Carlos M. Matias

Tetanically released zinc inhibits hippocampal mossy fiber calcium, zinc and synaptic responses

At the zinc-enriched mossy fiber synapses from hippocampal CA3 area, electrical or chemical stimulation evokes zinc release from glutamatergic synaptic vesicles that may cause different pre- or postsynaptic actions. Besides zinc that can be co-localized with glutamate and GABA, the mossy fibers contain a very high density of ATP-sensitive potassium channels that are activated by zinc. We have investigated the possibility that intensely released zinc inhibits presynaptic calcium changes and consequently zinc and glutamate release. The studies were made combining optical recording of fast presynaptic calcium and zinc signals, using the fluorescent indicators Fura-2 and N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide, respectively, with measurements of field potentials. We have observed that strong tetanic stimulation caused posttetanic depressions of electrically induced presynaptic calcium and zinc signals and of synaptic responses, the depressions being blocked by zinc chelators. These results suggest that endogenously released zinc has an inhibitory role, mediated by presynaptic ATP-sensitive potassium channels and/or presynaptic calcium channels, that leads to the depression of zinc and glutamate release.

Measurement of presynaptic zinc changes in hippocampal mossy fibers.

The hippocampal mossy fiber terminals of CA3 area contain high levels of vesicular zinc that is released in a calcium-dependent way, following high-frequency stimulation. However the properties of zinc release during normal synaptic transmission, paired-pulse facilitation and mossy fiber long-term potentiation are still unknown. Using the fluorescent zinc probe N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide, we measured fast mossy fiber zinc changes indicating that zinc is released following single and low levels of electrical stimulation. The observed presynaptic zinc signals are maintained during the expression of mossy fiber long-term potentiation, assumed to be mediated by an increase in transmitter release, and are enhanced during paired-pulse facilitation. This zinc enhancement is, like paired-pulse facilitation, reduced during established long-term potentiation. The correlation between the paired-pulse evoked zinc and field potential responses supports the idea that zinc is co-released with glutamate.

Effect of d-2 amino-5-phosphonopentanoate and nifedipine on postsynaptic calcium changes associated with long-term potentiation in hippocampal CA1 area

The induction of long-term potentiation (LTP) in CA1 hippocampal area requires a rise in intracellular postsynaptic calcium. Two major calcium mechanisms may mediate the transmembrane calcium influxes that contribute to this calcium accumulation: the N-methyl-D-aspartate (NMDA) receptor channels, which are voltage dependent and have large calcium permeability and voltage-dependent calcium channels (VDCCs). We have addressed the relative contribution of these routes of calcium entry before and during LTP expression, in synaptically evoked dendritic calcium transients from a population of CA1 pyramidal neurons. Combining the use of the fluorescent calcium indicator Fura-2 with field potential measurements, we observed that the calcium transients evoked by single stimuli, during the maintenance phase of LTP, were enhanced. These transients were not affected by D-2 amino-5-phosphonopentanoate (D-APV) (50 microM), an antagonist of NMDA receptors but were reduced by approximately one-quarter, in the presence of the L-type VDCCs blocker nifedipine (10 microM). During tetanic stimulation (100 Hz, 1 s) the components triggered by the activation of those two calcium mechanisms had comparable magnitudes representing the sum about half of the intracellular calcium accumulation. Thus, following both single and high frequency stimulation, a substantial fraction of calcium entry may occur through other types of VDCCs or be due to calcium release from intracellular stores.

Validation of TPEN as a Zinc Chelator in Fluorescence Probing of Calcium in Cells with the Indicator Fura-2

Fura-2 is widely used as a fluorescent probe to monitor dynamic changes in cytosolic free calcium in cells, where Ca2+ can enter through several types of voltage-operated or ligand-gated channels. However, Fura-2 is also sensitive to other metal ions, such as zinc, which may be involved in ionic channels and receptors. There is interest, in particular, in studying the synapses between mossy fibers and CA3 pyramidal cells which contain both calcium and high quantities of free or loosely bound zinc. We have found, through fluorescence probing, that endogenous zinc inhibits mossy fiber calcium transients. However, since these results might be explained by an effect of the zinc chelator N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) on the spectral properties of Fura-2, we have carried out a validation of the method through fluorescence excitation spectra of the complex Fura-2/calcium, and show that TPEN does not affect these spectra. This supports the idea that the observed calcium enhancement is related to a zinc inhibition of presynaptic calcium mechanisms, and confirms the use of the chelator TPEN as a general procedure for the biophysical study of Ca(II) in the presence of Zn(II) using Fura-2.

