Modulation of the expression of GABAA receptors in rat cerebellar granule cells by protein tyrosine kinases and protein kinase c

none3R. BALDUZZI; A. CUPELLO; M. ROBELLOR., Balduzzi; A., Cupello; Robello, Maur

GABA A Receptors of Cerebellar Granule in Culture: Interaction with Benzodiazepines

GABAA receptor mediated inhibition plays an important role in modulating the input/output dynamics of cerebellum. A characteristic of cerebellar GABAA receptors is the presence in cerebellar granule cells of subunits such as a6 and d which give insensitivity to classical benzodiazepines. In fact, cerebellar GABAA receptors have generally been considered a poor model for testing drugs which potentially are active at the benzodiazepine site. In this overview we show how rat cerebellar granule cells in culture may be a useful model for studying new benzodiazepine site agonists. This is based on the pharmacological separation of diazepam-sensitive a1 b2/3 c2 receptors from those which are diazepam-insensitive and contain the a6 subunit. This is achieved by utilizing furosemide/Zn2? which block a6 containing and incomplete receptor

Study of the Interaction of 1,4- and 1,5-Benzodiazepines with GABA<inf>A</inf> Receptors of Rat Cerebellum Granule Cells in Culture

The effects of a classical 1,4-benzodiazepine agonist, such as diazepam, its catabolite N-desmethyl-diazepam (nordiazepam), and 1,5-benzodiazepines such as clobazam and RL 214 (a triazolobenzodiazepine previously synthesized in our labs) were evaluated on native GABA(A) receptors of cerebellar granule cells in culture. The parameter studied was the increase of GABA-activated chloride currents caused by these substances. The contributions of alpha 6 beta 2/3 gamma 2 and alpha 1 alpha 6 beta 2/3 gamma 2 receptor subtypes to the increase of GABA-activated chloride current were investigated by comparing the effects of such substances in the presence vs. the absence of furosemide. Furosemide is in fact able to block such receptors. It was found that the percent enhancement of peak GABA-activated current doubled for diazepam, clobazam, and RL 214. However, it did not change for N-desmethyl-diazepam. These results indicate that diazepam, clobazam, and RL 214 interact exclusively with alpha 1 beta 2/3 gamma 2 receptors, while N-desmethyl-diazepam seems to interact with not only alpha 1-but also alpha 6-containing receptors

Two-Photon Photoactivation of Rubi-Gaba for Studying the Role of the Antisecretory Factor in the Modulation of the GABAA Receptor in Rat Cerebellar Granule Cells In Vitro

Caged compounds, widely diffused in neurophysiology, are molecules biologically or functionally inert until a short pulse of light enables them. Illumination releases the caged effector allowing the activation, manipulation, and control of selective biological functions and variations. in particular, 3D two-photon excitation microscopy, coupled with the electrophysiological technique of the patch-clamp in whole-cell configuration, provides a useful tool for studying several neurobiological processes such as the localized, accurate, and specific receptor response of a well-defined neuronal area. Here, we report this method to figure out the role of the protein Antisecretory Factor (AF) in the modulation of the GABA A receptor by using the caged neurotransmitter RuBi-GABA, rapidly photoreleased, in femtoliter volumes, in a precise and confined region of a neuron at a defined concentration. AF acts in vivo by inhibiting \u202

Electrophysiological study of the effects of side products of RuBi-GABA uncaging on GABA_A receptors in cerebellar granule cells

Abstract The study of the GABAA receptor itself and its pharmacology is of paramount importance for shedding light on the role of this receptor in the central nervous system. Caged compounds have emerged as powerful tools to support research in this field, as they allow to control, in space and time, the release of neurotransmitters enabling, for example, to map receptors' distribution and dynamics. Here we focus on γ-aminobutyric acid (GABA)-caged compounds, particularly on a commercial complex called RuBi-GABA, which has high efficiency of uncaging upon irradiation at visible wavelengths. We characterized, by electrophysiological measurements, the effects of RuBi-GABA on GABAA receptors of rat cerebellar granule cells in vitro. In particular, we evaluated the effects of side products obtained after RuBi-GABA photolysis. For this purpose, we developed a procedure to separate the "RuBi-cage" from GABA after uncaging RuBi-GABA with a laser source; then, we compared electrophysiological measurements acquired with and without administering the RuBi-cage in the perfusing bath. In conclusion, to investigate the role of the "cage" molecules both near and far from the cell soma, we compared experiments performed changing the distance of the uncaging point from the cell

Anticonvulsive Activity in Audiogenic DBA/2 Mice of 1,4-Benzodiazepines and 1,5-Benzodiazepines with Different Activities at Cerebellar Granule Cell GABAA Receptors

Herein, we tested in a model of generalized reflex epilepsy in mice different 1,4-benzodiazepines and 1,5-benzodiazepines with agonistic activity at the GABAA receptor population contributing to the peak component of the chloride current elicited by GABA in cerebellar granule cells (CGCs) in culture. The substances have all higher lipophilia than clobazam, an antiepileptic drug well known and used in human therapy. This ensures that they all can pass relatively easily the blood-brain barrier (BBB). The benzodiazepines were administered intraperitoneally (i.p.) and tested for their activity against sound-induced tonic and clonic seizures in a genetic model of experimental epilepsy, the DBA/2 mouse. Our data demonstrates an interesting inverse correlation between the ED50s and the efficacy (E %) of the drugs in increasing the peak chloride current elicited by GABA in cerebellar granule cells in culture. There is indication of the existence of a threshold of E % above which the increase of ED50 with increasing E % becomes linear. This is statistically significant for the clonic phase, whereas it is at the limit of significance for the tonic one. A possible interpretation of these results is that in this epilepsy model, projections from the cerebellum exert a convulsion prevention activity

Synthesis and pharmacological evaluation of functionalized isoindolinones on GABA-activated chloride currents in rat cerebellum granule cells in culture

A focused N-substituted 3-(2-piperazin-1-yl-2-oxoethyl)-2-(pyridin-2-yl)iso-indolin-1-ones small library was synthesized for modulation of GABA-A receptor function and compared to Zopiclone for the ability to increase GABA-activated chloride currents. All compounds were tested for their effects on GABA-activated chloride currents in rat cerebellar granule cells by use of the whole-cell patch clamp technique. Electrophysiological studies on cultured cerebellar granule cells revealed 3-[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-2-(5-nitropyridin-2-yl)iso-indolin-1-one (Id) as a partial agonist displaying 34% increase of the 10 \u3bcM GABA evoked peak chloride currents, antagonized by flumazenil. Moreover, a second group of compounds, with bulky functional groups at N-4 position of piperazine, have shown inverse agonist effects. The simple synthetic procedure and the possibility of modulating the efficacy of this class of ligands through additional structural modifications pave the way for further development of new molecules as a novel class of compounds able to interfere with benzodiazepine receptors