The Modulation Of Intracelluar Free Calcium Ions By Cholinergic Receptors And GABAergic Receptors In Rat Medial Vestibular Nucleus

PART 1: An Increase in Intracelluar Free Calcium Ions by Cholinergic Receptors in Rat Medial Vestibular NucleusObjective: To Study the modulations of intracellular free Ca2+ concentrations by cholinergic receptors in rat medial vestibular nucleus. Methods: The fluorescence intensity of medial vestibular nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca2+ measurement technique via confocal laser scanning microscope. Results: Acetylcholine was effective at increasing the fluorescence intensity of medial vestibular nucleus in a concentration dependent manner at a concentration range of 10-5~10-2 M, and the increase by acetylcholine was saturable with increasing concentrations of up to 1mM. The increase of intracelluar free calcium ions levels by 1mM acetylcholine could be inhibited by atropine and mecamylamine, and there is little difference between the inhibition between atropine and mecamylamine (P>0.05). Conclusion: The data provide the evidence that cholinergic receptors induce the increase of intracellular free Ca2+ levels, in a manner related to both muscarinic receptors and nicotinic receptors in rat medial vestibular nucleus. PART 2: An Increase in Intracelluar Free Calcium Ions by Muscarinic Receptors in Rat Medial Vestibular NucleusObjective: To Study the modulations of intracellular free Ca2+ concentrations by muscarinic receptors in rat medial vestibular nucleus, and the mechanisms of the modulations. Methods: The fluorescence intensity of medial vestibular nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca2+ measurement technique via confocal laser scanning microscope. Results: Muscarine chloride (a general muscarinic cholinergic agonist) induced a marked increase of fluorescence intensity in medial vestibular nucleus in a concentration dependent fashion at a concentration range of 5~100μM, and the increase by muscarine chloride was saturable with increasing concentrations of up to 50μM. The enhancement of fluorescence intensity by muscarine chloride was significantly reduced by thapsigargin (P<0.01), rather than Ca2+ free artifical cerebrospinal fluid (ACSF) and EGTA (P>0.05). And the increase of fluorescence intensity was also significantly inhibited by pirenzepine and 4-DAMP (P<0.05), rather than methoctramine and tropicamide (P>0.05). Conclusion: The data provide the evidence that muscarinic receptors induce the increase of intracellular free Ca2+ levels through the Ca2+ release of intracellular Ca2+ stores rather than influx of extracellular Ca2+, in a manner related to m1 and m3 subtypes rather than m2 and m4 subtypes of muscarinic receptors in rat medial vestibular nucleus. PART 3: An Increase in Intracelluar Free Calcium Ions by Nicotinic Receptors in Rat Medial Vestibular NucleusObjective: To Study the modulations of intracellular free Ca2+ concentrations by nicotinic receptors in rat medial vestibular nucleus, and the mechanisms of the modulations. Methods: The fluorescence intensity of medial vestibular nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca2+ measurement technique via confocal laser scanning microscope. Results: Fluorescence intensity of medial vestibular nucleus was markedly increased by nicotine (a general nicotinic cholinergic agonist) in a dose dependent manner at a concentration range of 10 to 500μM. and the increase by nicotine was saturable with increasing concentrations of up to 100μM. The enhancement of fluorescence intensity by nicotine was significantly reduced by EGTA, nifedipine and in Ca2+ free ASCF (P<0.01), but not in the presence of mibefradil or thapsigargin (P>0.05). The increase was also significantly reduced by Dihydro-β-erythroidine (P<0.01), rather than methyllycaconitine (P>0.05). Conclusion: The results suggest that nicotine increase intracellular free Ca2+ concentrations via the influx of extracellular Ca2+ at least across L-type voltage-gated Ca2+ channels rather than Ca2+ release from intracellular stores, in a manner related to theα4β2 subtype of nicotinic receptors, but notα7 subtype in rat medial vestibular nucleus. PART 4: A Change in Intracelluar Free Calcium Ions by GABAergic Receptors in Rat Medial Vestibular NucleusObjective: To Study the modulations of intracellular free Ca2+ concentrations by GABAergic receptors in rat medial vestibular nucleus, and the mechanisms of the modulations. Methods: The fluorescence intensity of medial vestibular nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca2+ measurement technique via confocal laser scanning microscope. Results: 5~500μM GABA could not induce change of the intracellular free Ca2+ concentrations (P>0.05), or inhibit the increase of the intracellular free Ca2+ concentrations by 50μM muscarine chloride (P>0.05). GABA induced the inhibition of increase by 100μM nicotine in a dose dependent manner at a concentration range of 5 to 500μM (P<0.05), and the inhibition by GABA was saturable with increasing concentrations of up to 100μM. 1μM~100μM baclofen (a selective GABAB agonist) could not change the increase in intracellular free Ca2+ levels by 50μM muscarine chloride or 100μM nicotine (P>0.05). Conclusion: GABAergic receptors induce the inhibition of the increase in intracellular free Ca2+ concentrations by nicotinic receptors, in a manner related to GABAA subtype, though blocking L-type voltage-gated Ca2+ channels possibly in rat medial vestibular nucleus.