In order to understand the phenomenon in a living cell correctly, it has been required to obtain intact responses from the cell membrane without disrupting the cytoplasmic circumstances. Gramicidin perforated patch configuration allows the electrical access to the whole cell with a minimal dialysis of cytoplasm and preventing the loss of native intracellular constituents, such as Cl-. Here, we would like to show the background of this method and the actual application of the gramicidin perforated patch recording mode on the dissociated neurons.
Cell Membrane ; Cytoplasm ; Dialysis ; Gramicidin* ; Neurons*

Cholinergic modulation of GABAergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) by the activation of muscarine receptors was investigated in mechanically dissociated rat nucleus basalis of the Meynert neurons using the conventional whole-cell patch recording configuration. Muscarine (10microM) reversibly and concentration-dependently decreased mIPSC frequency without affecting the current amplitude distribution. Muscarine action on GABAergic mIPSCs was completely blocked by 1microM methoctramine, a selective M2 receptor antagonist, but not by 1microM pirenzepine, a selective M1 receptor antagonist. NEM (10microM), a G-protein uncoupler, attenuated the inhibitory action of muscarine on GABAergic mIPSC frequency. Muscarine still could decrease GABAergic mIPSC frequency even in the Ca2+-free external solution. However, the inhibitory action of muscarine on GABAergic mIPSCs was completely occluded in the presence of forskolin. The results suggest that muscarine acts presynaptically and reduces the probability of spontaneous GABA release, and that such muscarine-induced inhibitory action seems to be mediated by G-protein-coupled M2 receptors, via the reduction of cAMP production. Accordingly, M2 receptor-mediated disinhibition of nBM neurons might play one of important roles in the regulation of cholinergic outputs from nBM neurons as well as the excitability of nBM neurons themselves.
Animals ; Cholinergic Neurons ; Colforsin ; gamma-Aminobutyric Acid ; GTP-Binding Proteins ; Inhibitory Postsynaptic Potentials ; Muscarine* ; Neurons* ; Pirenzepine ; Rats*