Purinergic modulation of [3H]GABA release from rat hippocampal nerve terminals
Introduction
Purines are important neuromodulatory substances in the hippocampus. Stimulation of neuronal preparations causes the release of purines, such as ATP, adenosine and diadenosine polyphosphates (Pintor and Miras-Portugal, 1995, Cunha et al., 1996a) in parallel with the release of neurotransmitters (e.g. Jo and Schlichter, 1999). Adenosine tonically inhibits hippocampal neuronal excitability by acting on inhibitory A1 receptors and can also activate facilitatory A2A receptors, enhancing hippocampal neurotransmitter release (Cunha et al., 1994a) and synaptic transmission (Cunha et al., 1994b). A1 Receptor mRNA is mostly expressed in granular and pyramidal cell bodies, with a pattern similar to that of A2A receptor mRNA although the latter is more diffuse (Cunha et al., 1994a). A1 Receptors are mostly located presynaptically and in axons (Swanson et al., 1995, Cunha et al., 1996b) and their density is higher than that of A2A receptors in the hippocampus (Cunha et al., 1996b). In spite of the different densities of A1 and A2A receptors, modulation of synaptic transmission and neurotransmitter release by adenosine in the hippocampus depends on a balance between A1 and A2A receptor activation (Cunha et al., 1994a, Cunha et al., 1994b). Adenosine can also activate two other receptors (A2B and A3) which have a diffuse and low density distribution in the hippocampus and that have not been demonstrated so far to modulate neurotransmitter release. ATP can also modulate neuronal excitability in the hippocampus, either directly through activation of P2 receptors (Pankratov et al., 1998) or indirectly upon its extracellular catabolism into adenosine through an ecto-nucleotidase cascade (Cunha et al., 1996a). Diadenosine polyphosphates also modulate synaptic transmission in the hippocampus (Klishin et al., 1994) either through P2 or P4 receptors (Pintor and Miras-Portugal, 1995) or through P1 receptors upon extracellular catabolism by ecto-nucleotidases (Pintor and Miras-Portugal, 1995).
Synaptic transmission in the hippocampus is processed by excitatory pathways tightly regulated by a network of inhibitory interneurons (Buckmaster and Soltesz, 1996). Purines can modulate neuronal excitability in the hippocampus either by directly modulating excitatory circuits, as seems to be the case in adenosine A1 inhibition (Lambert and Tyler, 1991, Yoon and Rothman, 1991), or by modification of GABAergic function. Although the modulation of inhibitory transmission is of fundamental importance for information processing in the hippocampus, it is generally assumed that the GABAergic system is not a target for purinergic modulation, since adenosine A1 agonists failed to affect inhibitory transmission in hippocampal slices (Lambert and Tyler, 1991, Yoon and Rothman, 1991). In the present work, we directly investigated the involvement of different purinergic receptors in the modulation of GABA release from hippocampal nerve terminals. We found that activation of adenosine A2A receptors enhances the evoked release of GABA via P-type calcium channel activation in a cAMP- and PKC-dependent manner.
Section snippets
Nerve terminal preparation
A synaptosomal fraction from the rat hippocampus was prepared as previously described (Cunha et al., 1997a). Briefly, male Wistar rats (130–140 g) were decapitated after anesthesia under halothane atmosphere, following the Portuguese guidelines for handling experimental animals. The brains were rapidly removed into ice-cold 0.32 M sucrose solution containing 1 mM EDTA, 1 mg/ml bovine serum albumin, and 5 mM HEPES buffered to pH 7.4 with 1 M NaOH. The brain was cut longitudinally and the two
Results
The stimulation of hippocampal synaptosomes with K+ (20 mM) for 2 min caused a nearly 3-fold increase in [3H]GABA release with an S2/S1 ratio of 0.74±0.02 (Fig. 1). The average basal release of 3H]GABA was 0.11±0.01% (n=25) and the peak of [3H]GABA release induced by K+ (20 mM) during S1 was 0.31±0.03% (n=25) of total tritium retained by the synaptosomes (Fig. 1). The K+-induced [3H]GABA release was mostly calcium-dependent, since omission of calcium from the extracellular medium and addition
Discussion
The present study shows that adenosine A2A receptors facilitate GABA release from hippocampal nerve terminals. The association of adenosine A2A receptors with GABAergic neurons was hypothesized based on in situ hybridization and on autoradiography studies with the prototypical A2A receptor agonist, [3H]CGS 21680, in the hippocampus (Cunha et al., 1994b) and in the medial septum (Cunha et al., 1995) which provides a major cholinergic and GABAergic projection into the hippocampus (Wainer et al.,
Acknowledgments
The technical assistance of M.D.Constantino is greatly acknowledged. RAC thanks A. Alves-Rodrigues for critically reviewing the manuscript. Supported by Fundação para a Ciência e Tecnologia (SAU/14012/1998).
References (54)
- et al.
Phosphorylation of an α1-like subunit of an ω-conotoxin-sensitive brain calcium channel by cAMP-dependent protein kinase A and protein kinase C
J. Biol. Chem.
(1991) - et al.
Inhibition of N-, P/Q- and other types of Ca2+ channels in rat hippocampal nerve terminals by adenosine Al receptor
Eur. J. Pharmacol.
(1997) - et al.
Evidence for functionally important adenosine A2a receptors in the rat hippocampus
Brain Res.
(1994) - et al.
Adenosine A2A receptors stimulate acetylcholine release from nerve terminals of the rat hippocampus
Neurosci. Lett.
(1995) - et al.
Inhibition of [3H]aminobutyric acid release by kainate receptor activation in rat hippocampal synaptosomes
Eur. J. Pharmacol.
(1997) - et al.
An adenosine agonist inhibits and a cyclic AMP analogue enhances the release of glutamate but not GABA from slices of rat dentate gyrus
Neurosci. Lett.
(1983) - et al.
Adenosine A2A receptors facilitate 45Ca2+ uptake through class A calcium channels in rat hippocampal CA3 but not CA1 synaptosomes
Neurosci. Lett.
(1997) - et al.
Protection against hippocampal kainate excitotoxicity by intracerebral administration of an adenosine A2A receptor antagonist
Brain Res.
(1998) - et al.
Possible functional role of diadenosine polyphosphates: negative feedback for excitation in hippocampus
Neuroscience
(1994) - et al.
Adenosine depresses excitatory but not fast inhibitory synaptic transmission in area CA1 of the rat hippocampus
Neurosci. Lett.
(1991)