Search

We previously reported that pre- and postsynaptic 5-hydroxytryptamine (5-HT) receptors effectively control glutamatergic transmission in adult rat cerebellum. To investigate where 5-HT acts in the glutamate ionotropic receptors/nitric oxide/guanosine 3',5'-cyclic monophosphate (cGMP) pathway, in the present study 5-HT modulation of the cGMP response to the nitric oxide donor S-nitroso-penicillamine (SNAP) was studied in adult rat cerebellar slices. While cGMP elevation produced by high-micromolar SNAP was insensitive to 5-HT, 1 microM SNAP, expected to release nitric oxide in the low-nanomolar concentration range, elicited cGMP production and endogenous glutamate release both of which could be prevented by activating presynaptic 5-HT1D receptors. Released nitric oxide appeared responsible for cGMP production and glutamate release evoked by 1 microM SNAP, as both the effects were mimicked by the structurally unrelated nitric oxide donor 2-(N,N-diethylamino)-diazenolate-2-oxide (0.1 microM). Dependency of the 1 microM SNAP-evoked release of glutamate on external Ca2+, sensitivity to presynaptic release-regulating receptors and dependency on ionotropic glutamate receptor functioning, suggest that nitric oxide stimulates exocytotic-like, activity-dependent glutamate release. Activation of ionotropic glutamate receptors/nitric oxide synthase/guanylyl cyclase pathway by endogenously released glutamate was involved in the cGMP response to 1 microM SNAP, as blockade of NMDA/non-NMDA receptors, nitric oxide synthase or guanylyl cyclase, abolished the cGMP response. To conclude, in adult rat cerebellar slices low-nanomolar exogenous nitric oxide could facilitate glutamate exocytotic-like release possibly from parallel fibers that subsequently activated the glutamate ionotropic receptors/nitric oxide/cGMP pathway. Presynaptic 5-HT1D receptors could regulate the nitric oxide-evoked release of glutamate and subsequent cGMP production.

By taking advantages of the main features of the microelectrode array (MEA) technology (i.e. multisite recordings, stable and long-term coupling with the biological preparation), we analyzed the changes in activity patterns induced by applying specific substances to dissociated cortical neurons from rat-embryos (E18). Data were recorded simultaneously from 60 electrodes, and the electrophysiological behavior was investigated during the third week in vitro, both at the spike and burst level. The analysis of the electrophysiological activity modulation, by applying agonists of the ionotropic glutamate receptors at low (i.e. 0.2–1–5 μM) and high (i.e. 50–100 μM) concentrations, is presented. Preliminary results show that the dynamics of the in vitro cortical neurons is very sensitive to pharmacological manipulation of the glutamatergic transmission and the effects on the network behavior are strictly dependent from the drug concentration. In particular, the addition of a high-dose of agonist determined a global and irreversible depression of the network activity, while, in the low-concentration case, the electrophysiological behavior showed different results, depending on the type of receptor involved. From these observations, we are encouraged to think of a more engineered system, based on in vitro cortical neurons, as a novel sensitive system for drug (pre)-screening and neuropharmacological evaluations.

Giuseppina Leo, Michele Guescini, Susanna Genedani⁎, Vilberto Stocchi, Chiara Carone, Monica Filaferro, Davide Sisti, Manuela Marcoli, Guido Maura, Pietro Cortelli, Diego Guidolin, Kjell Fuxe, Luigi Francesco Agnati a Department of Biomedical, Metabolic Sciences and Neuroscience, Physiology and Neuroscience Unit, University of Modena and Reggio Emilia, via Campi 287, Modena, Italy b Department of Biomolecular Sciences, University of Urbino Carlo Bo, via Aurelio Saffi, 2, Urbino, PU, Italy c Department of Diagnostic, Clinical Medicine and Public Health, University of Modena and Reggio Emilia, via Campi 287, Modena, Italy d Department of Pharmacy, University of Genova, viale Cembrano, 4, Genova, Italy e Department of Biomedical and NeuroMotor Sciences (DIBINEM), Alma Mater Studiorum, University of Bologna, Via Altura 3, Bologna, Italy f IRCCS Institute of Neurological Sciences, Ospedale Bellaria, Via Altura 3, Bologna, Italy g Department of Molecular Medicine, University of Padova, via Gabelli 65, Padova, Italy h Department of Neuroscience, Karolinska Institutet, Retzius vag 8, Stockholm, Sweden

Summary Male rats were subjected to chronic stress (motion, 100 Db sound and flashing lights) from age 10 days until they were 4 months of age. Systolic blood pressure rose to 146±2, 5 mmHg by week 8 and remained significantly elevated above control values until cessation of the stress. Liver COMT activity was increased after 3 weeks, but decreased after 7 and 15 weeks of stress. Brain, heart and adrenal COMT activity was scarcely affected by stress, if at all.

