Mossy Fiber Zinc Research Articles

A specific role for hippocampal mossy fiber's zinc in rapid storage of emotional memories.

We investigated the specific role of zinc present in large amounts in the synaptic vesicles of mossy fibers and coreleased with glutamate in the CA3 region. In previous studies, we have shown that blockade of zinc after release has no effect on the consolidation of spatial learning, while zinc is required for the consolidation of contextual fear conditioning. Although both are hippocampo-dependent processes, fear conditioning to the context implies a strong emotional burden. To verify the hypothesis that zinc could play a specific role in enabling sustainable memorization of a single event with a strong emotional component, we used a neuropharmacological approach combining a glutamate receptor antagonist with different zinc chelators. Results show that zinc is mandatory to allow the consolidation of one-shot memory, thus being the key element allowing the hippocampus submitted to a strong emotional charge to switch from the cognitive mode to a flashbulb memory mode. Individual differences in learning abilities have been known for a long time to be totally or partially compensated by distributed learning practice. Here we show that contextual fear conditioning impairments due to zinc blockade can be efficiently reduced by distributed learning practice.

Activation of group II metabotropic glutamate receptors blocks zinc release from hippocampal mossy fibers.

BackgroundThe hippocampal CA3 area contains large amounts of vesicular zinc in the mossy fiber terminals which is released during synaptic activity, depending on presynaptic calcium. Another characteristic of these synapses is the presynaptic localization of high concentrations of group II metabotropic glutamate receptors, specifically activated by DCG-IV. Previous work has shown that DCG-IV affects only mossy fiber-evoked responses but not the signals from associational-commissural afferents, blocking mossy fiber synaptic transmission. Since zinc is released from mossy fibers even for single stimuli and it is generally assumed to be co-released with glutamate, the aim of the work was to investigate the effect of DCG-IV on mossy fiber zinc signals.ResultsStudies were performed using the membrane-permeant fluorescent zinc probe TSQ, and indicate that DCG-IV almost completely abolishes mossy fiber zinc changes as it does with synaptic transmission.ConclusionsZinc signaling is regulated by the activation of type II metabotropic receptors, as it has been previously shown for glutamate, further supporting the corelease of glutamate and zinc from mossy fibers.

Quantitative analysis of zinc in rat hippocampal mossy fibers by nuclear microscopy

Zinc (Zn) is involved in regulating mental and motor functions of the brain. Previous approaches have determined Zn content in the brain using semi-quantitative histological methods. We present here an alternative approach to map and quantify Zn levels in the synapses from mossy fibers to CA3 region of the hippocampus. Based on the use of nuclear microscopy, which is a combination of imaging and analysis techniques encompassing scanning transmission ion microscopy (STIM), Rutherford backscattering spectrometry (RBS), and particle induced X-ray emission (PIXE), it enables quantitative elemental mapping down to the parts per million (μg/g dry weight) levels of zinc in rat hippocampal mossy fibers. Our results indicate a laminar-specific Zn concentration of 240±9μM in wet weight level (135±5μg/g dry weight) in the stratum lucidum (SL) compared to 144±6μM in wet weight level (81±3μg/g dry weight) in the stratum pyramidale (SP) and 78±10μM in wet weight level (44±5μg/g dry weight) in the stratum oriens (SO) of the hippocampus. The mossy fibers terminals in CA3 are mainly located in the SL. Hence the Zn concentration is suggested to be within this axonal presynaptic terminal system.

Hippocampal Poly(ADP-Ribose) Polymerase 1 and Caspase 3 Activation in Neonatal Bornavirus Infection

