Moleculary and electrophisologically being analysed effects of valproic acid, ziconotide and spinorphin to rat root ganglions nerve cells culturesl

The aim of this study was to investigate the effects of valproic acid,ziconotide and spinorphin on cultured rat dorsal root ganglion (DRG) neurones usingfluorescence calcium imaging. The effects of valproic acid was also investigated bypatch clamp trechnique. Dorsal root ganglia were dissected out and isolated as singlecells by enzymatic and physical procedures and placed in humidifıed incubatorcontaining 5% CO2 at 37 °C. Action potentials were recorded from these cells usingthe whole celi current clamp mode of the patch clamp technique. For thefluorescence calcium imaging studies, the DRG neurons were cultured on coatedcoverslips and loaded with 1 umol Fura-2 AM. Free intracellular Ca2+ ([Ca2+];)responses were quantifıed by the changes in 340/380 ratio for individual DRGneurons using fluorescence imaging system consisting of CCD camera coupled to aninverted microscope (with a 40x, 1.30 NA objective) and software. Ali data wereanalyzed by using unpaired t test, P <05 defıning statistical signifıcance.Patch clamp experiments were performed only for valproic acid andapplication of valproic acid (30 uM, 100 uM and 300 uM) action potentialparameters (n=16). Furthermore, in fluorescence calcium imaging experimentsvalproic acid (100 uM, n=10; 300uM, n=22 ve 1 mM, n=56) did not have anysignifıcant effect on [Ca2+]; responses to membrane depolarisation by HK+ (KC1, 30mM). Again in calcium imaging experiments, ziconotide (nM, n=12; 10 nM, n=18ve luM, n=14) strongly inhibited the [Ca2+]; increase due to application of HK+ in adose dependent manner. Additionally spinorphin (lOuM, n= 22; 100 uM, n= 25 ve300 uM, n=35) inhibited the Ca2+ transients evoked with HK+ in concentrationdependant manner in a subpopulation of small-sized sensory neurons. Furthermore,after application of spinorphin a signifıcant percent of small-diameter nociceptiveDRG neurones did not respond to stimulation by HK+ while the percentage of theresponse was not signifıcantly changed in large-diameter non-nociceptive DRGneurones. Results from this study indicates that spinorphin and ziconotide but notvalproic acid significantly inhibits calcium signalling, transient changes in freeintracellular Ca2+ concentration which are key for the modulation of celi membraneexcitability and neurotransmitter release, these agents may be effective analgesics.The effects of spinorphin and ziconotide on intracellular calcium signalling in thissensory neurones is established for the fırst time by this thesis study.Key words: Action potential, valproic acid, ziconotide, spinorphin, celiculture, patch clamp, ion channels.