Real Physiological Neuronal Responses Revealed by Gramicidin Perforated Patch Recording

Norio Akaike,Yasuhiro Kakazu 대한생리학회-대한약리학회 2001 The Korean Journal of Physiology & Pharmacology Vol.5 No.3

<P> 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<SUP>⁣</SUP>. 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.

Muscarine $M_2$ Receptor-mediated Presynaptic Inhibition of GABAergic Transmission in Rat Meynert Neurons

Jang, Il-Sung,Akaike, Norio The Korean Society of Pharmacology 2002 The Korean Journal of Physiology & Pharmacology Vol.6 No.2

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 $(10{\mu}M)$ reversibly and concentration-dependently decreased mIPSC frequency without affecting the current amplitude distribution. Muscarine action on GABAergic mIPSCs was completely blocked by $1{\mu}M$ methoctramine, a selective $M_2$ receptor antagonist, but not by $1{\mu}M$ pirenzepine, a selective $M_1$ receptor antagonist. NEM $(10{\mu}M),$ a G-protein uncoupler, attenuated the inhibitory action of muscarine on GABAergic mIPSC frequency. Muscarine still could decrease GABAergic mIPSC frequency even in the $Ca^{2+}-free$ 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 $M_2$ receptors, via the reduction of cAMP production. Accordingly, $M_2$ 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.

Muscarine M<SUB>2</SUB> Receptor-mediated Presynaptic Inhibition of GABAergic Transmission in Rat Meynert Neurons

Il-Sung Jang,Norio Akaike 대한생리학회-대한약리학회 2002 The Korean Journal of Physiology & Pharmacology Vol.6 No.2

<P> 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 (10μM) reversibly and concentration-dependently decreased mIPSC frequency without affecting the current amplitude distribution. Muscarine action on GABAergic mIPSCs was completely blocked by 1μM methoctramine, a selective M<SUB>2</SUB> receptor antagonist, but not by 1μM pirenzepine, a selective M<SUB>1</SUB> receptor antagonist. NEM (10μM), a G-protein uncoupler, attenuated the inhibitory action of muscarine on GABAergic mIPSC frequency. Muscarine still could decrease GABAergic mIPSC frequency even in the Ca<SUP>2⁢</SUP>-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 M<SUB>2</SUB> receptors, via the reduction of cAMP production. Accordingly, M<SUB>2</SUB> 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.

Modulation of glycine-induced chloride current in acutely dissociated rat periaqueductal gray neurons by μ-opioid agonist, DAGO

Kim,Chang Ju,Min,Byung Ii,Rhee,Jeong Seop,Akaike, Norio WHO COLLABORATING CENTRE FOR TRADITIONAL MEDICINE 1996 東西醫學硏究所 論文集 Vol.1996 No.-

Effect of a μ-opioid agonist (D-Ala², N-MePhe⁴, Gly -ol-enkephalin, DAGO), on glycine (Gly)-induced chloride current (l ) was investigated in the periaqueductal gray (PAG) neurons acutely dissociated 1-2-week-old wistar rats by the use of nystatin-perforated patch recording configuration under voltage-clamp condition. At a holding potential (V ) of-40 mV. DAGO caused a sustained potentiation of l at the low concentrations (10 -10 M) but reduced slightly the Gly response at the high concentration (10 M). The reversal potential of l was equal to the Cl?? equilibrium potential (E ) and was not changed in the presence of 10 M DAGO. The 10?? M Gly response was inhibited by the simultaneous treatment of forskolin and 3-isobutyl-1-methylxanthine (IBMX). H-89, a protein kinase A (PKA) inhibitor, increased the 10 M Gly response but had little effect on the 10 M Gly response. DAGO increased 10 M Gly response in the presence of forskolin and IBMX but, not more than in the absence of forskolin and IBMX. The 10 M Gly response augumented by DAGO was not affected by adding H-89. The present results suggest that the glycine-induced chloride current is cAMP dependent and is inhibited by PKA, and that the potentiation of the glycine response by DAGO is also cAMP dependent and is due to the inhibition of PKA as that of H-89. We conclude that the potentiation of glycine response by DAGO is mediated by an inhitition of cAMP-dependent PKA in the PAG neurons.

Antagonizing effect of protein kinase C activation on the μ-opioid agonist-induced inhibition of high voltage-activated calcium current in rat periaqueductal gray neuron

Cho, Young-Wuk,Han, Seung-Ho,Min, Byung-Il,Rhee, Jeong-Seop,Norio Akaike 경희대학교 동서의학연구소 2001 東西醫學硏究所 論文集 Vol.2001 No.-

Opioids have been thought to induce analgesia by activating the descending pain control system, especially at the level of periaquenductal gray, and regulate the neurotransmitter release through the inhibition of calcium channel. In the present study, the modulatory effects of protein kinase C and protein kinase A on the μ-opioid agonist-induced inhibition of the high-voltage activated calcium current were examined in the acutely dissociated rat periaqueductal gray neurons with the nystatin-perforated patch-clamp technique. Among 505 neurons tested, the barium current passing through the high-voltage activated calcium channels of 172 neurons (34%) were inhibited by 32±3% with the application of an μ-opioid agonist, [D-_Ala^2, N-MePhe^4, Gly^5 -ol]-enkephalin (DAMGO, 1 μM). The barium currents itself and the DAMGO-induced inhibitory effects were not affected by the application of either an adenylate cyclase activator (forskolin, 1μM) or a protein kinase inhibitor (staurosporin, 10nM) for 2 min. The DAMGO inhibition was completely and irreversibly antagonized by the application of a protein kinase C activator, phorbol-12-myristate-13-acetate (PMA, 1μM) for 2 min without any alteration of the barium current itself. However, the antagonizing effect of PMA was completely abolished by the application of 10 nM staurosporin for 2 min. After then, PMA did not show the antagonizing effect any more. Inversely, when staurosporin was applied before PMA, the antagonizing effect of PMA was also not shown. These results demonstrate that the μ-opiod agonist-induced inhibition of the periaqueductal gray neuronal high-voltage activated calcium current can be antagonized by protein kinase C activation. This finding may provide us a significant clue to understand the action mechanism of opioid-induced analgesia in the periaqueductal gray. ⓒ 2001 Elsevier Science B.V.All rights reserved.