In the present study we investigated the effect of in vivo intrastriatal injection of quinolinic acid (QA) on cytoskeletal proteins in astrocytes and neurons of young rats at early stage (30 min) after infusion. QA (150 nmoles/0.5 microL) significantly increased the in vitro phosphorylation of the low molecular weight neurofilament subunit (NFL) and the glial fibrillary acidic protein (GFAP) of neurons and astrocytes, respectively. This effect was mediated by cAMP-dependent protein kinase A (PKA), protein kinase C (PKC) and Ca(2+)/calmodulin-dependent protein kinase II (PKCaMII). In contrast, mitogen activated protein kinases were not activated by QA infusion. Furthermore, the specific N-methyl-D-aspartate (NMDA) antagonist MK-801 (0.25 mg/kg i.p), the antioxidant L-NAME (60 mg\kg\day), and diphenyldisselenide (PheSe)(2) (0.625 mg\kg\day) injected prior to QA infusion totally prevented QA-induced cytoskeletal hyperphosphorylation. We also observed that QA-induced hyperphosphorylation was targeted at the Ser55 phosphorylating site on NFL head domain, described as a regulatory site for NF assembly in vivo. This effect was fully prevented by MK801, by the PKA inhibitor H89 and by (PheSe)(2), whereas staurosporine (PKC inhibitor) only partially prevented Ser55 phosphorylation. The PKCaMII inhibitor (KN93) and the antioxidant L-NAME failed to prevent the hyperphosphorylation of Ser55 by QA infusion. Therefore, we presume that QA-elicited hyperphosphorylation of the neural cytoskeleton, and specially of NFLSer55, achieved by intrastriatal QA injection could represent an early step in the pathophysiological cascade of deleterious events exerted by QA in rat striatum. Our observations also indicate that NMDA-mediated Ca(2+) events and oxidative stress may be related to the altered protein cytoskeleton hyperphosphorylation observed with important implications for brain function.

Accumulating evidences indicate that endogenous modulators of excitatory synapses in the mammalian brain are potential targets for treating neuropsychiatric disorders. Indeed, glutamatergic and adenosinergic neurotransmissions were recently highlighted as potential targets for developing innovative anxiolytic drugs. Accordingly, it has been shown that guanine-based purines are able to modulate both adenosinergic and glutamatergic systems in mammalian central nervous system. Here, we aimed to investigate the potential anxiolytic-like effects of guanosine and its effects on the adenosinergic and glutamatergic systems. Acute/systemic guanosine administration (7.5 mg/kg) induced robust anxiolytic-like effects in three classical anxiety-related paradigms (elevated plus maze, light/dark box, and round open field tasks). These guanosine effects were correlated with an enhancement of adenosine and a decrement of glutamate levels in the cerebrospinal fluid. Additionally, pre-administration of caffeine (10 mg/kg), an unspecific adenosine receptors' antagonist, completely abolished the behavioral and partially prevented the neuromodulatory effects exerted by guanosine. Although the hippocampal glutamate uptake was not modulated by guanosine (both ex vivo and in vitro protocols), the synaptosomal K-stimulated glutamate release in vitro was decreased by guanosine (100 μM) and by the specific adenosine A receptor agonist, 2-chloro-N -cyclopentyladenosine (CCPA, 100 nM). Moreover, the specific adenosine A receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nM) fully reversed the inhibitory guanosine effect in the glutamate release. The pharmacological modulation of A receptors has shown no effect in any of the evaluated parameters. In summary, the guanosine anxiolytic-like effects seem closely related to the modulation of adenosinergic (A receptors) and glutamatergic systems.

Homocysteine (Hcy) is an excitatory amino acid which markedly enhances the vulnerability of neuronal cells to excitotoxicity and oxidative injury. Patients with severe hyperhomocysteinemia exhibit a wide range of clinical manifestations including neurological abnormalities such as mental retardation, cerebral atrophy, and seizures. In this study we treated cortical astrocytes and neurons in culture with 10 and 100 μM Hcy and after 24h exposure cytoskeletal remodeling was analyzed by immunocytochemistry. We observed dramatically altered actin cytoskeleton in astrocytes exposed to 100 μM Hcy, with concomitant change of morphology to fusiform and/or flattened cells with retracted cytoplasm. Moreover, we observed disruption of the glial fibrillary acidic protein (GFAP) meshwork, supporting misregulation of actin cytoskeleton. Induction of reactive oxygen species (ROS) in astrocytes showed fluctuating levels along 24h exposure to both Hcy concentrations. Actin remodeling induced by 100 μM Hcy was prevented by the antioxidants folate (5 μM) or trolox (80 μM). Unlike astrocyte cytoskeleton, results evidence little susceptibility of neuron cytoskeleton until 24h of treatment, since immunocytochemical analysis showed that 10 and 100 μM Hcy-treated neurons presented unaltered neurite arborization. Moreover, alterations in astrocyte and neuron viability were not observed along the 24h of exposure to Hcy. Neuron/astrocyte co-cultures evidence an anchorage dependence for neuronal survival over long exposure to Hcy. Taken together, these findings indicate, that the cytoskeleton of cortical astrocytes, but not of neurons in culture, is a target to Hcy and such effects are mediated by redox signaling. Astrocytes were able to respond to Hcy (100 μM) reorganizing their cytoskeleton, surviving, and protecting neurons from Hcy damage. Moreover our results suggest a protective role for astrocytes remodeling the cytoskelon, and probably generating signals that would assure neuronal survival in response to the damage induced by Hcy.