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

Background

The 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.

Results

Studies 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.

Conclusions

Zinc 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.     

Presynaptic facilitation of glutamate release from isolated hippocampal mossy fiber nerve endings by arachidonic acid

Hippocampal mossy fiber synaptosomes were used to investigate the role of arachidonic acid in the release of endogenous glutamate and the long-lasting facilitation of glutamate release associated with long-term potentiation. Exogenous arachidonate induced a dose-dependent efflux of glutamate from the hippocampal mossy fiber synaptosomes and this effect was mimicked by melittin. Neither treatment induced the release of occluded lactate dehydrogenase at the concentrations used in these experiments. In each case, removal of the biochemical stimulus allowed for glutamate efflux to return to spontaneous levels. However, there was a persistent effect of exposure to either arachidonate or melittin, since these compounds facilitated the glutamate release induced by the subsequent addition of 35 mM KCl. This facilitation of glutamate release resulted from an enhancement of both the magnitude and duration of the response to depolarization. Although exogenous prostanoids were also able to stimulate the release of glutamate, they appeared to play no direct role in secretion processes, since inhibition of eicosanoid synthesis potentiated the glutamate efflux in response to membrane depolarization or exogenous arachidonic acid. We suggest that the calcium-dependent accumulation of arachidonic acid in presynaptic membranes plays a central role in the release of endogenous glutamate and that the persistent effects of arachidonic acid may be related to the maintenance of long-term potentiation in the hippocampal mossy fiber-CA3 synapse.     

Modelling zinc changes at the hippocampal mossy fiber synaptic cleft

Zinc, a transition metal existing in very high concentrations in the hippocampal mossy fibers from CA3 area, is assumed to be co-released with glutamate and to have a neuromodulatory role at the corresponding synapses. The synaptic action of zinc is determined both by the spatiotemporal characteristics of the zinc release process and by the kinetics of zinc binding to sites located in the cleft area, as well as by their concentrations. This work addresses total, free and complexed zinc concentration changes, in an individual synaptic cleft, following single, short and long periods of evoked zinc release. The results estimate the magnitude and time course of the concentrations of zinc complexes, assuming that the dynamics of the release processes are similar to those of glutamate. It is also considered that, for the cleft zinc concentrations used in the model (≤ 1 μM), there is no postsynaptic zinc entry. For this reason, all released zinc ends up being reuptaken in a process that is several orders of magnitude slower than that of release and has thus a much smaller amplitude. The time derivative of the total zinc concentration in the cleft is represented by the difference between two alpha functions, corresponding to the released and uptaken components. These include specific parameters that were chosen assuming zinc and glutamate co-release, with similar time courses. The peak amplitudes of free zinc in the cleft were selected based on previously reported experimental cleft zinc concentration changes evoked by single and multiple stimulation protocols. The results suggest that following a low amount of zinc release, similar to that associated with one or a few stimuli, zinc clearance is mainly mediated by zinc binding to the high-affinity sites on the NMDA receptors and to the low-affinity sites on the highly abundant GLAST glutamate transporters. In the case of higher zinc release brought about by a larger group of stimuli, most zinc binding occurs essentially to the GLAST transporters, having the corresponding zinc complex a maximum concentration that is more than one order of magnitude larger than that for the high and low affinity NMDA sites. The other zinc complexes considered in the model, namely those formed with sites on the AMPA receptors, calcium and K_ATP channels and with ATP molecules, have much smaller contributions to the synaptic zinc clearance.     

The actions of synaptically released zinc at hippocampal mossy fiber synapses

Zn2+ is present at high concentrations in the synaptic vesicles of hippocampal mossy fibers. We have used Zn2+ chelators and the mocha mutant mouse to address the physiological role of Zn2+ in this pathway. Zn2+ is not involved in the unique presynaptic plasticities observed at mossy fiber synapses but is coreleased with glutamate from these synapses, both spontaneously and with electrical stimulation, where it exerts a strong modulatory effect on the NMDA receptors. Zn2+ tonically occupies the high-affinity binding site of NMDA receptors at mossy fiber synapses, whereas the lower affinity voltage-dependent Zn2+ binding site is occupied during action potential driven-release. We conclude that Zn2+ is a modulatory neurotransmitter released from mossy fiber synapses and plays an important role in shaping the NMDA receptor response at these synapses.     

