318 Brain Research, 535 (1990) 318-322 Elsevier BRES 24418 Nystatin-perforated patch recordings disclose NMDA-induced outward currents in cultured neocortical neurons Dinesh K. Mistry and John J. Hablitz Neurobiology Research Center and Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294 (U.S.A.) (Accepted 28 August 1990) Key words: Nystatin; N-Methyl-D-aspartate; Calcium; Potassium conductance; Neocortex Excitatory amino acid-induced responses were studied in cultured rat neocortical neurons using two types of whole-cell patch-clamp recordings. Conventional recording methods, using either KCI or CsCI in the patch pipet, showed N-methyl-D-aspartate (NMDA) currents to be biphasic, consisting of peak and steady-state currents with similar reversal potentials. Recordings obtained with the nystatin-perforated patch method and KCi as the principal intracellular cation disclosed an NMDA-evoked outward current. Outward currents were not seen with either recording method in response to kainate or quisqualate, nor in response to NMDA when CsCI was the major intracellular cation. The NMDA-evoked outward current is attributed to activation of a potassium current by calcium entering through the NMDA channel. This outward current may serve to limit neuronal depolarization during excessive NMDA-mediated excitation. Whole-cell voltage-clamp recordings have been used extensively to study excitatory amino acid responses in cultured neurons. Initial studies demonstrated that the agonist N-methyI-D-aspartate (NMDA) gated a cation channel that was permeable to sodium, potassium and calcium 1~. These studies, while important for understand- ing the molecular actions of NMDA, may not provide a clear picture of this agonist's effect on neuronal excit- ability in intact cells. A well-known complication of whole-cell recording is the run-down or wash-out of responses dependent upon cytoplasmic factors. This was originally noted in studies of voltage-gated calcium channels 2 but has been found to be a factor in transmit- ter-gated responses as well. In particular, NMDA recep- tors have been shown to require intracellular high-energy phosphates for maintenance of the normal functional state of the channel 1°'15. Moreover, NMDA responses can be influenced by the extent of calcium buffering and the nature of the intracellular cation employed TM. An alternative to whole-cell recording is the nystatin- perforated patch method 6. This approach uses nystatin, a pore-forming antibiotic, to permeabilize the membrane of a cell-attached patch, allowing electrical access to the cell interior. The pores formed allow passage of cations without appreciable dialysis of large proteins. For exam- ple, muscarine-activated currents recorded in rat lacrimal gland cells, which are G-protein dependent 3, do not show rundown when recorded in this manner 6. We have used the nystatin method to study NMDA responses in cultured rat neocortical neurons. Neocortical neurons were maintained in culture using modifications of techniques used previously 5. Cerebral cortices were removed from 18 day-old rat embryos, trypsinized and triturated with fire-polished pasteur pipettes. Cells were then plated on poly-L-lysine coated plastic culture dishes. Neurons were maintained in a medium consisting of basal Eagle's medium containing 5% bovine calf serum (HyClone) and 5% Hybri-max (Sigma), supplemented with 2 mM glutamine, 20 mM glucose and antibiotics. Cultures were kept at 37 °C in a humidified 5% CO 2 atmosphere and used after 5-25 days in vitro. Whole-cell patch-clamp recordings were obtained at room temperature (20-24 °C) with the neurons bathed in a saline containing (in mM): NaC1 140, CaC12 2, KCI 3, HEPES 10, glucose 10 (pH 7.3 adjusted with NaOH), Tetrodotoxin (1 /~g/ml) and glycine (1 ktM) were also present. For conventional whole-cell recording, patch pipettes contained (in mM): KCI 140, EGTA 11, CaCI2 1, HEPES 10 and glucose 10 (pH 7.3 adjusted with NaOH). The pipette solution used for the perforated- patch recordings contained (in mM): KCI 140, MgCI 2 7, HEPES 10 (pH 7.3 adjusted with KOH). In some perforated-patch experiments, KCI was replaced with equal amounts of CsCI. Stock solutions of nystatin (Squibb) containing 50 mg/ml were prepared daily by Correspondence: J.J. Hablitz, Neurobiology Research Center, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)