Stefania D'Agostino

Neurons and ECM regulate occludin localization in brain endothelial cells

We report that extracellular matrix and neurons modulate the expression of occludin, one of the main components of tight junctions, by rat brain endothelial cells (RBE4.B). Of the three extracellular matrix proteins which we tested (collagen I, collagen IV, and laminin), collagen IV stimulated at the best the expression of occludin mRNA. The corresponding protein, however, was not synthesized. Significant amounts of occludin accumulated only when RBE4.B cells were cultured on collagen IV-coated inserts, in the presence of cortical neurons, plated on laminin-coated companion wells. Finally, occludin segregated at the cell periphery, only when endothelial cells were co-cultured with neurons for ≥ 1 week.

Effects of nitric oxide-active drugs on the discharge of subthalamic neurons : microiontophoretic evidence in the rat

The presence of nitric oxide (NO) synthase and of soluble guanylyl cyclase, the main NO‐activated metabolic pathway, has been demonstrated in many cells of the subthalamic nucleus. In this study, the effects induced on the firing of 96 subthalamic neurons by microiontophoretically administering drugs modifying NO neurotransmission were explored in anaesthetized rats. Recorded neurons were classified into regularly and irregularly discharging on the basis of their firing pattern. Nω‐nitro‐l‐arginine methyl ester (L‐NAME; a NO synthase inhibitor), 3‐morpholino‐sydnonimin‐hydrocloride (SIN‐1; a NO donor), S‐nitroso‐glutathione (SNOG; another NO donor) and 8‐Br‐cGMP (a cell‐permeable analogue of cGMP, the main second‐messenger of NO neurotransmission) were iontophoretically applied while performing single‐unit extracellular recordings. The activity of most neurons was influenced in a statistically significant way: in particular, both current‐related inhibitory L‐NAME‐induced effects (20/39 tested cells) and excitatory effects of SIN‐1 (25/41 tested neurons), SNOG (19/32 tested cells) and 8‐Br‐cGMP (13/19 tested neurons) were demonstrated. Neither statistically significant differences between the responses of regularly and irregularly discharging cells, nor specific topographical clustering of responding neurons, were demonstrated. Neurons administered drugs oppositely modulating the NO neurotransmission often displayed responses to only one treatment. We hypothesize that NO neurotransmission could exert a modulatory influence upon subthalamic neurons, with a prevalent excitatory effect. However, in the light of the presence of some responses of opposite sign to the same drug displayed by different subthalamic neurons, more complex effects of NO neurotransmission could be suggested, probably due to interactions with other classical neurotransmitter systems.

Intensity of GABA-Evoked Responses Is Modified by Nitric Oxide-Active Compounds in the Subthalamic Nucleus of the Rat: A Microiontophoretic Study

We have previously described modulatory effects of nitric oxide (NO)–active drugs on subthalamic nucleus (STN) neurons. In this study, the effects of microiontophoretically applied NO‐active compounds on GABA‐evoked responses were investigated in subthalamic neurons extracellularly recorded from anesthetized rats: 45 of 62 cells were excited by S‐nitroso‐glutathione (SNOG), an NO donor, whereas 28 of 43 neurons were inhibited by Nω‐nitro‐L‐arginine methyl ester (L‐NAME), a NOS inhibitor. Nearly all neurons responding to SNOG and/or L‐NAME showed significant inhibitory responses to the administration of iontophoretic GABA. In these cells, the changes induced by NO‐active drugs in the magnitude of GABA‐evoked responses were used as indicators of NO modulation. In fact, when an NO‐active drug was co‐iontophoresed with GABA, significant changes in GABA‐induced responses were observed: generally, decreased magnitudes of GABA‐evoked responses were observed during continuous SNOG ejection, whereas the administration of L‐NAME enhanced GABA responses. In contrast, glutamate‐evoked responses were enhanced by SNOG and dampened by L‐NAME co‐iontophoresis. Furthermore, the iontophoretic administration of bicuculline (a GABAA receptor antagonist) completely abolished the GABA‐evoked inhibitory responses and reduced the magnitude of both the SNOG‐ and L‐NAME‐induced effects. The results suggest that the NO‐mediated modulation of subthalamic neurons could also be a result of an interaction between NO and GABAA neurotransmission. Increased NOS activity has been shown in the hyperactive STN neurons of parkinsonian patients; on the basis of our observations about the influence of NO‐active drugs on the baseline and GABA‐evoked activity of subthalamic cells, such hyperactivity suggests the involvement of increased NO levels and reduced sensitivity to GABA.