Recruitment of release sites underlies chemical presynaptic potentiation at hippocampal mossy fiber boutons

Synaptic plasticity is a cellular model for learning and memory. However, the expression mechanisms underlying presynaptic forms of plasticity are not well understood. Here, we investigate functional and structural correlates of presynaptic potentiation at large hippocampal mossy fiber boutons induced by the adenylyl cyclase activator forskolin. We performed 2-photon imaging of the genetically encoded glutamate sensor iGlu_u that revealed an increase in the surface area used for glutamate release at potentiated terminals. Time-gated stimulated emission depletion microscopy revealed no change in the coupling distance between P/Q-type calcium channels and release sites mapped by Munc13-1 cluster position. Finally, by high-pressure freezing and transmission electron microscopy analysis, we found a fast remodeling of synaptic ultrastructure at potentiated boutons: Synaptic vesicles dispersed in the terminal and accumulated at the active zones, while active zone density and synaptic complexity increased. We suggest that these rapid and early structural rearrangements might enable long-term increase in synaptic strength.

This study uses several high-resolution imaging techniques to investigate the structural correlates of presynaptic potentiation at hippocampal mossy fiber boutons, observing an increase in release sites and in release synchronicity accompanied by synaptic vesicle dispersion in the terminal and accumulation at release sites, but no modulation of the distance between calcium channel and release sites.     

Enhancement of hippocampal mossy fiber activity in zinc deficiency and its influence on behavior

The extracellular concentration of glutamate in the hippocampus is increased by hippocampal perfusion with CaEDTA, a membrane-impermeable zinc chelator, suggesting that the activity of glutamatergic neurons in the hippocampus are influenced by the extracellular concentrations of zinc. In the present study, the relationship between the extracellular concentrations of zinc and mossy fiber activity in the hippocampus was examined in mice and rats fed a zinc-deficient diet for 4 weeks. Timm's stain, by which histochemically reactive zinc in the presynaptic vesicles is detected, was attenuated in the hippocampus in zinc deficiency. The extracellular signal of ZnAF-2, a membrane-impermeable zinc indicator, was also lower in the hippocampal CA3, suggesting that the basal extracellular concentrations of zinc are lower maintained in zinc deficiency. To check mossy fiber activity after 4-week zinc deprivation, the decrease in the signal of FM4-64, an indicator of presynaptic activity (exocytosis), at mossy fiber synapses was measured under the condition of spontaneous depolarization. The decrease was significantly facilitated by zinc deficiency, suggesting that the basal exocytosis at mossy fiber synapses is enhanced by zinc deficiency. On the other hand, the increase in anxiety-like behavior was observed in the open-field test after 4-week zinc deprivation. The present study demonstrates that the decrease in the basal extracellular concentrations of zinc may be linked to the enhancement of the basal mossy fiber activity in zinc deficiency. This decrease seems to be also involved in neuropsychological behavior in zinc deficiency.     

Kappa opioid agonists inhibit transmitter release from guinea pig hippocampal mossy fiber synaptosomes

Opioid agonists specific for the , , and opioid receptor subtypes were tested for their ability to modulate potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity from guinea pig hippocampal mossy fiber synaptosomes. The opioid agonists U-62,066E and (–) ethylketocyclazocine, but not the agonist [D-Ala²,N-MePhe⁴,Gly⁵-ol]-enkephalin (DAGO) nor the agonist [D-Pen^2,5]enkephalin (DPDE), inhibited the potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity. U-62,066E, but not DAGO or DPDE, also inhibited the potassium-evoked rise in mossy fiber synaptosomal cytosolic Ca²⁺ levels, indicating a possible mechanism for agonist inhibition of transmitter release. DAGO and DPDE were found to be without any effect on cytosolic Ca²⁺ levels or transmitter release in this preparation. The U-62,066E inhibition of the potassium-evoked rise in synaptosomal cytosolic Ca²⁺ levels was partially attenuated by the opioid antagonist quadazocine and insensitive to the -opioid specific antagonist ICI 174,864 and the opioid-preferring antagonists naloxone and naltrexone. Quadazocine also reversed U-62,066E inhibition of the potassium-evoked release of L-glutamate, but not dynorphin B-like immunoreactivity. These results suggest that opioid agonists inhibit transmitter release from mossy fiber terminals through both opioid and non- opioid receptor mediated mechanisms.     

