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Address correspondence to Yuji Ikegaya, Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5841-4784. Fax: 81-3-5841-4784. E-mail: ikegaya{at}tk.airnet.ne.jp
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Abstract |
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Key Words: zinc; mossy fiber; hippocampus; synaptic plasticity; indicator
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Introduction |
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Despite numerous studies on Zn2+ action in the CNS, the physiological significance of synaptically released Zn2+ is largely unknown, one reason being that the spatiotemporal Zn2+ dynamics during synaptic activity remains unclear to date. To explore Zn2+ behavior, most of the previous studies have utilized such fluorescent Zn2+ indicators as Newport Green (Li et al., 2001b) and Mag-Fura-5 (Sensi et al., 1997; Canzoniero et al., 1999). These indicators have relatively low affinity for Zn2+, their Kd values being 1 and 27 nM, respectively. Considering that a very low concentration of extracellular Zn2+ ([Zn2+]o) is sufficient to inhibit the activity of NR2A-containing NMDA receptors, the major receptor form in the mature hippocampus (IC50 = 5 nM) (Paoletti et al., 1997), such low-sensitivity indicators cannot trace Zn2+ dynamics at a low but physiologically significant level. In addition, these indicators exhibit low selectivity for Zn2+ in the presence of other ions; e.g., Mag-Fura-5 shows affinity for Ca2+ and Mg2+ as well. Another problem is that Newport green shows high background fluorescence even in the absence of Zn2+, and a relatively small increase in fluorescence intensity after exposure to Zn2+. To overcome these problems, we employ ZnAF-2, a novel fluorescent indicator, to monitor Zn2+ dynamics. ZnAF-2 has a low Kd value of 2.7 nM for Zn2+ and its fluorescence is minimally changed in the presence of Ca2+, Mg2+, Cd2+, Ni2+, or other heavy metal ions (Hirano et al., 2000). Also, ZnAF-2 has no apparent toxicity to living cells (Hirano et al., 2000; 2002). These features allow us to assess physiologically relevant Zn2+ behavior in hippocampal slices without interference from other heavy metal ions.
Here we report that Zn2+ is released by MF synaptic terminals in an activity-dependent manner and diffuses extracellularly into the adjacent stratum radiatum after tens of seconds, thereby inhibiting NMDA receptormediated synaptic responses. Thus, the synaptically released Zn2+ may act as an activity-dependent, heterosynaptic modulator of hippocampal synaptic transmission.
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Results and discussion |
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To determine how the frequency of MF activity affects the spatiotemporal dynamics of synaptically released Zn2+, the MFs were activated by repetitive stimulation at 1 or 5 Hz. In either case, an apparent increase in [Zn2+]o was observed in stratum lucidum and proximal stratum radiatum, but the peak [Zn2+]o was smaller and the kinetics was slower as compared with those induced by a 100 Hz tetanus (Fig. 3, B and C). The time course of the [Zn2+]o changes was almost equivalent in both the subregions (Fig. 3, B and C). No MF stimulation induced no change of [Zn2+]o (Fig. 3 D).
Previous studies indicated that Zn2+ inhibits NMDA receptor function at very low concentrations (Paoletti et al., 1997). Zn2+ spread to stratum radiatum is, therefore, possible to modulate NMDA receptor function therein. To address the functional significance of Zn2+ spillover from MF synapses, NMDA receptormediated field excitatory postsynaptic potential (fEPSPNMDA) were extracellularly recorded at associational/commissural-CA3 pyramidal cell synapses. When a recording electrode was positioned in the proximal region of stratum radiatum (<100 µm from stratum lucidum), fEPSPNMDA declined transiently in response to MF tetanization (100 Hz for 2 s); the inhibition reached an apparent peak after 15 s and rapidly returned to baseline by 60 s (Fig. 4). This depression was completely relieved 15 min after bath application of 25 µM TPEN (Fig. 4 B), whereas TPEN alone did not affect baseline fEPSPNMDA (n = 7; P > 0.1; paired t test; unpublished data). The data indicate that endogenous Zn2+ mediates fEPSPNMDA-blocking action of MF tetanization but does not significantly work under basal conditions. As expected from the results of ZnAF-2, fEPSPNMDA recorded from the distal part of stratum radiatum (>200 µm far from stratum lucidum) was insensitive to the same stimulation of the MFs (Fig. 4).
