Paulo S. Pinheiro

Presynaptic glutamate receptors: physiological functions and mechanisms of action

Glutamate acts on postsynaptic glutamate receptors to mediate excitatory communication between neurons. The discovery that additional presynaptic glutamate receptors can modulate neurotransmitter release has added complexity to the way we view glutamatergic synaptic transmission. Here we review evidence of a physiological role for presynaptic glutamate receptors in neurotransmitter release. We compare the physiological roles of ionotropic and metabotropic glutamate receptors in short- and long-term regulation of synaptic transmission. Furthermore, we discuss the physiological conditions that are necessary for their activation, the source of the glutamate that activates them, their mechanisms of action and their involvement in higher brain function.

GluR7 is an essential subunit of presynaptic kainate autoreceptors at hippocampal mossy fiber synapses

Presynaptic ionotropic glutamate receptors are emerging as key players in the regulation of synaptic transmission. Here we identify GluR7, a kainate receptor (KAR) subunit with no known function in the brain, as an essential subunit of presynaptic autoreceptors that facilitate hippocampal mossy fiber synaptic transmission. GluR7−/− mice display markedly reduced short- and long-term synaptic potentiation. Our data suggest that presynaptic KARs are GluR6/GluR7 heteromers that coassemble and are localized within synapses. We show that recombinant GluR6/GluR7 KARs exhibit low sensitivity to glutamate, and we provide evidence that presynaptic KARs at mossy fiber synapses are likely activated by high concentrations of glutamate. Overall, from our data, we propose a model whereby presynaptic KARs are localized in the presynaptic active zone close to release sites, display low affinity for glutamate, are likely Ca2+-permeable, are activated by single release events, and operate within a short time window to facilitate the subsequent release of glutamate.

Zinc Dynamics and Action at Excitatory Synapses

Decades after the discovery that ionic zinc is present at high levels in glutamatergic synaptic vesicles, where, when, and how much zinc is released during synaptic activity remains highly controversial. Here we provide a quantitative assessment of zinc dynamics in the synaptic cleft and clarify its role in the regulation of excitatory neurotransmission by combining synaptic recordings from mice deficient for zinc signaling with Monte Carlo simulations. Ambient extracellular zinc levels are too low for tonic occupation of the GluN2A-specific nanomolar zinc sites on NMDA receptors (NMDARs). However, following short trains of physiologically relevant synaptic stimuli, zinc transiently rises in the cleft and selectively inhibits postsynaptic GluN2A-NMDARs, causing changes in synaptic integration and plasticity. Our work establishes the rules of zinc action and reveals that zinc modulation extends beyond hippocampal mossy fibers to excitatory SC-CA1 synapses. By specifically moderating GluN2A-NMDAR signaling, zinc acts as a widespread activity-dependent regulator of neuronal circuits.

Activation of neuropeptide Y receptors is neuroprotective against excitotoxicity in organotypic hippocampal slice cultures

Glutamate and NPY have been implicated in hippocampal neuropathology in temporal lobe epilepsy. Thus, we investigated the involvement of NPY receptors in mediating neuroprotection against excitotoxic insults in organotypic cultures of rat hippocampal slices. Exposure of hippocampal slice cultures to 2 μM AMPA (α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionate) induced neuronal degeneration, monitored by propidium iodide uptake, of granule cells and CA1 pyramidal cells. For dentate granule cells, selective activation of Y1, Y2, or Y5 receptors with 1 μM [Leu31,Pro34]NPY, 300 nM NPY13–36 or 1 μM 500 nM NPY(19–23)‐(Gly1,Ser3,Gln4,Thr6,Ala31,Aib32,Gln34)‐PP, respectively, had a neuroprotective effect against AMPA, whereas only the activation of Y2 receptors was effective for CA1 pyramidal cells. When the slice cultures were exposed to 6 μM kainate, the CA3 pyramidal cells displayed significant degeneration, and in this case the activation of Y1, Y2, and Y5 receptors was neuroprotective. For the kainic acid‐induced degeneration of CA1 pyramidal cells, it was again found that only the Y2 receptor activation was effective. Based on the present findings, it was concluded that Y1, Y2, and Y5 receptors effectively can modify glutamate receptor‐mediated neurodegeneration in the hippocampus.