The two sides of hippocampal mossy fiber plasticity

Two studies in this issue of Neuron (Kwon and Castillo and Rebola et al.) show that the mossy fiber-CA3 pyramidal neuron synapse, a hippocampal synapse well known for its presynaptic plasticity, exhibits a novel form of long-term potentiation of NMDAR-mediated currents, which is induced and expressed postsynaptically.     

Functional development of hippocampal mossy fiber synapses in rabbits

The course of functional maturation with age of mossy fiber synapses on pyramidal cells in areas CA_3,4 of the dorsal hippocampus was investigated by extracellular recording of focal potentials and single unit responses of the hippocampus to electrical stimulation of the dentate fascia in waking, unimmobilized rabbits aged from 1 to 14 days. After the 4th day of postnatal life focal potentials appeared in response to single stimulation, in the form of a biphasic short-latency wave, characteristic of responses of the mature hippocampus, accompanied by spike discharges with a latent period of 3 to 10 msec and inhibitory responses of the hippocampal neurons. During the next 10 days the amplitude of the focal potentials increased from several hundred millivolts, with the sharpest increase observed from the 4th through the 7th days. In early age periods global and unitary responses were shown to be capable of frequency potentiation and also of short-term after-potentiation.Brain Institute, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 12, No. 3, pp. 246–254, May–June, 1980.     

Patch-clamp recording from mossy fiber terminals in hippocampal slices

Rigorous analysis of synaptic transmission in the central nervous system requires access to presynaptic terminals. However, cortical terminals have been largely inaccessible to presynaptic patch-clamp recording, due to their small size. Using improved patch-clamp techniques in brain slices, we recorded from mossy fiber terminals in the CA3 region of the hippocampus, which have a diameter of 2-5 microm. The major steps of improvement were the enhanced visibility provided by high-numerical aperture objectives and infrared illumination, the development of vibratomes with minimal vertical blade vibrations and the use of sucrose-based solutions for storage and cutting. Based on these improvements, we describe a protocol that allows us to routinely record from hippocampal mossy fiber boutons. Presynaptic recordings can be obtained in slices from both rats and mice. Presynaptic recordings can be also obtained in slices from transgenic mice in which terminals are labeled with enhanced green fluorescent protein.     

Synaptic activation of presynaptic kainate receptors on hippocampal mossy fiber synapses

Kainate receptors (KARs) are a poorly understood family of ionotropic glutamate receptors. A role for these receptors in the presynaptic control of transmitter release has been proposed but remains controversial. Here, KAR agonists are shown to enhance fiber excitability, and a number of experiments show that this is a direct effect of KARs on the presynaptic fibers. In addition, KAR activation inhibits evoked transmitter release from mossy fiber synapses. Synaptic release of glutamate from either neighboring mossy fiber synapses or associational/commisural (A/C) synapses results in the activation of these presynaptic ionotropic KARs. These results, along with previous studies, indicate that KARs, through the endogenous release of glutamate, mediate excitatory postsynaptic potentials (EPSPs), alter presynaptic excitability, and modulate transmitter release.     

Long-term potentiation selectively expressed by NMDA receptors at hippocampal mossy fiber synapses

The mossy fiber to CA3 pyramidal cell synapse (mf-CA3) provides a major source of excitation to the hippocampus. Thus far, these glutamatergic synapses are well recognized for showing a presynaptic, NMDA receptor-independent form of LTP that is expressed as a long-lasting increase of transmitter release. Here, we show that in addition to this "classical" LTP, mf-CA3 synapses can undergo a form of LTP characterized by a selective enhancement of NMDA receptor-mediated transmission. This potentiation requires coactivation of NMDA and mGlu5 receptors and a postsynaptic calcium rise. Unlike classical LTP, expression of this mossy fiber LTP is due to a PKC-dependent recruitment of NMDA receptors specifically to the mf-CA3 synapse via a SNARE-dependent process. Having two mechanistically different forms of LTP may allow mf-CA3 synapses to respond with more flexibility to the changing demands of the hippocampal network.     