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Although the role of Zn2+ in MF terminals has been unclear, the development of the high-affinity, Zn2+-specific indicator ZnAF-2 has enabled us to precisely map the extracellular fate of synaptically released Zn2+. We have shown for the first time that Zn2+ released from MF terminals is distributed over the surrounding areas (up to 100 µm far from the released site) within tens of seconds, and also that the Zn2+ spillover causes a heterosynaptic inhibition of NMDA receptor function. Therefore, Zn2+ is likely to serve as an intersynaptic mediator in etching the history of MF activity into neighboring synapses in hippocampal circuits.
Recent evidence showed that Zn2+ plays a role in synaptic transmission and plasticity at MF-CA3 synapses. The baseline level of Zn2+ yields a tonic inhibition of NMDA receptors at MF synapses, and MF tetanization results in a further inhibition by bulk release of Zn2+ (Vogt et al., 2000). The endogenous Zn2+ may also be involved in the induction of NMDA receptorindependent long-term potentiation at MF synapses (Weiss et al., 1989; Lu et al., 2000; Vogt et al., 2000; Li et al., 2001a), in which Zn2+ may behave like a second messenger after entering into presynaptic or postsynaptic neurons (Li et al., 2001a). Thus, past studies on mossy fiber Zn2+ have focused mainly on its homosynaptic action. However, if Zn2+ could only coact with neurotransmitters at the released site, the role of Zn2+ would be limited to a monotonous modulation. Here we found that Zn2+ influences NMDA receptor function even at neighboring synapses in stratum radiatum as well. Similarly, Zn2+ probably exerts its heterosynaptic action at adjacent MF synapses in stratum lucidum. Therefore, we consider that this metal ion is assigned a highly dynamic role in regulating the physiological function of hippocampal CA3 local circuits.
Zn2+ is shown to inhibit NMDA currents and potentiate AMPA currents (Rassendren et al., 1990), but we found no evidence that fEPSPAMPA was increased after MF activation. Some reports indicated that AMPA receptors have different subunit compositions including splicing variants, thereby showing different responsiveness to Zn2+ (Dreixler and Leonard, 1994; Shen and Yang, 1999). Indeed, only half of the CA3 neurons are sensitive to Zn2+ (Lin et al., 2001). This may account for no change in AMPA responses in our experiments. However, a more plausible explanation is a difference in the Zn2+ sensitivity of NMDA and AMPA receptors. The concentrations giving a half-maximal response are 5 nM for NMDA receptors (Paoletti et al., 1997) and 30 µM for AMPA receptors (Rassendren et al., 1990); AMPA receptors are nearly 104-fold less sensitive to Zn2+. The peak [Zn2+]o in stratum radiatum may be in the range of 530 µM.
There was an apparent discrepancy in time course between the increase of ZnAF-2 signal and the inhibition of fEPSPNMDA in stratum radiatum. Both peaked about 15 s after MF stimulation. However, after the peak, ZnAF-2 signal was kept high for >60 s while fEPSPNMDA returned to baseline within 60 s. Because of the high-affinity of ZnAF-2 (Kd = 2.7 nM), the indicator may interfere with intrinsic Zn2+ uptake system, and Zn2+ may remain in the extracellular space as a stable complex with ZnAF-2. Therefore, we cannot exclude the possibility that ZnAF-2 signal does not strictly reflect naturally occurring Zn2+ dynamics, particularly in the decay kinetics. Nonetheless, this does not disclaim the fact that Zn2+ diffuses from the released site. The result of ZnAF-2 photobleaching and the TPEN effect on fEPSPNMDA provide unambiguous evidence for a significant spread of Zn2+ beyond the MF region.