Antagonism of recombinant and native GluK3-containing kainate receptors

A number of kainate receptor antagonists have shown selectivity for receptors containing the GluK1 subunit. Here, we analyze the effects of these GluK1 antagonists on currents mediated by recombinant homomeric GluK3 and heteromeric GluK2/3 receptors expressed in HEK 293 cells and activated by fast application of glutamate. We show that, amongst these compounds, UBP302, UBP310 and UBP316 effectively block recombinant homomeric GluK3 receptors. However, these antagonists are ineffective in blocking homomeric GluK2 or heteromeric GluK2/3 receptors. In addition, these antagonists do not affect presynaptic kainate receptors at mouse hippocampal mossy fibre synapses, which are thought to be composed of GluK2 and GluK3 subunits. Moreover, the AMPA receptor-selective non-competitive antagonist GYKI 53655 blocks, at high concentrations, GluK3-containing receptors and decreases short-term plasticity at mossy fibre synapses. These results expand the range of targets of kainate receptor antagonists and provide pharmacological tools to study the elusive mechanisms of neurotransmitter control by presynaptic kainate receptors.

Synaptotagmin Interaction with SNAP-25 Governs Vesicle Docking, Priming, and Fusion Triggering

SNARE complex assembly constitutes a key step in exocytosis that is rendered Ca2+-dependent by interactions with synaptotagmin-1. Two putative sites for synaptotagmin binding have recently been identified in SNAP-25 using biochemical methods: one located around the center and another at the C-terminal end of the SNARE bundle. However, it is still unclear whether and how synaptotagmin-1 × SNARE interactions at these sites are involved in regulating fast neurotransmitter release. Here, we have used electrophysiological techniques with high time-resolution to directly investigate the mechanistic ramifications of proposed SNAP-25 × synaptotagmin-1 interaction in mouse chromaffin cells. We demonstrate that the postulated central binding domain surrounding layer zero covers both SNARE motifs of SNAP-25 and is essential for vesicle docking, priming, and fast fusion-triggering. Mutation of this site caused no further functional alterations in synaptotagmin-1-deficient cells, indicating that the central acidic patch indeed constitutes a mechanistically relevant synaptotagmin-1 interaction site. Moreover, our data show that the C-terminal binding interface only plays a subsidiary role in triggering but is required for the full size of the readily releasable pool. Intriguingly, we also found that mutation of synaptotagmin-1 interaction sites led to more pronounced phenotypes in the context of the adult neuronal isoform SNAP-25B than in the embryonic isoform SNAP-25A. Further experiments demonstrated that stronger synaptotagmin-1 × SNAP-25B interactions allow for the larger primed vesicle pool supported by SNAP-25 isoform B. Thus, synaptotagmin-1 × SNARE interactions are not only required for multiple mechanistic steps en route to fusion but also underlie the developmental control of the releasable vesicle pool.