Infection of neonatal rats with Borna disease virus results in a characteristic behavioral syndrome and apoptosis of subsets of neurons in the hippocampus, cerebellum, and cortex (neonatal Borna disease [NBD]). In the NBD rat hippocampus, dentate gyrus granule cells progressively degenerate. Apoptotic loss of granule cells in NBD is associated with accumulation of zinc in degenerating neurons and reduced zinc in granule cell mossy fibers. Excess zinc can trigger poly(ADP-ribose) polymerase 1 (PARP-1) activation, and PARP-1 activation can mediate neuronal death. Here, we evaluate hippocampal PARP-1 mRNA and protein expression levels, activation, and cleavage, as well as apoptosis-inducing factor (AIF) nuclear translocation and executioner caspase 3 activation, in NBD rats. PARP-1 mRNA and protein levels were increased in NBD hippocampi. PARP-1 expression and activity were increased in granule cell neurons and glia with enhanced ribosylation of proteins, including PARP-1 itself. In contrast, levels of poly(ADP-ribose) glycohydrolase mRNA were decreased in NBD hippocampi. PARP-1 cleavage and AIF expression were also increased in astrocytes in NBD hippocampi. Levels of activated caspase 3 protein were increased in NBD hippocampi and localized to nuclei, mossy fibers, and dendrites of granule cell neurons. These results implicate aberrant zinc homeostasis, PARP-1, and caspase 3 activation as contributing factors in hippocampal neurodegeneration in NBD.

Response of hippocampal mossy fiber zinc to excessive glutamate release

The response of hippocampal mossy fiber zinc to excessive glutamate release was examined to understand the role of the zinc in excessive excitation in the hippocampus. Extracellular zinc and glutamate concentrations during excessive stimulation with high K + were compared between the hippocampal CA3 and CA1 by the in vivo microdialysis. Zinc concentration in the CA3 was more increased than that in the CA1, while glutamate concentration in the CA3 was less increased than that in the CA1. It is likely that more increase in extracellular zinc is linked with less increase in extracellular glutamate in the CA3. To see zinc action in mossy fiber synapses during excessive excitation, furthermore, 1 mM glutamate was regionally delivered to the stratum lucidum in the presence of zinc or CaEDTA, a membrane-impermeable zinc chelator, and intracellular calcium signal was measured in the CA3 pyramidal cell layer. The persistent increase in calcium signal during stimulation with glutamate was significantly attenuated in the presence of 100 μM zinc, while significantly enhanced in the presence of 1 mM CaEDTA. These results suggest that zinc released from mossy fibers attenuates the increase in intracellular calcium signal in mossy fiber synapses and postsynaptic CA3 neurons after excessive inputs to dentate granular cells.

Effect of the zinc chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) on hippocampal mossy fiber calcium signals and on synaptic transmission

An important pool of chelatable zinc is present in the synaptic vesicles of mossy fiber terminals from hippocampal CA3 area, being zinc released following single or repetitive electrical stimulation. Previous studies have suggested different synaptic roles for released mossy fiber zinc, including the inhibition of presynaptic calcium and of postsynaptic N-methyl-D-aspartate (NMDA) and gamma amino-butyric acid (GABAA) receptors. The effect of endogenously released zinc on mossy fiber long-term potentiation (LTP) induction also is not yet established. We have investigated the effect of the permeant zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) on mossy fiber calcium and on synaptic transmission, before and during the application of LTP-inducing stimulation. We have found, using the calcium indicator Fura-2, that single and tetanically-evoked mossy fiber calcium signals are both enhanced in the presence of 20 microM TPEN, while the single field potentials are unaffected. As expected, no effect was observed on the single calcium signals or field potentials obtained at the CA3-CA1 synapses, from the CA1 area, which has a lower concentration of vesicular zinc. These results support the idea that at the hippocampal mossy fiber synapses, released zinc inhibits presynaptic calcium mechanisms. A higher concentration of TPEN (100 microM) significantly reduced mossy fiber synaptic transmission but did not prevent the induction of mossy fiber LTP, suggesting that zinc is not required for the formation of this form of LTP.

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.

Hippocampal mossy fiber calcium transients are maintained during long-term potentiation and are inhibited by endogenous zinc