In conclusion, the present study has established that the metal ion Zn2+ is an activity-dependent, spatiotemporal modulator of NMDA receptor function in hippocampal CA3 local circuits and that the extracellular Zn2+ gradient made after MF activation reaches 100 µm but eliminates within tens of seconds. The spillover range is probably variable along with MF presynaptic release probability, which is known to increase after the induction of long-term potentiation (Toth et al., 2000). Considering that NMDA receptors serve as a coincidence detector in synaptic plasticity and learning and memory (Bliss and Collingridge, 1993; Martin et al., 2000), the Zn2+ gradient may yield different learning rules along the apical dendrite of a CA3 pyramidal cell, and therefore MF activation may emphasize a difference in information processing between the distal and proximal segments of the postsynaptic dendrite. This work predicts a novel form of experience-dependent modulation of synaptic plasticity, i.e., Zn2+-mediated, heterosynaptic metaplasticity, and thus provides new insights into information processing of the hippocampus.
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Materials and methods |
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Hippocampal slice preparation
Postnatal 1727-d-old Wistar/ST rats (SLC) were anesthetized with ether and decapitated, according to the Japanese Pharmacological Society guide for the care and use of laboratory animals. The brain was quickly removed and immersed in ice-cold artificial cerebrospinal fluid (ACSF) containing 124 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 1.24 mM KH2PO4, 1.4 mM MgSO4, 2.2 mM CaCl2, and 10 mM glucose, continuously bubbled with 95% O2 and 5% CO2. Horizontal hippocampal slices of 300350 µm in thickness were prepared using the vibratome ZERO-1 (Dosaka).
Extracellular Zn2+ imaging
ZnAF-2 is incapable of permeating the cell membrane, but its diacetylated form can be passively loaded into cells where it is cleaved to cell-impermeant products by intracellular acetylase (Hirano et al., 2000). Therefore, for intracellular Zn2+ fluorescence imaging, hippocampal slices were preloaded with 10 µM ZnAF-2 diacetate (ZnAF-2-DA) in the dark for 90 min at room temperature, and washed with ACSF for at least 30 min to remove unincorporated ZnAF-2-DA from the intercellular space. For extracellular Zn2+ detection, slices were loaded with 10 µM ZnAF-2 for at least 90 min. Zn2+ imaging was performed at 2732°C with the confocal microscopic system BioRad MRC-1000 equipped with the inverted microscope ECLIPSE TE300 (Nikon) and an argon ion laser (monochromator set to 492 nm). Emitted light images at 514 nm or greater were acquired at rates of 0.2-1 Hz through a 10x objective (0.45 of numerical aperture) with an intensified CCD camera and digitized with Laser Sharp Acquisition (Bio-Rad Laboratories). Autofluorescence was below the detection limits of the camera, and photobleaching was negligible under these conditions; neither was subtracted from the data.
Electrical stimulation and extracellular recording
To induce the release of Zn2+ from MF terminals, bipolar tungsten electrodes were placed in the stratum granulosum of the dentate gyrus, and trains of stimuli (at 1, 5, or 100 Hz, each rectangular pulse with a 60-µs duration and 500-µA intensity) were delivered. For extracellular recording, slices were preincubated in a 95% O25% CO2-saturated ACSF for at least 1 h at 32°C, placed in an interface recording chamber, and perfused with ACSF equilibrated with 95% O2 and 5% CO2 at 32°C. Test stimuli were delivered every 10 s through the bipolar tungsten electrodes positioned across the associational/commissural fibers in the middle part of CA3 stratum radiatum. The fEPSPs were recorded from CA3 stratum radiatum by a glass microelectrode filled with 0.15 M NaCl (1 M
of resistance). To check whether the fEPSPs were contaminated with MF responses, single-pulse stimulation was applied to the MFs. We could easily confirm that this stimulation induced a positive field response in stratum radiatum if we obtained a complete separation of the two inputs. When the MF stimulation evoked a negative response like associational/commissural stimulation, the experiment was discarded. AMPA receptormediated response (fEPSPAMPA) was recorded in the presence of 50 µM D-2-amino-5-phosphonopentanoic acid and evaluated by its amplitude. fEPSPNMDA was isolated in Mg2+-free ACSF containing 20 µM CNQX and evaluated by the area under the curve from 4 to 45 ms after test stimulus. The stimulus intensity was set to produce fEPSPAMPA with an amplitude of 50% of maximum or fEPSPNMDA with an area of 70% of maximum. The baseline was recorded for at least 10 min to ensure the stability of the response.
