Introduction those involving kinase and phosphatase enzymes, which

Brain Research 882 2000 86–94 www.elsevier.com locate bres Research report Mechanisms underlying H O -mediated inhibition of synaptic 2 2 transmission in rat hippocampal slices Marat V. Avshalumov, Billy T. Chen, Margaret E. Rice Departments of Physiology and Neuroscience and Neurosurgery , New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA Accepted 8 August 2000 Abstract Hydrogen peroxide H O inhibits the population spike PS evoked by Schaffer collateral stimulation in hippocampal slices. 2 2 21 Proposed mechanisms underlying this effect include generation of hydroxyl radicals ?OH and inhibition of presynaptic Ca entry. We have examined these possible mechanisms in rat hippocampal slices. Inhibition of the evoked PS by H O was sharply concentration- 2 2 dependent: 1.2 mM H O had no effect, whereas 1.5 and 2.0 mM H O reversibly depressed PS amplitude by roughly 80. The iron 2 2 2 2 chelator, deferoxamine 1 mM, and the endogenous ?OH scavenger, ascorbate 400 mM, prevented PS inhibition, confirming ?OH involvement. Isoascorbate 400 mM, which unlike ascorbate is not taken up by brain cells, also prevented PS inhibition, indicating an extracellular site of ?OH generation or action. We then investigated whether H O -induced PS depression could be overcome by 2 2 21 prolonged stimulation, which enhances Ca entry. During 5-s, 10-Hz trains under control conditions, PS amplitude increased to over 200 during the first three–four pulses, then stabilized. In the presence of H O , PS amplitude was initially depressed, but began to 2 2 recover after 2.5 s of stimulation, finally reaching 80 of the control maximum. In companion experiments, we assessed the effect of 21 21 21 H O on presynaptic Ca entry by monitoring extracellular Ca concentration [Ca ] during train stimulation in the presence of 2 2 o 21 21 postsynaptic receptor blockers. Evoked [Ca ] shifts were apparently unaltered by H O , suggesting a lack of effect on Ca entry. o 2 2 Taken together, these findings suggest new ways in which reactive oxygen species ROS might act as signaling agents, specifically as modulators of synaptic transmission.  2000 Elsevier Science B.V. All rights reserved. Theme : Excitable membranes and synaptic transmission Topic : Presynaptic mechanisms 21 Keywords : Hydrogen peroxide; Oxidative stress; Transmitter release; Ion-selective microelectrode; Ca ; Ascorbate