Therapeutic Potential of Metabotropic Glutamate Receptor Modulators

Glutamate is the main excitatory neurotransmitter in the central nervous system (CNS) and is a major player in complex brain functions. Glutamatergic transmission is primarily mediated by ionotropic glutamate receptors, which include NMDA, AMPA and kainate receptors. However, glutamate exerts modulatory actions through a family of metabotropic G-protein-coupled glutamate receptors (mGluRs). Dysfunctions of glutamatergic neurotransmission have been implicated in the etiology of several diseases. Therefore, pharmacological modulation of ionotropic glutamate receptors has been widely investigated as a potential therapeutic strategy for the treatment of several disorders associated with glutamatergic dysfunction. However, blockade of ionotropic glutamate receptors might be accompanied by severe side effects due to their vital role in many important physiological functions. A different strategy aimed at pharmacologically interfering with mGluR function has recently gained interest. Many subtype selective agonists and antagonists have been identified and widely used in preclinical studies as an attempt to elucidate the role of specific mGluRs subtypes in glutamatergic transmission. These studies have allowed linkage between specific subtypes and various physiological functions and more importantly to pathological states. This article reviews the currently available knowledge regarding the therapeutic potential of targeting mGluRs in the treatment of several CNS disorders, including schizophrenia, addiction, major depressive disorder and anxiety, Fragile X Syndrome, Parkinson’s disease, Alzheimer’s disease and pain.

Selective Block of Postsynaptic Kainate Receptors Reveals Their Function at Hippocampal Mossy Fiber Synapses

Progress in understanding the roles of kainate receptors (KARs) in synaptic integration, synaptic networks, and higher brain function has been hampered by the lack of selective pharmacological tools. We have found that UBP310 and related willardiine derivatives, previously characterized as selective GluK1 and GluK3 KAR antagonists, block postsynaptic KARs at hippocampal mossy fiber (MF) CA3 synapses while sparing AMPA and NMDA receptors. We further show that UBP310 is an antagonist of recombinant GluK2/GluK5 receptors, the major population of KARs in the brain. Postsynaptic KAR receptor blockade at MF synapses significantly reduces the sustained depolarization, which builds up during repetitive activity, and impacts on spike transmission mediated by heterosynaptic signals. In addition, KARs present in aberrant MF synapses in the epileptic hippocampus were also blocked by UBP310. Our results support a specific role for postsynaptic KARs in synaptic integration of CA3 pyramidal cells and describe a tool that will be instrumental in understanding the physiopathological role of KARs in the brain.

Calpain activation is involved in early caspase-independent neurodegeneration in the hippocampus following status epilepticus

Evidence for increased calpain activity has been described in the hippocampus of rodent models of temporal lobe epilepsy. However, it is not known whether calpains are involved in the cell death that accompanies seizures. In this work, we characterized calpain activation by examining the proteolysis of calpain substrates and in parallel we followed cell death in the hippocampus of epileptic rats. Male Wistar rats were injected with kainic acid (10 mg/kg) intraperitoneally and killed 24 h later, after development of grade 5 seizures. We observed a strong Fluoro‐Jade labeling in the CA1 and CA3 areas of the hippocampus in the rats that received kainic acid, when compared with saline‐treated rats. Immunohistochemistry and western blot analysis for the calpain‐derived breakdown products of spectrin showed evidence of increased calpain activity in the same regions of the hippocampus where cell death is observed. No evidence was found for caspase activation, in the same conditions. Treatment with the calpain inhibitor MDL 28170 significantly prevented the neurodegeneration observed in CA1. Taken together, our data suggest that early calpain activation, but not caspase activation, is involved in neurotoxicity in the hippocampus after status epilepticus.

Proteolysis of NR2B by calpain in the hippocampus of epileptic rats.

Overactivation of N-methyl-D-aspartate receptors is known to mediate excitotoxicity due to excessive entry of calcium, leading to the activation of several calcium-dependent enzymes. Calpains are calcium-activated proteases that appear to play a role in excitotoxic neuronal death. Several cellular proteins are substrates for these proteases, particularly the N-methyl-D-aspartate receptor. Recently, cleavage of NR2B subunits has been implicated in excitotoxic neurodegeneration in ischemia. In this work, we investigated the proteolysis by calpains of NR2B subunits of the N-methyl-D-aspartate receptor in the hippocampus of epileptic rats. Our results show that cleaved forms of NR2B subunits are formed after status epilepticus, in the same areas of the hippocampus where calpain activation was detected by immunohistochemical staining of calpain-specific spectrin breakdown products.