Attenuation of abnormal glutamate release in zinc deficiency by zinc and Yokukansan

The mechanism of the abnormal increase in extracellular glutamate concentration in the hippocampus induced with 100 mM KCl in zinc deficiency is unknown. In the present study, the changes in glutamate release (exocytosis) and GLT-1, a glial glutamate transporter, expression were studied in young rats fed a zinc-deficient diet for 4 weeks. Exocytosis at mossy fiber boutons was enhanced as reported previously and GLT-1 protein was increased in the hippocampus. The enhanced exocytosis is thought to increase extracellular glutamate concentration. However, the basal concentration of extracellular glutamate in the hippocampus was not increased by zinc deficiency, suggesting that GLT-1 protein increased serves to maintain the basal concentration of extracellular glutamate. The enhanced exocytosis was attenuated in the presence of 100 μM ZnCl₂, which attenuated the abnormal increase in extracellular glutamate induced with high K⁺ in zinc deficiency. The present study indicates that zinc attenuates abnormal glutamate release in zinc deficiency. The enhanced exocytosis was also attenuated in slices from zinc-deficient rats administered Yokukansan, a herbal medicine, in which the abnormal increase in extracellular glutamate induced with high K⁺ was attenuated. It is likely that Yokukansan is useful for prevention or cure of abnormal glutamate release. The enhanced exocytosis in zinc deficiency is a possible mechanism on abnormal increase in extracellular glutamate in the hippocampus induced with high K⁺.     

Attenuation of abnormal glutamate release in zinc deficiency by zinc and Yokukansan

The mechanism of the abnormal increase in extracellular glutamate concentration in the hippocampus induced with 100 mM KCl in zinc deficiency is unknown. In the present study, the changes in glutamate release (exocytosis) and GLT-1, a glial glutamate transporter, expression were studied in young rats fed a zinc-deficient diet for 4 weeks. Exocytosis at mossy fiber boutons was enhanced as reported previously and GLT-1 protein was increased in the hippocampus. The enhanced exocytosis is thought to increase extracellular glutamate concentration. However, the basal concentration of extracellular glutamate in the hippocampus was not increased by zinc deficiency, suggesting that GLT-1 protein increased serves to maintain the basal concentration of extracellular glutamate. The enhanced exocytosis was attenuated in the presence of 100 μM ZnCl₂, which attenuated the abnormal increase in extracellular glutamate induced with high K⁺ in zinc deficiency. The present study indicates that zinc attenuates abnormal glutamate release in zinc deficiency. The enhanced exocytosis was also attenuated in slices from zinc-deficient rats administered Yokukansan, a herbal medicine, in which the abnormal increase in extracellular glutamate induced with high K⁺ was attenuated. It is likely that Yokukansan is useful for prevention or cure of abnormal glutamate release. The enhanced exocytosis in zinc deficiency is a possible mechanism on abnormal increase in extracellular glutamate in the hippocampus induced with high K⁺.     

Long-term rearrangements of hippocampal mossy fiber terminal connectivity in the adult regulated by experience

We investigated rearrangements of connectivity between hippocampal mossy fibers and CA3 pyramidal neurons. We found that mossy fibers establish 10-15 local terminal arborization complexes (LMT-Cs) in CA3, which exhibit major differences in size and divergence in adult mice. LMT-Cs exhibited two types of long-term rearrangements in connectivity in the adult: progressive expansion of LMT-C subsets along individual dendrites throughout life, and pronounced increases in LMT-C complexities in response to an enriched environment. In organotypic slice cultures, subsets of LMT-Cs also rearranged extensively and grew over weeks and months, altering the strength of preexisting connectivity, and establishing or dismantling connections with pyramidal neurons. Differences in LMT-C plasticity reflected properties of individual LMT-Cs, not mossy fibers. LMT-C maintenance and growth were regulated by spiking activity, mGluR2-sensitive transmitter release from LMTs, and PKC. Thus, subsets of terminal arborization complexes by mossy fibers rearrange their local connectivities in response to experience and age throughout life.     

