Saffron reduces ATP-induced retinal cytotoxicity by targeting P2X7 receptors.

P2X7-type purinergic receptors are distributed throughout the nervous system where they contribute to physiological and pathological functions. In the retina, this receptor is found in both inner and outer cells including microglia modulating signaling and health of retinal cells. It is involved in retinal neurodegenerative disorders such as retinitis pigmentosa and age-related macular degeneration (AMD). Experimental studies demonstrated that saffron protects photoreceptors from light-induced damage preserving both retinal morphology and visual function and improves retinal flicker sensitivity in AMD patients. To evaluate a possible interaction between saffron and P2X7 receptors (P2X7Rs), different cellular models and experimental approaches were used. We found that saffron positively influences the viability of mouse primary retinal cells and photoreceptor-derived 661W cells exposed to ATP, and reduced the ATP-induced intracellular calcium increase in 661W cells. Similar results were obtained on HEK cells transfected with recombinant rat P2X7R but not on cells transfected with rat P2X2R. Finally, patch-clamp experiments showed that saffron inhibited cationic currents in HEK-P2X7R cells. These results point out a novel mechanism through which saffron may exert its protective role in neurodegeneration and support the idea that P2X7-mediated calcium signaling may be a crucial therapeutic target in the treatment of neurodegenerative diseases.

Neuropeptide S inhibits release of 5-HT and glycine in mouse amygdala and frontal/prefrontal cortex through activation of the neuropeptide S receptor.

Neuropeptide S (NPS) is a neurotransmitter/neuromodulator that has been identified as the natural ligand of G protein-coupled receptors termed NPS receptors (NPSRs). The NPS-NPSR system is involved in the control of numerous centrally-mediated behaviours, including anxiety. As several classical transmitters play a role in fear/anxiety, we here investigated the regulation by NPS of the exocytotic release of 5-hydroxytryptamine (5-HT) and glycine in nerve terminals isolated from mouse frontal/prefrontal cortex and amygdala. Synaptosomes, prelabelled with the tritiated neurotransmitters, were depolarized in superfusion with 12-15 mM KCl and exposed to varying concentrations of NPS. The evoked release of [(3)H]5-HT in frontal/prefrontal cortex was potently inhibited by NPS (maximal effect about 25% at 0.1 nM). Differently, the neuropeptide exhibited higher efficacy but much lower potency in amygdala (maximal effect about 40% at 1 µM). NPS was an extremely potent inhibitor of the K(+)-evoked release of [(3)H]glycine in frontal/prefrontal nerve endings (maximal effect about 25% at 1 pM). All the inhibitory effects observed were counteracted by the NPSR antagonist SHA 68, indicating that the neuropeptide acted at NPSRs. In conclusion, NPS can inhibit the exocytosis of 5-HT and of glycine through the activation of presynaptic NPSRs situated on serotonergic and glycinergic terminals in areas involved in fear/anxiety behaviours. The possibility exists that the NPSRs in frontal/prefrontal cortex are high-affinity receptors involved in non-synaptic transmission, whereas the NPSRs on amygdala serotonergic terminals are low-affinity receptors involved in axo-axonic synaptic communication.

Dual effect of beta-amyloid on α7 and α4ß2 nicotinic receptors controlling the release of glutamate, aspartate and GABA in rat hippocampus.

