm), low temperature (12 degrees C), and the GABA uptake blocker nipecotic acid (1 mm) prevented ATP-induced [3 H]GABA efflux. Brilliant blue G and PPADS also reduced electrical field stimulation-induced [3 H]GABA efflux. In conclusion, P2X7 Rs are localized to the excitatory terminals in the hippocampus, and their activation regulates the release of glutamate and GABA from themselves and from their target cells.