The neurochemical and autonomic pharmacological profile of 1,2,3,4,10, 14b-hexahydro-2-methyl-pyrazino[2,1-a]pyrido[2,3-c]pyrido[2, 3-c] [2] benzazepine [+/-)Org 3770) and the related antidepressant drug, mianserin, have been compared. The uptake of [3H]noradrenaline ([3H]NA) in vitro was weakly affected by (+/-)Org 3770 (pKi = 5.6) in contrast to mianserin (pKi = 7.4). Both (+/-)Org 3770 and mianserin facilitated the release of [3H]NA in slices of cortex. The effects of NA mediated by alpha 2-adrenoceptors on the release of both [3H]NA or [3H]serotonin ([3H]5-HT) were antagonized by (+)Org 3770 with pKi values of 8.4 and 8.1, respectively. However, (-)Org 3770 only antagonized the effect of NA on the release of [3H]5-HT (pA2 = 7.7). The binding of [3H]rauwolscine to alpha 2-adrenoceptors was inhibited by (+/-)Org 3770 and mianserin with identical affinity (pKi = 7.0), whereas the binding of [3H]prazosin to alpha 1-adrenoceptors was less potently affected by (+/-)Org 3770 (pKi = 6.4) than by mianserin (pKi = 7.1). A similar difference was found for alpha 1- and alpha 2-adrenoceptors in vas deferens of the rat. The binding of [3H]mianserin to 5-HT2 receptors was less potently blocked by (+/-)Org 3770 (pKi = 8.1) than by mianserin (pKi = 9.4) while the binding of [3H]mepyramine to histamine-1 receptors was more potently affected by (+/-)Org 3770 (pKi = 9.3) than by mianserin (pKi = 8.75). The binding of [3H]quinuclidinylbenzilate to muscarinic cholinergic receptors was blocked equally by (+/-)Org 3770 (pKi = 6.1) and mianserin (pKi = 6.3). Similar data on tryptamine-D, histamine-1 and muscarinic cholinergic receptors in isolated organs were obtained. A prominent role for the blockade of alpha 2-adrenoceptors in the therapeutic effects of mianserin and (+/-)Org 3770 in depression is suggested, probably excluding a role of inhibition of the uptake of NA.

Summary The influx and efflux of the extracellular marker 14 C-inulin in the isolated gastric-fundal smooth muscle follow multiexponential kinetics. Inulin appeared to enter not only the extracellular spaces (ESP) but also less accessible compartments after a long incubation. Pre-incubation with a range of non-agonistic drugs affected both influx and efflux kinetics. The total inulin spaces (IS) during influx were increased by papaverine, TH (a papaverine-derivative), morphine and codeine, but they were decreased by atropine, dibenamine, promethazine, chlorpnomazine, LSD, psilocybin and methysergide. The analysis of the efflux curves showed that most of drugs induced a shift of inulin from the slow (intracellular?) to the fast (ESP) compartment, thus reflecting the increases of IS during influx. Promethazine and chlorpromazine decreased the swelling and acted mainly on the slow compartment and the non-exchangeable inulin (bound fraction), thus the decrease of IS appeared to be related to a loss of water from tissues and/or to the membrane-stabilizing action of both drugs. Psilocybin and, to a lesser extent, LSD and methysergide, increased the radioactivity exchanged in the fast compartment, but decreased the IS, probably due to less inulin-acoessible water available for diffusion rather than to a true effect on ESP.

The effects of (−)-propranolol, (+)-propranolol and (±)cyanopindolol on the release of [ 3 H]5-hydroxytryptamine ([ 3 H]5-HT) were investigated in synaptosomes from the hippocampus of the rat, depolarized in superfusion with 15mM KCl. (−)-Propranolol, but not (+)-propranolol, inhibited in a concentration-dependent way the K + -evoked release of [ 3 H]5-HT. (±)-Cyanopindolol behaved similarly but was about 10 times more potent than (−)-propranolol. The inhibitory effects of (−)-propranolol and (±)-cyanopindolol were prevented by the autoreceptor antagonist methiothepin. Both β-adrenoceptor antagonists antagonized the inhibition by exogenous 5-HT of the K + -evoked release of [ 3 H]5-HT. The data suggest that some β-adrenoceptor antagonists may behave as mixed agonists-antagonists at the 5-HT autoreceptor. Synaptosomes in superfusion appear to be particularly suitable to study separately agonistic compared to antagonistic activity of compounds having a mixed agonist-antagonist profile.