Presynaptic adenosine A2A receptors dampen cannabinoid CB1 receptor-mediated inhibition of corticostriatal glutamatergic transmission.

BACKGROUND AND PURPOSE: Both cannabinoid CB1 and adenosine A2A receptors (CB1 receptors and A2A receptors) control synaptic transmission at corticostriatal synapses, with great therapeutic importance for neurological and psychiatric disorders. A postsynaptic CB1 -A2A receptor interaction has already been elucidated, but the presynaptic A2A receptor-mediated control of presynaptic neuromodulation by CB1 receptors remains to be defined. Because the corticostriatal terminals provide the major input to the basal ganglia, understanding the interactive nature of converging neuromodulation on them will provide us with novel powerful tools to understand the physiology of corticostriatal synaptic transmission and interpret changes associated with pathological conditions. EXPERIMENTAL APPROACH: Pharmacological manipulation of CB1 and A2A receptors was carried out in brain nerve terminals isolated from rats and mice, using flow synaptometry, immunoprecipitation, radioligand binding, ATP and glutamate release measurement. Whole-cell patch-clamp recordings were made in horizontal corticostriatal slices. KEY RESULTS: Flow synaptometry showed that A2A receptors were extensively co-localized with CB1 receptor-immunopositive corticostriatal terminals and A2A receptors co-immunoprecipitated CB1 receptors in these purified terminals. A2A receptor activation decreased CB1 receptor radioligand binding and decreased the CB1 receptor-mediated inhibition of high-K(+) -evoked glutamate release in corticostriatal terminals. Accordingly, A2A receptor activation prevented CB1 receptor-mediated paired-pulse facilitation and attenuated the CB1 receptor-mediated inhibition of synaptic transmission in glutamatergic synapses of corticostriatal slices. CONCLUSIONS AND IMPLICATIONS: Activation of presynaptic A2A receptors dampened CB1 receptor-mediated inhibition of corticostriatal terminals. This constitutes a thus far unrecognized mechanism to modulate the potent CB1 receptor-mediated presynaptic inhibition, allowing frequency-dependent enhancement of synaptic efficacy at corticostriatal synapses.

Functional interaction between pre-synaptic α6ß2-containing nicotinic and adenosine A2A receptors in the control of dopamine release in the rat striatum.

BACKGROUND AND PURPOSE: Pre-synaptic nicotinic ACh receptors (nAChRs) and adenosine A2A receptors (A2A Rs) are involved in the control of dopamine release and are putative therapeutic targets in Parkinson's disease and addiction. Since A2A Rs have been reported to interact with nAChRs, here we aimed at mapping the possible functional interaction between A2A Rs and nAChRs in rat striatal dopaminergic terminals. EXPERIMENTAL APPROACH: We pharmacologically characterized the release of dopamine and defined the localization of nAChR subunits in rat striatal nerve terminals in vitro and carried out locomotor behavioural sensitization in rats in vivo. KEY RESULTS: In striatal nerve terminals, the selective A2A R agonist CGS21680 inhibited, while the A2A R antagonist ZM241385 potentiated the nicotine-stimulated [(3) H]dopamine ([(3) H]DA) release. Upon blockade of the α6 subunit-containing nAChRs, the remaining nicotine-stimulated [(3) H]DA release was no longer modulated by A2A R ligands. In the locomotor sensitization experiments, nicotine enhanced the locomotor activity on day 7 of repeated nicotine injection, an effect that no longer persisted after 1 week of drug withdrawal. Notably, ZM241385-injected rats developed locomotor sensitization to nicotine already on day 2, which remained persistent upon nicotine withdrawal. CONCLUSIONS AND IMPLICATIONS: These results provide the first evidence for a functional interaction between nicotinic and adenosine A2A R in striatal dopaminergic terminals, with likely therapeutic consequences for smoking, Parkinson's disease and other dopaminergic disorders.

Functional identification of cell phenotypes differentiating from mice retinal neurospheres using single cell calcium imaging.

Degeneration of neural retina causes vision impairment and can lead to blindness. Neural stem and progenitor cells might be used as a tool directed to regenerative medicine of the retina. Here, we describe a novel platform for cell phenotype-specific drug discovery and screening of proneurogenic factors, able to boost differentiation of neural retinal progenitor cells. By using single cell calcium imaging (SCCI) and a rational-based stimulation protocol, a diversity of cells emerging from differentiated retinal neurosphere cultures were identified. Exposure of retinal progenitor cultures to KCl or to α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) stimulated Ca(2+) transients in microtubule-associated protein 2 (MAP-2) positive neurons. Doublecortin (DCX) and polysialated neural cell adhesion molecule (PSA-NCAM) positive neuroblasts were distinguished from differentiated neurons on the basis of their response to muscimol. Ca(2+) fluxes in glial fibrillary acidic protein (GFAP) or glutamine synthetase (GS) positive cells were induced by ATP. To validate the platform, neurospheres were treated with brain-derived neurotrophic factor (BDNF) (proneurogenic) or ciliary neurotrophic factor (CNTF) (gliogenic factor). BDNF increased the percentage of differentiated cells expressing Tuj-1 sensitive to KCl or AMPA and reduced the population of cells responding to muscimol. CNTF exposure resulted in a higher number of cells expressing GFAP responding to ATP. All together, our data may open new perspectives for cell type-specific discovery of drug targets and screening of novel proneurogenic factors to boost differentiation of neural retina cells to treat degenerative retinal diseases.