The hippocampal mossy fiber long-term potentiation (LTP) is an N-methyl- d-aspartate (NMDA) receptor-independent form of long-lasting synaptic plasticity characteristic of the zinc-enriched mossy fiber synapses. Its expression is generally considered to have a presynaptic locus and to be mediated by a persistent increase of evoked transmitter release. Because the release process is calcium-dependent, the observed increase in synaptic efficacy could be due to a persistent modification of presynaptic calcium mechanisms, triggered by the large calcium influx associated with long-term potentiation induction. Alternatively, it might be caused by an enhancement in the sensitivity to calcium of some components of the synaptic vesicle release system, following the large intraterminal calcium accumulation. We investigated the first hypothesis by measuring presynaptic Fura-2 calcium signals associated with electrically induced mossy fiber long-term potentiation. We have observed that like residual calcium, single presynaptic calcium changes are not enhanced during the maintenance phase of mossy fiber long-term potentiation. This result supports the idea that this form of long-term potentiation may be mediated by persistent changes of some process occurring after calcium entry. It has been established that voltage-dependent calcium channels are inhibited by zinc and that endogenous zinc is released in a calcium-dependent way following intense mossy fiber activation. Because there is evidence that at these synapses zinc is also released following single electrical stimulation, we investigated the effect of endogenous zinc on single presynaptic calcium signals and on field potentials associated with mossy fiber LTP. We have observed that this form of LTP could be induced in the presence of the permeant heavy metal chelator N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and that application of this chelator, during LTP, caused an enhancement of the presynaptic calcium signals without affecting synaptic transmission. This enhancement is consistent with the idea that mossy fiber zinc, released following individual stimuli, inhibits presynaptic calcium mechanisms.

Lack of vesicular zinc in mossy fibers does not affect synaptic excitability of CA3 pyramidal cells in zinc transporter 3 knockout mice

Zinc is found throughout the CNS in synaptic vesicles of glutamatergic neurons and has been suggested to have a modulatory role in the brain because of its interaction with voltage- and ligand-gated ion channels. We took advantage of zinc transporter 3 knockout mice, which lack vesicular zinc, to study the possible physiological role of this heavy metal in hippocampal mossy fiber neurotransmission. We examined postsynaptic responses evoked by mossy fiber activation, recorded in CA3 pyramidal cells in hippocampal slices prepared from zinc transporter 3 knockout and wild-type mice. Field-potential response threshold and amplitude, input-output curves, and paired-pulse evoked responses were the same in slices from zinc transporter 3 knockout and wild-type mice. Furthermore, neither amplitude nor duration of pharmacologically isolated N-methyl- d-aspartate, non- N-methyl- d-aspartate, GABA A, and GABA B receptor-mediated postsynaptic potentials differed between zinc transporter 3 knockout and wild-type mice. There was no difference in the magnitude of epileptiform discharges evoked by repetitive stimulation or kainic acid application. However, in slices from zinc transporter 3 knockout mice, there was greater attenuation of GABA A-mediated inhibitory postsynaptic potentials during tetanic stimulation compared with slices from wild-type animals. We conclude that lack of vesicular zinc in mossy fibers does not significantly affect the mossy fiber-associated synaptic excitability of CA3 pyramidal cells; however, zinc may modulate GABAergic synaptic transmission under conditions of intensive activation.

Mossy fiber zinc and temporal lobe epilepsy: pathological association with altered "epileptic" gamma-aminobutyric acid A receptors in dentate granule cells.

Temporal lobe epilepsy is associated with circuit rearrangements within the hippocampus. Mossy fibers sprout and pathologically innervate the inner molecular layer of the dentate gyrus, providing a recurrent excitatory pathway not present in the control brain. In addition to releasing glutamate, these recurrent collaterals also release zinc, which can accumulate in high concentrations in the extracellular space. Accompanying these dentate gyrus circuit rearrangements are alterations in the subunit expression patterns and pharmacology of gamma-aminobutyric acid A (GABAA) receptors in dentate granule cells. In normal, control granule cells, GABAA receptors are zinc insensitive as a result of high levels of expression of the alpha1 subunit in these cells. In epileptic brain, expression of alpha1 subunits decreases and expression of alpha4 and delta subunits increases, leading to the assembly of GABAA receptors that are exquisitely zinc sensitive. This temporal and spatial association of the expression of zinc-sensitive GABAA receptors and the emergence of a zinc-delivery system unique to the epileptic hippocampus has led to the formulation of an hypothesis that suggests that zinc release during repetitive activation of the dentate gyrus may lead to a catastrophic failure of inhibition under conditions mediating seizure initiation. This could contribute to the limbic hyperexcitability characteristic of temporal lobe epilepsy.