BACKGROUND: We previously showed that beta-amyloid (Aß), a peptide considered as relevant to Alzheimer's Disease, is able to act as a neuromodulator affecting neurotransmitter release in absence of evident sign of neurotoxicity in two different rat brain areas. In this paper we focused on the hippocampus, a brain area which is sensitive to Alzheimer's Disease pathology, evaluating the effect of Aß (at different concentrations) on the neurotransmitter release stimulated by the activation of pre-synaptic cholinergic nicotinic receptors (nAChRs, α4ß2 and α7 subtypes). Particularly, we focused on some neurotransmitters that are usually involved in learning and memory: glutamate, aspartate and GABA. METHODOLOGY/FINDINGS: WE USED A DUAL APPROACH: in vivo experiments (microdialysis technique on freely moving rats) in parallel to in vitro experiments (isolated nerve endings derived from rat hippocampus). Both in vivo and in vitro the administration of nicotine stimulated an overflow of aspartate, glutamate and GABA. This effect was greatly inhibited by the highest concentrations of Aß considered (10 µM in vivo and 100 nM in vitro). In vivo administration of 100 nM Aß (the lowest concentration considered) potentiated the GABA overflow evoked by nicotine. All these effects were specific for Aß and for nicotinic secretory stimuli. The in vitro administration of either choline or 5-Iodo-A-85380 dihydrochloride (α7 and α4ß2 nAChRs selective agonists, respectively) elicited the hippocampal release of aspartate, glutamate, and GABA. High Aß concentrations (100 nM) inhibited the overflow of all three neurotransmitters evoked by both choline and 5-Iodo-A-85380 dihydrochloride. On the contrary, low Aß concentrations (1 nM and 100 pM) selectively acted on α7 subtypes potentiating the choline-induced release of both aspartate and glutamate, but not the one of GABA. CONCLUSIONS/SIGNIFICANCE: The results reinforce the concept that Aß has relevant neuromodulatory effects, which may span from facilitation to inhibition of stimulated release depending upon the concentration used.

Presynaptic nicotinic α7 and non-α7 receptors stimulate endogenous GABA release from rat hippocampal synaptosomes through two mechanisms of action.

BACKGROUND: Although converging evidence has suggested that nicotinic acetylcholine receptors (nAChR) play a role in the modulation of GABA release in rat hippocampus, the specific involvement of different nAChR subtypes at presynaptic level is still a matter of debate. In the present work we investigated, using selective α7 and α4ß2 nAChR agonists, the presence of different nAChR subtypes on hippocampal GABA nerve endings to assess to what extent and through which mechanisms they stimulate endogenous GABA release. METHODOLOGY/FINDINGS: All agonists elicited GABA overflow. Choline (Ch)-evoked GABA overflow was dependent to external Ca(2+), but unaltered in the presence of Cd(2+), tetrodotoxin (TTX), dihydro-ß-erythroidine (DHßE) and 1-(4,4-Diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride SKF 89976A. The effect of Ch was blocked by methyllycaconitine (MLA), α-bungarotoxin (α-BTX), dantrolene, thapsigargin and xestospongin C, suggesting that GABA release might be triggered by Ca(2+) entry into synaptosomes through the α7 nAChR channel with the involvement of calcium from intracellular stores. Additionally, 5-Iodo-A-85380 dihydrochloride (5IA85380) elicited GABA overflow, which was Ca(2+) dependent, blocked by Cd(2+), and significantly inhibited by TTX and DHßE, but unaffected by MLA, SKF 89976A, thapsigargin and xestospongin C and dantrolene. These findings confirm the involvement of α4ß2 nAChR in 5IA85380-induced GABA release that seems to occur following membrane depolarization and opening calcium channels. CONCLUSIONS/SIGNIFICANCE: Rat hippocampal synaptosomes possess both α7 and α4ß2 nAChR subtypes, which can modulate GABA release via two distinct mechanisms of action. The finding that GABA release evoked by the mixture of sub-maximal concentration of 5IA85380 plus sub-threshold concentrations of Ch was significantly larger than that elicited by the sum of the effects of the two agonists is compatible with the possibility that they coexist on the same nerve terminals. These findings would provide the basis for possible selective pharmacological strategies to treat neuronal disorders that involve the dysfunction of hippocampal cholinergic system.

Alterations of glutamate release in the spinal cord of mice with experimental autoimmune encephalomyelitis.