Anandamide and NADA bi-directionally modulate presynaptic Ca2+ levels and transmitter release in the hippocampus.

BACKGROUND AND PURPOSE: Inhibitory CB(1) cannabinoid receptors and excitatory TRPV(1) vanilloid receptors are abundant in the hippocampus. We tested if two known hybrid endocannabinoid/endovanilloid substances, N-arachidonoyl-dopamine (NADA) and anandamide (AEA), presynapticaly increased or decreased intracellular calcium level ([Ca(2+)](i)) and GABA and glutamate release in the hippocampus. EXPERIMENTAL APPROACH: Resting and K(+)-evoked levels of [Ca(2+)](i) and the release of [(3)H]GABA and [(3)H]glutamate were measured in rat hippocampal nerve terminals. KEY RESULTS: NADA and AEA per se triggered a rise of [Ca(2+)](i) and the release of both transmitters in a concentration- and external Ca(2+)-dependent fashion, but independently of TRPV(1), CB(1), CB(2), or dopamine receptors, arachidonate-regulated Ca(2+)-currents, intracellular Ca(2+) stores, and fatty acid metabolism. AEA was recently reported to block TASK-3 potassium channels thereby depolarizing membranes. Common inhibitors of TASK-3, Zn(2+), Ruthenium Red, and low pH mimicked the excitatory effects of AEA and NADA, suggesting that their effects on [Ca(2+)](i) and transmitter levels may be attributable to membrane depolarization upon TASK-3 blockade. The K(+)-evoked Ca(2+) entry and Ca(2+)-dependent transmitter release were inhibited by nanomolar concentrations of the CB(1) receptor agonist WIN55212-2; this action was sensitive to the selective CB(1) receptor antagonist AM251. However, in the low micromolar range, WIN55212-2, NADA and AEA inhibited the K(+)-evoked Ca(2+) entry and transmitter release independently of CB(1) receptors, possibly through direct Ca(2+) channel blockade. CONCLUSIONS AND IMPLICATIONS: We report here for hybrid endocannabinoid/endovanilloid ligands novel dual functions which were qualitatively similar to activation of CB(1) or TRPV(1) receptors, but were mediated through interactions with different targets.

Distinct mechanisms underlying alpha1-adrenoceptor and P2x purinoceptor operated ATP release and contraction in the guinea-pig vas deferens.

The temperature-dependence of ATP release and contraction response evoked by different agonists were investigated in superfused guinea-pig vas deferens. Alpha-adrenoceptor agonists, i.e. noradrenaline (300 microM), and alpha-methyl-noradrenaline (300 microM), increased the basal ATP outflow, measured by the luciferin-luciferase assay, and induced biphasic contractile response. Cooling the bath temperature to 12 degrees C almost completely inhibited ATP release and twitch contraction evoked by alpha-adrenoceptor agonists, whereas the phasic contraction remained unaffected. In contrast, twitch contraction and subsequent ATP release induced by beta,gamma-methylene-ATP, a selective P2 receptor agonist (100 microM), was not reduced by low temperature. The ectoATPase activity, measured by HPLC technique was not significantly different at 37 degrees C and 12 degrees C. Nifedipine (1 microM), the voltage sensitive Ca2+ channel blocker eliminated beta,gamma-methylene-ATP evoked twitch contraction but not ATP release. In conclusion, alpha-adrenoceptor and P2 receptor agonists utilize distinct mechanisms to elicit ATP release and contraction: alpha-adrenoceptor-mediated ATP release and contraction is temperature-dependent, indicating the involvement of a carrier-mediated process in it, whereas P2x purinoceptor evoked ATP release and twitch is mediated by a different mechanism.

Excessive release of [3H] noradrenaline by veratridine and ischemia in spinal cord.

