Direct patch recording from identified presynaptic terminals mediating glutamatergic EPSCs in the rat CNS, in vitro.
1. An in vitro brainstem slice preparation of the superior olivary complex has been developed permitting patch recording from a presynaptic terminal (calyx of Held) and from its postsynaptic target--the principal neurone of the medial nucleus of the trapezoid body (MNTB). 2. The fluorescent stain DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) was used in fixed tissue and Lucifer Yellow in living slices, to identify calices enclosing single MNTB neuronal somata. 3. Whole-cell recording from the MNTB neurone shows evoked EPSCs preceded by a prespike, corresponding to the presynaptic action potential (AP). In some cases one patch pipette recorded from both pre- and postsynaptic elements, but confirmation of exclusively presynaptic recording was obtained using pipettes containing Lucifer Yellow in a further eleven cases. 4. Under current clamp, the pre- and postsynaptic sites could be distinguished by their response to step depolarizations; presynaptic terminals generated a train of APs at frequencies up to 200 Hz, while MNTB neurones gave a single AP. Each presynaptic AP had an after-hyperpolarization lasting less than 2 ms. 5. Under voltage clamp, step depolarizations of presynaptic terminals generated a tetrodotoxin-sensitive inward current followed by rapidly activating outward potassium currents at potentials more positive than -60 mV. The outward current exhibited little inactivation over the 150 ms steps and 4-aminopyridine (200 microM) blocked 63.0 +/- 14.5% (mean +/- S.D., n = 3) of the sustained current at 0 mV. Like the squid giant synapse, mammalian terminals express rapidly activating 'delayed rectifier'-type potassium currents.
Department of Cell Physiology and Pharmacology, University of Leicester, UK.
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Forsythe
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D001921:Brain / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D002232:Carbocyanines; D004594:Electrophysiology; D005456:Fluorescent Dyes; D018698:Glutamic Acid / Q000378:metabolism / Q000502:physiology*; D007546:Isoquinolines; D018613:Microscopy, Confocal; D009474:Neurons / Q000502:physiology; D018408:Patch-Clamp Techniques; D011149:Pons / Q000166:cytology / Q000378:metabolism; D017729:Presynaptic Terminals / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D018091:Receptors, AMPA / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D013779:Tetrodotoxin / Q000494:pharmacology
D002232:Carbocyanines; D005456:Fluorescent Dyes; D007546:Isoquinolines; D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid; C024286:3,3'-dioctadecylindocarbocyanine; D013779:Tetrodotoxin; C017475:lucifer yellow