Perinatal Hypothyroidism Decreases Hippocampal Mossy Fiber Zinc Density in Rats

The effect of perinatal hypothyroidism on hippocampal mossy fiber zinc density was examined in rats. Timed pregnant Sprague-Dawley rat dams were given water containing either 0.02% propylthiouracil (PTU) or vehicle from gestational day 18 until their litters were weaned on postnatal day 31. Hippocampal mossy fiber zinc density was reduced by 75% in both the dorsal and ventral hippocampal formation CA3 stratum lucidum region of 31-day-old PTU-treated rats compared to untreated controls. Perinatal hypothyroidism did not alter hippocampal tissue zinc concentration, indicating that the PTU-induced reduction in mossy fiber zinc was not a consequence of reduced hippocampal zinc concentration. At 120 days of age, 3 months after discontinuation of PTU treatment, hippocampal mossy fiber zinc density remained significantly reduced by 33-45% in PTU-treated rats compared to control. These data indicate that perinatal hypothyroidism causes a long-lasting reduction in hippocampal mossy fiber zinc density.

Hippocampal mossy fiber zinc deficit in mice genetically selected for ethanol withdrawal seizure susceptibility

Hippocampal mossy fiber zinc was examined in mice selectively bred for differences in susceptibility to handling-induced convulsions during ethanol withdrawal. The density of mossy fiber zinc in the CA3 stratum lucidum was significantly decreased in the duplicate lines of untreated withdrawal seizure prone (WSP) mice compared to untreated withdrawal seizure resistant (WSR) mice. Mossy fiber zinc densities in randomly bred control lines of mice (WSC) were intermediate to WSP and WSR mice. Serum, whole brain and whole hippocampal zinc were not significantly different between WSP and WSR mice, indicating that the reduction in the chelatable pool of hippocampal mossy fiber zinc was not a consequence of deficits in brain or whole body zinc nutrition. A highly significant correlation between hippocampal mossy fiber zinc density and handling-induced convulsion indices suggests that a reduction in mossy fiber zinc may be one contributing factor in the expression of seizure susceptibility in WSP mice.

Quantitative histofluorescence of hippocampal mossy fiber zinc

N-(6-methoxy-8-quinolyl)- p-toluenesulfonamide (TS-Q) is a fluorescent probe for histochemically detecting a chelatable pool of zinc in the hippocampal formation and other brain regions. Utilization of zinc:TS-Q histofluorescence as a quantitative tool required further characterization of zinc:TS-Q fluorescence and the development of appropriate zinc standards. Studies of zinc:TS-Q fluorescence in aqueous solutions were conducted to optimize the reaction conditions for TS-Q chelation with zinc. Possible interference of zinc:TS-Q fluorescence by chelation of other biologically significant cations with zinc:TS-Q was shown to be negligible. Zinc standards were developed using a water soluble plastic embedding medium. Varying the concentration of zinc in the plastic sections yielded a standard curve exhibiting a log-linear relationship between 0.15 and 11 fg zinc/100 μm 2. Zinc:TS-Q histofluorescence was characterized in horizontal sections of rat ventral hippocampal formation using a computerized image analysis system. Section thickness and TS-Q concentration were optimized. The concentration of zinc varied between 1.5 and 17 fg/100 μm 2 in different regions of the mossy fiber projection of the ventral hippocampal formation. The development of this quantitative histofluorescence technique should facilitate our understanding of the functional role of zinc in the mossy fiber projection and can be utilized to investigate changes in mossy fiber zinc in a variety of pathological states.

Prenatal ethanol exposure decreases hippocampal mossy fiber zinc in 45-day-old rats.

The long-term consequences of prenatal ethanol exposure on histochemically detectable hippocampal mossy fiber zinc was examined using a recently developed quantitative histofluorescence procedure. Pregnant Sprague-Dawley rats were maintained throughout gestation on one of three dietary regimens: (a) a liquid diet containing either 3.35% ethanol, (b) an isocalorically matched liquid diet pair-fed to the 3.35% ethanol group, or (c) lab chow ad libitum. At 45 days of age, offspring from each of the three diet groups were sacrificed for determination of hippocampal mossy fiber zinc and zinc analysis of selected tissues by atomic absorption spectroscopy. Hippocampal mossy fiber zinc was reduced by 36% in dorsal and 20% in ventral hippocampal formation stratum lucidum of rats exposed to the 3.35% ethanol diet compared to the offspring of the pair-fed control and ad libitum control dams. No significant differences in zinc:TS-Q histofluorescence were observed between the ad libitum and pair-fed control groups. No significant differences were observed among groups in tissue wet weight or tissue zinc concentration in any of the brain or other body regions analyzed. These results indicate a long lasting prenatal ethanol exposure-induced reduction in hippocampal mossy fiber zinc in the absence of changes in any indices of total body zinc nutriture. These results suggest that prenatal exposure to relatively low blood ethanol levels (30-40 mg/dl) has subtle, yet long-lasting effects in the hippocampal formation, a brain region important in the process of memory consolidation.