Nicotine-induced and depolarisation-induced glutamate release from hippocampus mossy fibre synaptosomes: two distinct mechanisms

Hippocampus mossy fibre terminals activate CA3 pyramidal neurons via two distinct mechanisms, both quantal and glutamatergic: (i) rapid excitatory transmission in response to afferent action potentials and (ii) delayed and prolonged release following nicotinic receptor activation. These processes were analysed here using rat hippocampus mossy fibres synaptosomes. The relationships between synaptosome depolarisation and glutamate release were established in response to high-KCl and gramicidin challenges. Half-maximal release corresponded to a 52 mV depolarisation step. KCl-induced release was accompanied by transient dissipation of the proton gradient across synaptic vesicle membrane. Nicotine elicited a substantial glutamate release from mossy fibre synaptosomes (EC₅₀ 3.14 μM; V _max 12.01 ± 2.1 nmol glutamate/mg protein; Hill's coefficient 0.99). However, nicotine-induced glutamate release was not accompanied by any change in the membrane potential or in the vesicular proton gradient. The effects of acetylcholine (200 μM) were similar to those of nicotine (25 μM). Nicotinic α7 receptors were evidenced by immuno-cytochemistry on the mossy fibre synaptosome plasma membrane. Therefore, the same terminals can release glutamate in response to two distinct stimuli: (i) rapid neurotransmission involving depolarisation-induced activation of voltage-gated Ca²⁺ channels and (ii) a slower nicotinic activation which does not involve depolarisation or dissipation of the vesicular proton gradient.     

Metabotropic glutamate receptors modulate GABA release from mouse hippocampal slices

The effects of metabotropic glutamate receptor agonists on the basal and potassium (50 mM K⁺)-stimulated release of [³H]GABA from mouse hippocampal slices were investigated using a superfusion system. The group I agonist (1±)-1-aminocyclopentane-trans-1,3-dicarboxylate enhanced the basal GABA release and reduced the K⁺-evoked release by a mechanism antagonized by (RS)-1-aminoindan-1,5-dicarboxylate in both cases. The group II agonist (2S,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine failed to have any effect on the basal release, but inhibited the stimulated release. This inhibition was not affected by the antagonist (2S)-2-ethylglutamate. The group III agonists L(+)-amino-4-phosphonobutyrate and O-phospho-L-serine inhibited the basal GABA release, which effects were blocked by the antagonist (RS)-2-cyclopropyl-4-phosphonophenylglycine. Moreover, the suppression of the K⁺-evoked release by L(+)2-amino-4-phosphonobutyrate was apparently receptor-mediated, being blocked by (RS)-2-cyclopropyl-4-phosphonophenylglycine. The results show that activation of metabotropic glutamate receptors of group I is able to potentiate the basal release of GABA, whereas activation of groups I and III receptors reduce K⁺-stimulated release in mouse hippocampal slices.     

Cytosolic acidification and intracellular zinc release in hippocampal neurons

In neurons exposed to glutamate, Ca2? influx triggers intracellular Zn2? release via an as yet unclear mechanism. As glutamate induces a Ca2?-dependent cytosolic acidification, the present work tested the relationships among intracellular Ca2? concentration ([Ca2?](i)), intracellular pH (pH(i) ), and [Zn2?](i). Cultured hippocampal neurons were exposed to glutamate and glycine (Glu/Gly), while [Zn2?](i), [Ca2?](i) and pH(i) were monitored using FluoZin-3, Fura2-FF, and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Glu/Gly applications decreased pH(i) to 6.1 and induced intracellular Zn2? release in a Ca2?-dependent manner, as expected. The pH(i) drop reduced the affinity of FluoZin-3 and Fura-2-FF for Zn2?. The rate of Glu/Gly-induced [Zn2?](i) increase was not correlated with the rate of [Ca2?](i) increase. Instead, the extent of [Zn2?](i) elevations corresponded well to the rate of pH(i) drop. Namely, [Zn2?](i) increased more in more highly acidified neurons. Inhibiting the mechanisms responsible for the Ca2?-dependent pH(i) drop (plasmalemmal Ca2? pump and mitochondria) counteracted the Glu/Gly-induced intracellular Zn2? release. Alkaline pH (8.5) suppressed Glu/Gly-induced intracellular Zn2? release whereas acidic pH (6.0) enhanced it. A pH(i) drop to 6.0 (without any Ca2? influx or glutamate receptor activation) led to intracellular Zn2? release; the released Zn2? (free Zn2? plus Zn2?) bound to Fura-2FF and FluoZin-3) reached 1 μM.