RAPID COMMUNICATION Zn Blocks the NMDA- and Ca-Triggered Postexposure Current Ipe in Hippocampal Pyramidal Cells

Chen, Qiang X., Katherine L. Perkins, and Robert K. S. Wong. Whole cell voltage-clamp was performed following the procedure described by Hamill et al. (1981) with the use of a List EPCZn blocks the NMDAand Ca-triggered postexposure current Ipe in hippocampal pyramidal cells. J. Neurophysiol. 79: 1124– 7 patch-clamp amplifier and pClamp software (Axon Instruments) . Access resistances were Ç10 MV. Good recordings were ensured 1126, 1998. Whole cell voltage-clamp recordings from acutely isolated hippocampal CA1 pyramidal cells from adult guinea pigs by discarding cells that had seal resistances õ20 GV, which took more than four gentle sucks to break the membrane, or which did were used to evaluate divalent cations as possible blockers of the postexposure current (Ipe ) . Ipe is a cation current that is triggered not maintain a stable input resistance during the first 5 min of recording before NMDA exposure. The holding potential was 050 by the rise in intracellular Ca concentration that occurs after the application of a toxic level of N-methyl-D-aspartate (NMDA). or 055 mV. Cells were in a 1-ml bath that was perfused continuously with Once triggered, Ipe continues to grow until death of the neuron occurs. Ipe may be a critical link between transient NMDA exposure extracellular control solution at a rate of 1–2 ml per min. Extracellular control solution contained (in mM) 140 NaCl, 2 KCl, 2 and cell death. Ipe was blocked by micromolar concentrations of Zn . The Zn effect had an IC50 of 64 mM and saturated at 500 CaCl2 , 10 N-2-hydroxyethylpiperazine-N *-2-ethanesulfonic acid (HEPES), 0.01 glycine, 25 glucose, pH adjusted to 7.4 with NaOH. mM. Prolonged Zn block of Ipe revealed that the maintenance of a steady Ipe is not dependent on Ipe-mediated Ca influx but that Applications of NMDA and divalent cations were achieved with a seven-barrel flow tube (Celentano and Wong 1994) or a threethe continuous growth in Ipe is dependent on Ipe-mediated Ca influx. The availability of an effective blocker of Ipe should facilibarrel continuous flow system (Chen and Wong 1995b). NMDA solution was made by dissolving NMDA (100 mM) in extracellular tate the investigation of the intracellular activation pathway of Ipe and the role of Ipe in neuronal death. control solution. The divalent cation solutions were made by adding the chloride salt of the divalent cation to extracellular control solution. In one set of experiments intracellular perfusion was used to I N T R O D U C T I O N switch from control intracellular solution to high calcium intracelN-methyl-D-aspartate (NMDA) exposure can lead to the lular solution while recording from a single cell (Chen et al. 1990). activation of a cation current called the postexposure current Control intracellular solution contained (in mM) 20 CsCl, 100 CsOH, 0.5 bis-(o-aminophenoxy)-N ,N ,N*,N*-tetraacetic acid (Ipe ) in hippocampal neurons (Chen et al. 1997). The con(BAPTA), 10 HEPES, pH adjusted to 7.2 with methanesulfonic ductance underlying Ipe shows a high Ca 2/ permeability. Ipe acid. High calcium intracellular solution was made by adding 1 is triggered by the increase in intracellular Ca concentramM Ca to the control intracellular solution (Chen et al. 1990; tion caused by Ca influx through the NMDA receptor Chen et al. 1997). The dose-response curve fitting was performed channel. Once triggered, Ipe continues to increase in ampliwith Sigmaplot (Jandel Scientific) . tude (in the absence of NMDA) until death of the neuron occurs. Procedures that prevent the induction of Ipe or supR E S U L T S press it after induction also reduce neuronal death (Chen et al. 1997). This paper identifies Zn as an effective blocker As shown previously (Chen et al. 1997), prolonged appliof Ipe . The availability of a blocker should facilitate further cation of NMDA (10 min, 100 mM) triggers Ipe , which is studies into the intracellular activation pathway of Ipe and associated with an increase in conductance (Fig. 1A) . Figure the role of Ipe in NMDA toxicity. 1A shows that Ipe was blocked during short applications of Zn (25 s, 500 mM) but otherwise continued to grow in amplitude once it was triggered. The conductance associated M E T H O D S with Ipe was greatly reduced during the Zn 2/ block (Fig. Acutely isolated hippocampal CA1 neurons from adult guinea 2B) . A shorter (2 min) application of NMDA could also pigs were prepared according to the Kay and Wong (1986) method activate Ipe , but there was a delay after the end of the NMDA with several modifications to increase the harvest of healthy neuapplication before Ipe was evident (Fig. 1B) . The Ipe trigrons and preserve NMDA responses (see Chen et al. 1997). gered by these shorter applications of NMDA was also Healthy neurons were selected by choosing those that were uniblocked during short applications of Zn (Fig. 1B) . formly bright under phase contrast microscopy. These neurons have All twelve cells tested with a 10 min NMDA exposure a normal (around 060 mV) and stable resting potential within the first hour of recording, have an ability to fire action potentials, and developed Ipe . In all twelve cases, the Ipe became evident show reversible receptor-channel modulation by second messenger during the NMDA exposure. Eight of 10 cells tested with a systems (Chen and Wong 1995a,b; Chen et al. 1990). 2-min NMDA exposure developed Ipe . In these eight cells, the membrane current returned to baseline after NMDA ex-