In this study, the properties of ischemic condition-induced and veratridine-evoked [3H]noradrenaline ([3H]NA) release from rat spinal cord slices were compared. It was expected that ischemia mimicked by oxygen and glucose deprivation results in the impairment of Na+/K+ -ATPase with a consequent elevation of the intracellular Na+ -level which reverses the NA carrier and promotes excessive NA release, and veratridine, by the activation of Na+ channels, releases NA both carrier-mediated and Ca2+ -dependent, i.e. vesicular manner. In our experiments, veratridine (1-100 microM) dose-dependently increased the resting [3H]NA release, and its effect was only partially blocked by low temperature or the lack of external calcium, whereas the sodium channel inhibitor tetrodotoxin (TTX, 1 microM) completely prevented it, indicating that veratridine induces NA release via axonal depolarization and reversing the transporters by eliciting Na+ -influx. In contrast to TTX, the local anesthetic lidocaine (100 microM) only partially blocked the veratridine-induced [3H]NA release due to its inhibitory action on K+ channels. The ischemia-induced [3H]NA release was abolished at 12 degrees C, a temperature known to block only the transporter-mediated release of transmitters. However, lidocaine was also partially effective to reverse the action of ischemia on the NA release, indicating that lidocaine is not a useful compound in the treatment of spinal cord-injured patients against the excessive excytotoxic NA release.

Long-lasting facilitation of 4-amino-n-[2,3-(3)H]butyric acid ([(3)H]GABA) release from rat hippocampal slices by nicotinic receptor activation.

In this study we explored the effect of the stimulation of nicotinic acetylcholine receptors located on interneurons by measuring 4-amino-n-[2,3-(3)H]butyric acid ([(3)H]GABA) release and monitoring [Ca (2+)](i) in superfused hippocampal slices. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione, (+/-)-2-amino-5-phosphonopentanoic acid, and atropine, i.e., under the blockade of N-methyl-D-aspartate and non-N-methyl-D-aspartate glutamate and muscarinic receptors, nicotine did not alter the spontaneous outflow of [(3)H]GABA, but significantly increased the stimulation-evoked [(3)H]GABA efflux. This effect of nicotine depended on the time interval between nicotine treatment and electrical stimulus, the concentration of nicotine (1-100 microM), and the parameters of electrical depolarization. Acetylcholine (0.03-3 mM), and the alpha 7 subtype-selective agonist choline (0.1-10 mM), also potentiated stimulus-evoked release of [(3)H]GABA, whereas 1,1-dimethyl-4-phenilpiperazinium iodide failed to increase the tritium outflow significantly. The effect of nicotine treatment was prevented by tetrodotoxin (1 microM) and by the nicotinic acetylcholine receptor antagonist mecamylamine (10 microM), and the alpha 7 subtype-selective antagonists alpha-bungarotoxin (100 nM) and methyllycaconitine (10 nM), whereas dihidro-beta-erythroidine (20 nM) was without effect. Perfusion of 100 microM nicotine caused a [Ca(2+)](i) transient in about one-third of the tested interneurons; however, the response to subsequent electrical stimulation remained unchanged. Inhibition of the GABA transporter system by nipecotic acid (1 mM) or by decreasing the bath temperature to 12 degrees C abolished completely the effect of nicotine to potentiate the stimulation-evoked release of GABA. These findings indicate that the activation of alpha 7-type nicotinic receptors of hippocampal interneurons results in a long-lasting ability of these cells to respond to depolarization with an increased release of GABA mediated by the transporter system.

GABAergic interneurons are the targets of cannabinoid actions in the human hippocampus.

Cannabinoids have been shown to disrupt memory processes in mammals including humans. Although the CB1 neuronal cannabinoid receptor was identified several years ago, neuronal network mechanisms mediating cannabinoid effects are still controversial in animals, and even more obscure in humans. In the present study, the localization of CB1 receptors was investigated at the cellular and subcellular levels in the human hippocampus, using control post mortem and epileptic lobectomy tissue. The latter tissue was also used for [3H]GABA release experiments, testing the predictions of the anatomical data. Detectable expression of CB1 was confined to interneurons, most of which were found to be cholecystokinin-containing basket cells. CB1-positive cell bodies showed immunostaining in their perinuclear cytoplasm, but not in their somadendritic plasmamembrane. CB1-immunoreactive axon terminals densely covered the entire hippocampus, forming symmetrical synapses characteristic of GABAergic boutons. Human temporal lobectomy samples were used in the release experiments, as they were similar to the controls regarding cellular and subcellular distribution of CB1 receptors. We found that the CB1 receptor agonist, WIN 55,212-2, strongly reduced [3H]GABA release, and this effect was fully prevented by the specific CB1 receptor antagonist SR 141716A. This unique expression pattern and the presynaptic modulation of GABA release suggests a conserved role for CB1 receptors in controlling inhibitory networks of the hippocampus that are responsible for the generation and maintenance of fast and slow oscillatory patterns. Therefore, a likely mechanism by which cannabinoids may impair memory and associational processes is an alteration of the fine-tuning of synchronized, rhythmic population events.