L-AP3 blocks rises in intracellular calcium associated with hippocampal CA1 LTP.

The involvement of type I metabotropic glutamate receptors in hippocampal CA1 long-term potentiation (LTP) depends on the applied tetanic stimulation protocol. Activation of these receptors may cause an elevation of intracellular calcium via the formation of the second messenger inositol triphosphate (IP3) and subsequent intracellular calcium release. It has been shown that the type I metabotropic receptors antagonist L-2-amino-3-phosphonopropionate (L-AP3) blocks CA1 LTP. Combining dendritic calcium and field potential measurements in CA1 hippocampal area, we found that L-AP3 did not affect single calcium transients but reduced the calcium changes evoked by a single tetanus, preventing the long-lasting calcium enhancements associated with CA1 LTP. These findings suggest that the formation of this type of LTP requires calcium release from IP3-sensitive stores.

Sulfamethoxazole induces zinc changes at hippocampal mossy fiber synapses from pregnant rats

The accumulation of intracellular ionic zinc and pharmaceutical compounds, like the antibiotic sulfamethoxazole, may contribute to various neuropathologies. Sulfamethoxazole and the drug trimethoprim, are inhibitors of enzymes involved in the synthesis of tetrahydrofolate and also of carbonic anhydrases. The inhibition of the latter enzymes, which are localized both intra- and extracellularly and have a key role in pH regulation, causes alkalinization that is associated with higher spontaneous transmitter release. Intense synaptic stimulation causes the entry of released zinc into postsynaptic neurons, through glutamate receptor channels or voltage dependent calcium channels. The aim of this study was to evaluate the effect of sulfamethoxazole (180 μM) on basal postsynaptic zinc and to compare it with that caused by two depolarizing media, containing high potassium or tetraethylammonium, which may induce long term synaptic plasticity. The studies were performed in brain slices from gestating rats, at the mossy fiber synapses from hippocampal CA3 area, using the zinc indicator Newport Green. In the presence of KCl (20 mM) and sulfamethoxazole (180 μM) the zinc signals were enhanced, unlike in tetraethylammonium (25 mM). After sulfamethoxazole the tetraethylammonium evoked zinc signal had reduced amplitude. Thus, the data suggests that sulfamethoxazole enhances transmitter release affecting synaptic zinc physiology.

Postsynaptic zinc potentiation elicited by KCl depolarization at hippocampal mossy fiber synapses

The hippocampal mossy fibers contain a substantial quantity of loosely-bound zinc in their glutamatergic presynaptic vesicles, which is released in synaptic transmission processes. Despite the large number of studies about this issue, the zinc changes related to short and long-term forms of potentiation are not totally understood. This work focus on zinc signals associated with chemically-induced mossy fiber synaptic plasticity, in particular on postsynaptic zinc signals evoked by KCl depolarization. The signals were detected using the medium affinity fluorescent zinc indicator Newport Green. The application of large concentrations of KCl, 20 mM and 60 mM, in the extracellular medium evoked zinc potentiations that decreased and remained stable after washout of the first and the second media, respectively. These short and long-lasting enhancements are considered to be due to zinc entry into postsynaptic neurons. We have also observed that following established zinc potentiation, another application of 60 mM KCl only elicited further enhancement when combined with external zinc. These facts support the idea that the KCl-evoked presynaptic depolarization causes higher zinc release leading to zinc influx into the postsynaptic region.

Effect of tolbutamide on tetraethylammonium-induced postsynaptic zinc signals at hippocampal mossy fiber-CA3 synapses

The application of tetraethylammonium (TEA), a blocker of voltage-dependent potassium channels, can induce long-term potentiation (LTP) in the synaptic systems CA3-CA1 and mossy fiber-CA3 pyramidal cells of the hippocampus. In the mossy fibers, the depolarization evoked by extracellular TEA induces a large amount of glutamate and also of zinc release. It is considered that zinc has a neuromodulatory role at the mossy fiber synapses, which can, at least in part, be due to the activation of presynaptic ATP-dependent potassium (KATP) channels. The aim of this work was to study properties of TEA-induced zinc signals, detected at the mossy fiber region, using the permeant form of the zinc indicator Newport Green. The application of TEA caused a depression of those signals that was partially blocked by the KATP channel inhibitor tolbutamide. After the removal of TEA, the signals usually increased to a level above baseline. These results are in agreement with the idea that intense zinc release during strong synaptic events triggers a negative feedback action. The zinc depression, caused by the LTP-evoking chemical stimulation, turns into potentiation after TEA washout, suggesting the existence of a correspondence between the observed zinc potentiation and TEA-evoked mossy fiber LTP.