1. Introduction those involving kinase and phosphatase enzymes, which

are sensitive to this ROS [10,23,30,51]. Reactive oxygen species ROS occur naturally during It has also been shown that H O can modulate neuronal 2 2 cell metabolism and are strongly regulated by the intracel- activity. The effect of H O on hippocampus slice physi- 2 2 lular antioxidant network. Under pathological conditions, ology has been particularly well-studied [13,21,23,29,35– including ischemia, hyperoxia, and exposure to certain 37,46]. Both the primary effect and the mechanism of toxins, ROS can cause damage to the central nervous action of H O in hippocampal slices, however, remain 2 2 system [14,17]. Recent studies, however, have suggested uncertain. Several reports in the literature indicate that that ROS, particularly hydrogen peroxide H O , might H O causes a reversible depression of the population 2 2 2 2 also play a role in cellular signaling [12]. Among the most spike PS recorded in CA1 stratum pyramidale during important signaling pathways modulated by H O are stimulation of the Schaffer collaterals [13,35–37], although 2 2 an augmentation in hippocampal PS amplitude has also been reported [21]. In addition, it is not yet certain whether Corresponding author. Tel.: 11-212-263-5438; fax: 11-212-689- PS depression is mediated directly by H O or by hy- 0334. 2 2 E-mail address : margaret.ricenyu.edu M.E. Rice. droxyl radicals ?OH that can be produced from the 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 8 3 5 - 3 M .V. Avshalumov et al. Brain Research 882 2000 86 –94 87 interaction of H O with tissue iron or copper through the tobarbital sodium. After decapitation, the brain was rapidly 2 2 Fenton reaction [7,16,17]: removed and placed in ice-cold, oxygenated 95 O 5 2 CO artificial cerebrospinal fluid ACSF for about 1 min, 2 n 1 n 11 1 2 H O 1 Me → Me 1 ? OH 1 OH , then bisected, blocked and the tissue mounted on the stage 2 2 of a Vibratome Ted Pella, St. Louis, MO, USA. Trans- n 1 where Me is the reduced form of the metal ion. Pellmar verse hippocampal slices were cut in ice-cold ACSF that and colleagues concluded that PS depression was mediated contained in mM: 120 NaCl, 5 KCl, 35 NaHCO , 1.25 3 by ?OH, because deferoxamine, an iron chelator, prevented NaH PO , 1.5 CaCl , 1.3 MgCl , and 10 glucose. Slices 2 4 2 2 H O -induced changes in PS amplitude [37]. In other 2 2 were maintained in an incubation chamber at room tem- experiments, however, although with lower concentrations perature for at least 1 h before recording. of deferoxamine, Katsuki and colleagues [21] concluded that ?OH was not involved in the effects of H O on 2 2 2.2. Extracellular recording hippocampal slice physiology. The site of action of H O in mediating PS depression is 2 2 For the measurement of evoked potentials, an individual also not yet clear, although there is strong evidence for a slice was transferred to a submersion-recording chamber presynaptic location: H O had no effect on antidromically 2 2 Warner Instrument, Hamden, CT, USA, where it was evoked PS in CA1 stratum pyramidale [34]; nor did it alter 21 continuously superfused with ACSF at 1.5 ml min at EPSPs evoked by glutamate iontophoresis [35]. These data 318C. Extracellular PSs were elicited by stimulating the led Pellmar to propose that H O might inhibit transmitter 2 2 21 Schaffer collaterals and recorded in the stratum pyramidale release by decreasing presynaptic Ca entry [35]. Recent- of CA1 with conventional glass electrodes 2–7 MV tip ly, our laboratory showed that H O can decrease stimu- 2 2 21 resistance, backfilled with 1 M NaCl connected to an lated dopamine release in striatal slices in a Ca -depen- Axoprobe 1A amplifier Axon Instruments, Foster City, dent manner, consistent with a presynaptic site of action of CA, USA. Pulse duration was 100 ms, with the stimulus H O [6]. 2 2 intensity adjusted to the lowest level 0.2–1.3 V required In the present studies, we tested the hypothesis that to evoke a PS of maximal amplitude. The stimulating generation of ?OH is required for the action of H O and 2 2 21 electrode was a twisted bipolar electrode, made from investigated whether decreased presynaptic Ca entry Teflon-insulated platinum–iridium wire. In some experi- might be the mode of action. To ascertain whether PS ments, 10-Hz pulse trains of 5-s duration were used, with depression is mediated by ?OH, the effect of H O was 2 2 the same pulse duration and amplitude as in single pulse assessed in the presence of deferoxamine [37], with experiments. In all slices, the evoked PS was monitored for concomitant determination of H O concentration in the 2 2 25–30 min in normal ACSF to ascertain that the response presence of this chelator. In addition, we tested whether was stable; only slices with stable PS responses during this the endogenous ?OH scavenger, ascorbate [2,4,8,40], could control period were tested further. For each experimental modulate the effect of H O . To determine whether the 2 2 condition tested with single pulse stimulation, three evoked action of ascorbate was intracellular or extracellular, H O 2 2 PS records were stored and averaged using locally written was applied in the presence of the stereoisomer of ascor- software. The amplitude in mV of these averaged PS bate, isoascorbate D -ascorbate, which has similar electro- records was measured from the mean of the positive peak chemical properties to ascorbate, but is not a substrate for preceding and the positive peak following the negative PS. the stereoselective ascorbate transporter [48]. Consequent- Acquisition of pulse train experiments data was controlled ly, isoascorbate is not taken up by cells and remains in the by CLAMPEX 7.0 software Axon Instruments which im- extracellular compartment [4]. In companion experiments, 21 ported PS records in the computer via a DigiData 1200B we evaluated the effect of H O on presynaptic Ca entry 2 2 21 21 DA board Axon Instruments. by monitoring extracellular Ca concentration [Ca ] o using ion-selective microelectrodes ISMs [31,42]. 21 2.3. Ca ion-selective microelectrode recording 21 Stimulated [Ca ] shifts in hippocampal slices were 2. Materials and methods o 21 monitored using Ca -sensitive ion-selective microelec- 21 21 2.1. Hippocampal slice preparation trodes Ca -ISMs. Ca -ISMs were positioned in 21 stratum radiatum of CA1; Ca entry was monitored Hippocampal slices 400-mm thickness were prepared during 10-Hz pulse trains of 5 s duration in the presence of from young adult 50–60 days old, male Long–Evans postsynaptic receptor blockers: picrotoxin, 50 mM; AP5, 21 rats. All animal experimentation was conducted following 50 mM; and CNQX, 25 mM. Double-barreled Ca -ISMs NIH guidelines and with approval by the NYU School of were prepared using theta glass Warner Instrument and Medicine Institutional Animal Care and Use Committee. calibrated as described previously [31,42]. The ion-sensing 21 Rats were deeply anaesthetized with 50 mg kg pen- barrel contained Fluka 21048 Fluka Chemical, Ronkon- 88 M koma, NY, USA as the ion exchanger and was backfilled Students t-test or one-way ANOVA followed by Student– with 100 mM CaCl . The reference barrel was backfilled Newman–Keuls test was used as appropriate. The thres- 2 with 150 mM NaCl. Extracellular potentials recorded with hold of statistical significance was considered to be P , the reference barrel were subtracted from the ion signals 0.05. 21 using the AxoProbe 1A amplifier. Stimulated [Ca ] o shifts were recorded on a chart recorder and stored also on 21 computer. The maximum amplitude of [Ca ] shifts was

3. Results