We have investigated the spontaneous and the depolarisation-induced release of [(3)H]D-aspartate ([(3)H]D-ASP), a non-metabolisable analogue of glutamate, in spinal cord slices, synaptosomes and gliosomes from mice with experimental autoimmune encephalomyelitis (EAE) at 13, 21 and 55 days post-immunisation (d.p.i.), representing onset, peak and chronic phases of the pathology. At 13 and 21 d.p.i., the KCl-evoked, calcium-dependent overflow of [(3)H]D-ASP in spinal cord slices was significantly lower (30-40%), whereas at 55 d.p.i. it was significantly higher (30%), than that elicited in matched controls. When the release was measured from spinal cord synaptosomes and gliosomes in superfusion, a different picture emerged. The spontaneous and the KCl(15 mM)-induced release of [(3)H]D-ASP were significantly increased both in synaptosomes (17% and 45%, respectively) and gliosomes (26% and 25%, respectively) at 21, but not at 13, d.p.i. At 55 d.p.i., the KCl-induced [(3)H]D-ASP release was significantly increased (40%) only in synaptosomes. Finally, uptake of [(3)H]D-ASP was markedly (50-60%) increased in spinal cord synaptosomes, but not in gliosomes, obtained from EAE mice at 21 d.p.i., whereas no differences could be detected at 13 d.p.i. Our data indicate that glutamatergic neurotransmission is altered in the spinal cord of EAE mice.

L-aspartate as an amino acid neurotransmitter: mechanisms of the depolarization-induced release from cerebrocortical synaptosomes.

The role of L-aspartate as a classical neurotransmitter of the CNS has been a matter of great debate. In this study, we have characterized the main mechanisms of its depolarization-induced release from rat purified cerebrocortical synaptosomes in superfusion and compared them with those of the well known excitatory neurotransmitter L-glutamate. High KCl and 4-aminopyridine were used as depolarizing agents. At 15 mM KCl, the overflows of both transmitters were almost completely dependent on external Ca2+. At 35 and 50 mM KCl, the overflows of L-aspartate, but not those of L-glutamate, became sensitive to DL-threo-b-benzyloxy aspartic acid (DL-TBOA), an excitatory amino acid transporter inhibitor. In the presence of DL-TBOA, the 50 mM KCl-evoked release of L-aspartate was still largely external Ca2+-dependent. The DL-TBOA insensitive,external Ca2+-independent component of the 50 mM KCl-evoked overflows of L-aspartate and L-glutamate was significantly decreased by the mitochondrial Na+/Ca2+ exchanger blocker CGP 37157. The Ca2+-dependent, KCl-evoked overflows of L-aspartate and L-glutamate were diminished by botulinum neurotoxin C, although to a significantly different extent. The 4-aminopyridine-induced L-aspartate and L-glutamate release was completely external Ca2+-dependent and never affected by DL-TBOA. Superimposable results have been obtained by pre-labeling synaptosomes with [3H]D aspartate and [3H]L-glutamate. Therefore, our data showing that L-aspartate is released from nerve terminals by calcium dependent,exocytotic mechanisms support the neurotransmitter role of this amino acid.

In vivo effects of phosphodiesterase inhibition on basal cyclic guanosine monophosphate levels in the prefrontal cortex, hippocampus and cerebellum of freely moving rats.

We have characterized the various phosphodiesterases (PDE) that degrade cyclic GMP in the prefrontal cortex, hippocampus, and cerebellum using the microdialysis technique to measure in vivo extracellular cyclic GMP in awake rats. The following PDE blockers were used (100 and 1,000 microM): 8-methoxymethyl-IBMX (8-MM-IBMX), erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), milrinone, rolipram, and zaprinast. For solubility reasons, sildenafil was tested only at 100 microM. All drugs were administered locally in the brain regions through the dialysis probe. At 100 microM, 8-MM-IBMX enhanced the cyclic nucleotide extracellular levels in the prefrontal cortex and hippocampus but not in the cerebellum; EHNA and milrinone were active only in the hippocampus; rolipram was devoid of any effect; zaprinast and sildenafil were effective in all three brain areas. At 1 mM, 8-MM-IBMX, milrinone, and zaprinast increased extracellular cyclic GMP in all the brain regions examined, EHNA became active also in the prefrontal cortex and rolipram showed a significant effect only in the cerebellum. This is the first in vivo functional study showing that, in cortex, PDE1, -2, and -5/9 degrade cGMP, with PDE9 probably playing a major role; in hippocampus, PDE5/9 and PDE1 are mainly involved and seem almost equally active, but PDE2 and -3 also contribute; in cerebellum, PDE5/9 are the main cGMP hydrolyzing enzymes, but also PDE1 and -4 significantly operate.