|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1993 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1992 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Property of the muscarinic response in freshly dissociated neurons from frontal cortex and hippocampal CA1 pyramidal layr of Wistar rats was studied by use of the nystatin-perforated patch-clamp technique and the fura 2 fluorometry of [Ca^<2+>]_i. Application of muscarinic agonists and antagonists was performed with the Y-tube technique which allowed rapid excahnge (<10ms) of solutions surrounding the tested neuron.
Under voltage-clamp at -40 mV, majority of the cortex neurons responded to ACh, generating an inward current by closing a kind of K^+ channels (M-channel). Comparison of the effects of a variety of muscarinic agonists, including ACh, oxotremorine-M, McN-A-343, muscarine and oxotremorine and antagonists, including atropine, 4-DAMP, pirenzepine and AF-DX-116 suggested that the inward current was mediated by the M1 type of muscarinic receptor.
The CA1 pyramidal neurones were classified into three groups of which the ACh responses jncluded only an inward current, only a transient outward current or a complex of the both currents. Pharmacological identification of the receptor subtype with those muscarininic agonists and antagonists employed for the cortex neurons also suggested that the inward current in CA1 neurons was mediated by the M1 receptor but the transient outward current was by the M3 subtype. The fura 2 fluorometry revealed that the muscarinic receptor-operated transient outward current was highly dependent on [Ca^<2+>]_i. Comparing the effects of pertussis toxin, W-7, chlorpromazine, trifluoroperazine, H-7 and KN-62 on the outward current, the involvement of pertussis toxin-insensitive GTP-binding protein and Ca^<2+>-calmodulin-dependent proteinkinase II in the muscarinic response was also suggested.