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Footnotes |
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Acknowledgments |
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Submitted: 15 April 2002
Revised: 29 May 2002
Accepted: 4 June 2002
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References |
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Bliss, T.V., and G.L. Collingridge. 1993. A synaptic model of memory: long-term potentiation in the hippocampus. Nature. 361:3139.[CrossRef][Medline]
Canzoniero, L.M., D.M. Turetsky, and D.W. Choi. 1999. Measurement of intracellular free zinc concentrations accompanying zinc-induced neuronal death. J. Neurosci. 19:RC31.[Medline]
Dreixler, J.C., and J.P. Leonard. 1994. Subunit-specific enhancement of glutamate receptor responses by zinc. Mol. Brain Res. 22:144150.[Medline]
Eom, S.J., E.Y. Kim, J.E. Lee, H.J. Kang, J. Shim, S.U. Kim, B.J. Gwag, and E.J. Choi. 2001. Zn2+ induces stimulation of the c-Jun N-terminal kinase signaling pathway through phosphoinositide 3-kinase. Mol. Pharmacol. 59:981986.
Hirano, T., K. Kikuchi, Y. Urano, T. Higuchi, and T. Nagano. 2002. Improvement and biological applications of fluorescent probes for zinc, ZnAFs. J. Am. Chem. Soc. In press.
Hubbard, S.R., W.R. Bishop, P. Kirschmeier, S.J. George, S.P. Cramer, and W.A. Hendrickson. 1991. Identification and characterization of zinc binding sites in protein kinase C. Science. 254:17761779.[Medline]
Kamiya, H., H. Shinozaki, and C. Yamamoto. 1996. Activation of metabotropic glutamate receptor type 2/3 suppresses transmission at rat hippocampal mossy fibre synapses. J. Physiol. 493:447455.[Abstract]
Koh, J.Y., S.W. Suh, B.J. Gwag, Y.Y. He, C.Y. Hsu, and D.W. Choi. 1996. The role of zinc in selective neuronal death after transient global cerebral ischemia. Science. 272:10131016.[Abstract]
Li, Y., C.J. Hough, C.J. Frederickson, and J.M. Sarvey. 2001a. Induction of mossy fiber Ca3 long-term potentiation requires translocation of synaptically released Zn2+. J. Neurosci. 21:80158025.
Li, Y., C.J. Hough, S.W. Suh, J.M. Sarvey, and C.J. Frederickson. 2001b. Rapid translocation of Zn2+ from presynaptic terminals into postsynaptic hippocampal neurons after physiological stimulation. J. Neurophysiol. 86:25972604.
Lin, D.D., A.S. Cohen, and D.A. Coulter. 2001. Zinc-induced augmentation of excitatory synaptic currents and glutamate receptor responses in hippocampal CA3 neurons. J. Neurophysiol. 85:11851196.
Martin, S.J., P.D. Grimwood, and R.G. Morris. 2000. Synaptic plasticity and memory: an evaluation of the hypothesis. Annu. Rev. Neurosci. 23:649711.[CrossRef][Medline]
Peters, S., J. Koh, and D.W. Choi. 1987. Zinc selectively blocks the action of N-methyl-d-aspartate on cortical neurons. Science. 236:589593.[Medline]
Paoletti, P., P. Ascher, and J. Neyton. 1997. High-affinity zinc inhibition of NMDA NR1-NR2A receptors. J. Neurosci. 17:57115725.
Sensi, S.L., L.M. Canzoniero, S.P. Yu, H.S. Ying, J.Y. Koh, G.A. Kerchner, and D.W. Choi. 1997. Measurement of intracellular free zinc in living cortical neurons: routes of entry. J. Neurosci. 17:95549564.
Shumilla, J.A., K.E. Wetterhahn, and A. Barchowsky. 1998. Inhibition of NF-kappa B binding to DNA by chromium, cadmium, mercury, zinc, and arsenite in vitro: evidence of a thiol mechanism. Arch. Biochem. Biophys. 349:356362.[CrossRef][Medline]
Toth, K., G. Suares, J.J. Lawrence, E. Philips-Tansey, and C.J. McBain. 2000. Differential mechanisms of transmission at three types of mossy fiber synapse. J. Neurosci. 20:82798289.
Weiss, J.H., J.Y. Koh, C.W. Christine, and D.W. Choi. 1989. Zinc and LTP. Nature. 338:212.[Medline]