Research ReportZinc differentially acts on components of long-term potentiation at hippocampal CA1 synapses
Introduction
The hippocampus plays an important role in learning, memory and recognition of novelty. The most widely accepted mechanisms of memory formation are synaptic plasticity (Bliss and Lomo, 1973, Bliss and Collingridge, 1993). The mechanisms of plasticity at the Schaffer collateral/commissural synapses in the hippocampal CA1 region have been extensively studied in the brain (Bliss and Collingridge, 1993, Malenka and Nicoll, 1999, Luscher et al., 2000). It is well established that long-term potentiation (LTP) at this pathway involves the synaptic activation of N-methyl-d-aspartate (NMDA) receptors (Nicoll and Malenka, 1999, Malenka and Bear, 2004). This activation plays a key role for the increase in postsynaptic Ca2+ concentration.
The activation of NMDA receptors, which are heterogeneous with multiple subclasses (Mayer and Armstrong, 2004), is blocked by divalent heavy metals such as zinc (IC50 for the low-affinity site, approximately 20 μM at − 40 mV) and copper (IC50, 0.27 μM) (Vlachova et al., 1996, Paoletti et al., 2009). Zinc and copper are trace metals that play essential roles in the brain function as well as brain development (Prohaska, 1987, Mathie et al., 2006). They are present at high levels in the brain, especially in the hippocampus (Hanig and Aprison, 1967, Donaldson et al., 1974, Tarohda et al., 2004). A major portion of both metals serves as key components in many proteins and co-factors for the activity of many enzymes that are critical for brain function (Vallee and Falchuk, 1993). Both ions can also function as signaling molecules; zinc and copper, which are histochemically reactive as revealed by Timm's sulfide-silver staining method, are concentrated in synaptic vesicles, especially in some glutamatergic neurons (Frederickson, 1989).
It is estimated that the basal concentrations of zinc and copper in the brain extracellular space are < 0.5 μM and 0.2–1.7 μM, respectively (Weiss et al., 2000, Mathie et al., 2006). Because they are co-released with neurotransmitters during neuronal excitation (Vogt et al., 2000, Hopt et al., 2003), it is possible that the extracellular concentrations of zinc and copper in the brain are changed spatially and temporally. However, the changes in their concentrations are poorly understood. The extracellular concentration of zinc in the hippocampus, which is stained at high densities by Timm's method, is estimated to be less than 1 μM, judging from the data on in vivo microdialysis experiments (Takeda et al., 2003). Although the extracellular concentration of zinc reached during LTP induction is a matter of debate, the data that zinc (5 μM) multi-functionally modulates LTP induction in the hippocampus imply that it is very low micromolar (Takeda et al., 2008, Takeda et al., 2009a).
It is likely that other heavy metals such as manganese exist in the synaptic vesicles (Takeda 2003). Manganese can be released into the extracellular space by neuronal excitation (Takeda et al., 1998), although extracellular manganese concentration after tetanic stimulation is estimated to be much less than zinc and copper. Cadmium is believed to be unnecessary for brain function. When cadmium is transported into brain extracellular space after exposure to cadmium, it is taken up into neurons and also released into the extracellular space by the excitation (Minami et al., 2001). Manganese and cadmium can serve as NMDA receptor blockers (IC50, approximately 36 μM and 48 μM for manganese and cadmium, respectively) (Mayer et al., 1989, Guilarte and Chen, 2007) and voltage-dependent calcium channel (VDCC) blockers (Kostyuk, 1986, Castelli et al., 2003). However, the action of manganese and cadmium in synaptic neurotransmission remains to be clarified (Minami et al., 2001, Takeda, 2003).
On the basis of the evidence that ZnCl2 (5 μM) potentiates NMDA receptor-dependent CA1 LTP (Takeda et al., 2009a), the effect of zinc, copper, manganese and cadmium on NMDA receptor-dependent CA1 LTP was compared in the present study. The potentiation of NMDA receptor-dependent CA1 LTP was zinc-specific in heavy metals tested. CA1 LTP is also induced in NMDA receptor-independent pathways (Harris and Cotman, 1986, Grover and Teyler, 1990, Cavuş and Teyler, 1996). Thus, the effect of zinc on NMDA receptor-independent forms was examined.
Section snippets
Effect of heavy metals on NMDA receptor-dependent CA1 LTP
Extracellular zinc concentration is increased by the release from the Schaffer collateral/commissural terminals in the CA1 after tetanic stimulation (Takeda et al., 2007). This increase seems to be involved in CA1 LTP, because CaEDTA, a zinc chelator, inhibits it (Izumi et al., 2006, Takeda et al., 2009a). CA1 LTP induced by tetanic stimulation (100 Hz, 1 s) is abolished in the presence of 2-amino-5-phosphonovalerate (APV), a NMDA receptor antagonist, and is potentiated in the presence of 5 μM ZnCl
Discussion
In hippocampal slices prepared from young animals, the components of CA1 LTP are changed with the strength of tetanic stimulation. A 100-Hz tetanus applied to the Schaffer collateral/commissural inputs to CA1 cells induces a NMDA receptor-dependent form, which is sensitive to APV, while a 200-Hz tetanus induces NMDA receptor-independent forms, which are insensitive to APV (Grover and Teyler, 1990, Shankar et al., 1998, Li et al., 2006). In any form of CA1 LTP, intracellular calcium mobilization
Hippocampal slice preparation
Male Wistar rats (6 weeks old, Japan SLC, Hamamatsu, Japan) were anesthetized with ether and decapitated in accordance with 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 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 1.0 mM NaH2PO4, 2.5 mM CaCl2, 26.2 mM NaHCO3, and 11 mM d-glucose (pH 7.3). Transverse hippocampal slices (400 μm) were prepared using a vibratome ZERO-1
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