The effect of dietary zinc deficiency on the mossy fiber zinc content of the rat hippocampus. A microbeam PIXE study. Particle Induced X-Ray Emission.

The effect of dietary zinc deficiency on the mossy fiber zinc content of the rat hippocampus was investigated using PIXE (Particle Induced X-Ray Emission) spectroscopy. Using the proton microbeam (60 X 60 microns), 2 mm line-scans were made on hippocampal sections and the data were expressed as absolute zinc concentrations. Values of 55 and 136 ppm (dry weight) were found for the mean background zinc level and the maximum mossy fiber zinc level, respectively, in animals fed a control diet containing 50 ppm zinc. Treatment of these animals with dithizone caused about 50% reduction in the maximum mossy fiber zinc level. Feeding a zinc-deficient diet for 28 days did not cause a decrease in the mossy fiber zinc level, however, feeding the zinc-deficient diet for 90 days reduced the maximum mossy fiber zinc level by about 30%. The results are discussed in relation to the behavioral abnormalities that have been observed in zinc-deficient animals.

Effects of alcohol ingestion on zinc content of human and rat central nervous systems

The abundance of zinc in hippocampal mossy fibers has stimulated investigation of zinc status in various pathologic states in which behavioral or anatomic deficits involving the hippocampus are known to occur. Limited autopsy studies of chronic alcoholic humans have suggested that the content of zinc might be reduced in several brain regions whereas reported attempts to replicate these findings in ethanol-exposed experimental animals have produced inconsistent results. In this comparative study, the zinc concentration in 10 brain regions, all spinal cord segments, and microdissected hippocampal subfields was measured by isotope ratio mass spectrometry. A widespread 15 to 20% reduction in zinc content was observed in all regions of chronic alcoholics compared with controls but a selective involvement of hippocampus was not detected. In the experimental studies, groups of rats were exposed to ethanol by one of three routes: inhalation for 2 weeks, as an ethanol/liquid diet for 3 months, or a single intoxicating i.p. dose. Determinations of tissue uptake of radiozinc and of total zinc content were made. In contrast to human pathologic material, zinc pool size and tissue uptakes were not affected by experimental ethanol administration by any route. This study confirmed that a reduction in zinc concentration occurs in the central nervous system of chronic alcoholics. The animal studies indicated, however, that simple ethanol exposure, even for prolonged periods, does not perturb zinc metabolism in brain. Together, these observations argue that the abnormalities of zinc metabolism in chronic alcoholics are possibly secondary to homeostatic alterations associated with hepatic failure.

Cholinergic denervation-induced increase of chelatable zinc in mossy-fiber region of the hippocampal formation

Chelatable zinc has repeatedly been associated with hippocampal mossy fibers, but the neurobiological functions of the zinc have remained unclear. Zinc is a constituent of the 7S Nerve Growth Factor (NGF) molecule. The hippocampal area is rich in neurotrophic factors and cholinergic deafferentation of the hippocampus appears to activate these neurotrophic factors. In this paper we report that cholinergic deafferentation of the hippocampus causes a transient rise in mossy-fiber zinc, as measured by zinc-dithizonate histochemistry. The changes are not significant until 10 days after the septal lesion, and the zinc returns to control levels by 21–28 days. Dithizonate has an association constant of 10 −15 with zinc, while NGF has an association constant of 10 −11. Thus densitometric measurement of zinc dithizone within the hippocampus may be a quantitative histochemical marker of a zinc-associated nerve growth-like substance. The data support recent findings that nerve growth-like factors increase in the hippocampal area following deafferentation, and in addition suggest that zinc is a critical factor in the regulation of trophic phenomena in the hippocampal formation.