Glycine response in acutely dissociated ventromedial hypothalamic neuron of the rat: new approach with gramicidin perforated patch-clamp technique.
1. We investigated the glycine-induced response in ventromedial hypothalamic (VMH) neurons freshly dissociated from 8- to 12-day-old rats using the nystatin and gramicidin perforated patch recording modes. The nystatin-formed pores in the plasma membrane are permeable for both monovalent cations and anions, whereas those formed by gramicidin are permeable only to monovalent cations. Therefore, when the patch-pipette contains 150 mM Cl- and gramicidin, the physiological intracellular Cl- concentration ([Cl-]i) is undisturbed in the cell-attached condition of the pipette. 2. At holding potentials of -40 to -60 mV, glycine induced inward currents and outward currents in the nystatin and gramicidin perforated patch recording modes, respectively. The values of the half-maximum effective concentration (EC50) and the Hill coefficient in the concentration-response relationships of the glycine responses were 2.9 x 10(-5) M, 1.1, and 4.2 x 10(-5) M, 1.4, respectively. These values were quite similar in both recording modes. 3. The reversal potentials of the glycine responses (EGly) were -1.5 mV in the nystatin perforated patch recording and -75.0 to -24.8 mV in the gramicidin perforated patch recording. 4. Strychnine (3 x 10(-8) M) inhibited the glycine-induced outward currents in a competitive manner and the half-inhibition concentration (IC50) of strychnine on the 10(-4) M glycine-induced response was 1.9 x 10(-8) M. 5. The physiological [Cl-]i in the VMH neurons calculated from the EGly obtained by the gramicidin perforated patch mode ranged from 6.0 to 43.8 mM (n = 28).
Abe
Y
Y
Department of Neurophysiology, Tohoku University School of Medicine, Sendai, Japan.
Furukawa
K
K
Itoyama
Y
Y
Akaike
N
N
D000818:Animals; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects*; D002712:Chlorides / Q000378:metabolism; D005998:Glycine / Q000494:pharmacology*; D006096:Gramicidin / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D009474:Neurons / Q000187:drug effects; D009761:Nystatin / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D009435:Synaptic Transmission / Q000187:drug effects*; D014697:Ventromedial Hypothalamic Nucleus / Q000187:drug effects*
D018118:Chloride Channels; D002712:Chlorides; D009761:Nystatin; D006096:Gramicidin; D005998:Glycine