ABSTRACT
Title
Atypical regulation of glutamate release by endocannabinoids in rat cortical astrocytes
Authors
Giribaldi F.1
1Department of Experimental Medicine and Center of Excellence for Biomedical Research, University of Genova, Italy.
1Department of Experimental Medicine and Center of Excellence for Biomedical Research, University of Genova, Italy.
Abstract
Endogenous cannabinoids rapresent an important family of substances playing a critical role in numerous physiological processes, mostly in the central nervous system (CNS). Endocannabinoids can modulate neuronal excitability by interacting with two types of cannabinoid receptors termed CB1R and CB2R. Anandamide, an endogenous ligand for CB1R, can also acts as an agonist at the transient potential vanilloid receptor 1 (TRPV1), expressed both in the periphery and in the CNS. Overwhelming evidences support the role of endocannabinoids in neurons, although several data have lately proved their activity also in astrocytes. In this context, recent studies have demonstrated a bidirectional communication between neurons and astrocytes, based on the expression by astrocytes of different cannabinoid receptors that can be activated by neuron-released endocannabinoids. Despite this evidence, the presence of CBRs in astrocytes and their involvement in the neuron-astrocyte communication remain largely unknown.
In the present study we investigated on the precence of the endocannabinoid machinery and on the effects of cannabinoid receptor activation on glutamate release in sub-cellular astroglial particles, named gliosomes, purified from rat cerebral cortex, a preparation that appears well suited to investigate the functional neurochemistry of mature astrocytes.
Gliosomes contained the most studied endocannabinoids anandamide and 2-arachidonoylglycerol, as well their major biosynthetic and hydrolytic enzymes. Gliosomes express also CB1Rs, CB2Rs, and TRPV1, as ascertained by binding experiments, Western blotting and confocal microscopy. The modulation of the depolarization-evoked release of the glutamate analogue [3H]D-aspartate by endocannabinoid receptor activation was measured in cortical gliosomes. Methanandamide, a stable analogue of anandamide that acts as CB1R, CB2R and TRPV1 agonist, exhibited a biphasic activity: it increased the depolarization-evoked [3H]D-aspartate release at lower concentrations (around 0.1 µM) but decreased it at higher concentration (around 3 µM). Experiments made using ACEA, JWH133 or capsaicin, selective agonists at CB1R, CB2R and TRPV1 respectively, demonstrated that the potentiation of [3H]D-aspartate release was due to CB1R activation, while the inhibition was mediated by CB2Rs and TRPV1. These findings were confirmed by antagonizing methanandamide using antagonists selective for CB1R (SR141716), CB2R (SR144528) or TRPV1 (5-IRTX). Both the selective CB1 or vanilloid receptor activation by ACEA or JWH133 evoked cytoplasmic IP3 augmentation; on the contrary, cAMP levels were unchanged, suggesting phospholipase C involvement as the main signalling transduction mechanism in gliosomes.
To conclude, this investigation demonstrates that gliosomes are fully equipped with a functional endocannabinoid system and support the existence of a neuron-astrocyte communication route mediated by endocannabinoid-glutamate signalling in the cerebral cortex.
In the present study we investigated on the precence of the endocannabinoid machinery and on the effects of cannabinoid receptor activation on glutamate release in sub-cellular astroglial particles, named gliosomes, purified from rat cerebral cortex, a preparation that appears well suited to investigate the functional neurochemistry of mature astrocytes.
Gliosomes contained the most studied endocannabinoids anandamide and 2-arachidonoylglycerol, as well their major biosynthetic and hydrolytic enzymes. Gliosomes express also CB1Rs, CB2Rs, and TRPV1, as ascertained by binding experiments, Western blotting and confocal microscopy. The modulation of the depolarization-evoked release of the glutamate analogue [3H]D-aspartate by endocannabinoid receptor activation was measured in cortical gliosomes. Methanandamide, a stable analogue of anandamide that acts as CB1R, CB2R and TRPV1 agonist, exhibited a biphasic activity: it increased the depolarization-evoked [3H]D-aspartate release at lower concentrations (around 0.1 µM) but decreased it at higher concentration (around 3 µM). Experiments made using ACEA, JWH133 or capsaicin, selective agonists at CB1R, CB2R and TRPV1 respectively, demonstrated that the potentiation of [3H]D-aspartate release was due to CB1R activation, while the inhibition was mediated by CB2Rs and TRPV1. These findings were confirmed by antagonizing methanandamide using antagonists selective for CB1R (SR141716), CB2R (SR144528) or TRPV1 (5-IRTX). Both the selective CB1 or vanilloid receptor activation by ACEA or JWH133 evoked cytoplasmic IP3 augmentation; on the contrary, cAMP levels were unchanged, suggesting phospholipase C involvement as the main signalling transduction mechanism in gliosomes.
To conclude, this investigation demonstrates that gliosomes are fully equipped with a functional endocannabinoid system and support the existence of a neuron-astrocyte communication route mediated by endocannabinoid-glutamate signalling in the cerebral cortex.