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.

Hypoxia-induced desensitization and internalization of adenosine A1 receptors in the rat hippocampus.

Activation of A1 adenosine receptors is important for both the neuromodulatory and neuroprotective effects of adenosine. However, short periods of global ischemia decrease A1 adenosine receptor density in the brain and it is not known if a parallel loss of functional efficiency of A1 adenosine receptors occurs. We now tested if hypoxia leads to changes in the density and efficiency of A1 adenosine receptors to inhibit excitatory synaptic transmission in rat hippocampal slices. In control conditions, the adenosine analog 2-chloroadenosine, inhibited field excitatory post-synaptic potentials with an EC50 of 0.23 microM. After hypoxia (95% N2 and 5% CO2, for 60 min) and reoxygenation (30 min), the EC50 increased to 0.73 microM. This EC50 shift was prevented by the presence of the A1 adenosine receptor antagonist 8-phenyltheophyline, but not by the A(2A)R antagonist 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine, during the hypoxic period. This decreased efficiency of A1 adenosine receptors was not paralleled by a global change of A1 adenosine receptor density or affinity (as evaluated by the binding parameters obtained in nerve terminal membranes). However, the density of biotinylated A1 adenosine receptors at the plasma membrane of nerve terminals was reduced by 30% upon hypoxia/reoxygenation, in a manner prevented by the A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine and mimicked by prolonged (60 min) supra-maximal activation of A1 adenosine receptors with 2-chloroadenosine (10 microM). These results indicate that hypoxia leads to a rapid (<90 min) homologous desensitization of A1 adenosine receptor-mediated inhibition of synaptic transmission that is likely due to an internalization of A1 adenosine receptors in nerve terminals.

Adenosine A2A receptors and metabotropic glutamate 5 receptors are co-localized and functionally interact in the hippocampus: a possible key mechanism in the modulation of N-methyl-D-aspartate effects.

Hippocampal metabotropic glutamate 5 receptors (mGlu5Rs) regulate both physiological and pathological responses to glutamate. Because mGlu5R activation enhances NMDA-mediated effects, and given the role played by NMDA receptors in synaptic plasticity and excitotoxicity, modulating mGlu5R may influence both the physiological and the pathological effects elicited by NMDA receptor stimulation. We evaluated whether adenosine A2A receptors (A(2A)Rs) modulated mGlu5R-dependent effects in the hippocampus, as they do in the striatum. Co-application of the A(2A)R agonist CGS 21680 with the mGlu5R agonist (RS)-2-chloro-s-hydroxyphenylglycine(CHPG) synergistically reduced field excitatory postsynaptic potentials in the CA1 area of rat hippocampal slices. Endogenous tone at A(2A)Rs seemed to be required to enable mGlu5R-mediated effects, as the ability of CHPG to potentiate NMDA effects was antagonized by the selective A(2A)R antagonist ZM 241385 in rat hippocampal slices and cultured hippocampal neurons, and abolished in the hippocampus of A(2A)R knockout mice. Evidence for the interaction between A(2A)Rs and mGlu5Rs was further strengthened by demonstrating their co-localization in hippocampal synapses. This is the first evidence showing that hippocampal A(2A)Rs and mGlu5Rs are co-located and act synergistically, and that A(2A)Rs play a permissive role in mGlu5R receptor-mediated potentiation of NMDA effects in the hippocampus.

Presynaptic kainate receptors are localized close to release sites in rat hippocampal synapses.

The subsynaptic distribution of kainate receptors is still a matter of much debate given its importance to understand the way they influence neuronal communication. Here, we show that, in synapses of the rat hippocampus, presynaptic kainate receptors are localized within the presynaptic active zone close to neurotransmitter release sites. The activation of these receptors with low concentrations of agonists induces the release of [(3)H]glutamate in the absence of a depolarizing stimulus. Furthermore, this modulation of [(3)H]glutamate release by kainate is more efficient when compared with a KCl-evoked depolarization that causes a more than two-fold increase in the intra-terminal calcium concentration but no apparent release of [(3)H]glutamate, suggesting a direct receptor-mediated process. Using a selective synaptic fractionation technique that allows for a highly efficient separation of presynaptic, postsynaptic and non-synaptic proteins we confirmed that, presynaptically, kainate receptors are mainly localized within the active zone of hippocampal synapses where they are expected to be in a privileged position to modulate synaptic phenomena.

Adenosine A1 and A2A receptors are co-expressed in pyramidal neurons and co-localized in glutamatergic nerve terminals of the rat hippocampus.

Adenosine is a neuromodulator that controls neurotransmitter release through inhibitory A1 and facilitatory A2A receptors. Although both adenosine receptor-mediated inhibition and facilitation of glutamate release have been observed, it is not clear whether both A1 and A2A receptors are located in the same glutamatergic nerve terminal or whether they are located on different populations of these terminals. Thus, we have tested if single pyramidal glutamatergic neurons from the hippocampus simultaneously expressed A1 and A2A receptor mRNA and if A1 and A2A receptors were co-localized in hippocampal glutamatergic nerve terminals. Single cell PCR analysis of visually identified pyramidal neurons revealed the simultaneous presence of A1 and A2A receptor mRNA in four out 16 pyramidal cells possessing glutamatergic markers but not GABAergic or astrocytic markers. Also, A1 and A2A receptor immunoreactivities were co-localized in 26 +/- 4% of nerve terminals labeled with antibodies against vesicular glutamate transporters type 1 or 2, i.e. glutamatergic nerve terminals. This indicates that glutamatergic neurons in the hippocampus co-express A1 and A2A receptors and that these two receptors are co-localized in a subset of glutamatergic nerve terminals.

Different synaptic and subsynaptic localization of adenosine A2A receptors in the hippocampus and striatum of the rat.

Adenosine A(2A) receptors are most abundant in the striatum where they control the striatopallidal pathway thus controlling locomotion. Extra-striatal A(2A) receptors are considerably less abundant but their blockade confers robust neuroprotection. We now have investigated if striatal and extra-striatal A(2A) receptors have a different neuronal location to understand their different functions. The binding density of the A(2A) antagonist, [(3)H]-7-(2-phenylethyl)-5-amino-2-(2-furyl)pyrazolo[4,3e][1,2,4]triazolo[1,5-c]pyrimidine ([(3)H]SCH 58261), was enriched in nerve terminals membranes (B(max)=103+/-12 fmol/mg protein) compared with total membranes (B(max)=29+/-4 fmol/mg protein) from the hippocampus, the same occurring with A(2A) receptor immunoreactivity. In contrast, there was no enrichment of [(3)H]SCH 58261 binding or A(2A) receptor immunoreactivity in synaptosomal compared with total membranes from the striatum. Further subcellular fractionation of hippocampal nerve terminals revealed that A(2A) receptor immunoreactivity was enriched in the active zone of presynaptic nerve terminals, whereas it was predominantly located in the postsynaptic density in the striatum, although a minority of striatal A(2A) receptors were located in the presynaptic active zone. These results indicate that A(2A) receptors in the striatum are not enriched in synapses in agreement with the preponderant role of A(2A) receptors in signal processing in striatopallidal neurons. In contrast, hippocampal A(2A) receptors are enriched in synapses, mainly in the active zone, in accordance with their role in controlling neurotransmitter release. This regional variation in the neuronal distribution of A(2A) receptors reinforces the care required to extrapolate our knowledge from striatal A(2A) receptors to other brain preparations.