Molecular Mechanisms of Epilepsy: The Role of the Chloride Transporter KCC2.

Epilepsy is a neurological disease characterized by abnormal or synchronous brain activity causing seizures, which may produce convulsions, minor physical signs, or a combination of symptoms. These disorders affect approximately 65 million people worldwide, from all ages and genders. Seizures apart, epileptic patients present a high risk to develop neuropsychological comorbidities such as cognitive deficits, emotional disturbance, and psychiatric disorders, which severely impair quality of life. Currently, the treatment for epilepsy includes the administration of drugs or surgery, but about 30% of the patients treated with antiepileptic drugs develop time-dependent pharmacoresistence. Therefore, further investigation about epilepsy and its causes is needed to find new pharmacological targets and innovative therapeutic strategies. Pharmacoresistance is associated to changes in neuronal plasticity and alterations of GABAA receptor-mediated neurotransmission. The downregulation of GABA inhibitory activity may arise from a positive shift in GABAA receptor reversal potential, due to an alteration in chloride homeostasis. In this paper, we review the contribution of K+-Cl--cotransporter (KCC2) to the alterations in the Cl- gradient observed in epileptic condition, and how these alterations are coupled to the increase in the excitability.

GRASP1 ubiquitination regulates AMPA receptor surface expression and synaptic activity in cultured hippocampal neurons.

The synaptic expression of glutamate receptors of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) type is dynamically controlled by interaction with binding partners and auxiliary proteins. These proteins can be regulated by posttranslational modifications, including ubiquitination. In this work, we investigated the regulation of glutamate receptor interacting protein-associated protein 1 (GRASP1) by ubiquitin-dependent mechanisms and its impact on surface expression and activity of synaptic AMPA receptors. Cotransfection of GFP-ubiquitin decreased myc-GRASP1 protein levels in HEK293T cells, and this effect was inhibited upon transfection of an ubiquitin mutant that cannot be ubiquitinated on Lys48. In addition, transfection of cultured hippocampal neurons with GFP-ubiquitin reduced the dendritic levels of endogenous GRASP1 and decreased the surface expression of GluA1 AMPA receptor subunits, an effect that was partly reversed by cotransfection with GRASP1. Similarly, transfection of hippocampal neurons with GFP-ubiquitin decreased the amplitude of miniature excitatory postsynaptic currents (mEPSCs) mediated by Ca2+ -impermeable AMPA receptors, and this effect was abrogated by cotransfection of GRASP1. Together, the results show a role for ubiquitination in the regulation of the postsynaptic protein GRASP1, which has an impact on the surface distribution of AMPA receptors and on their activity at the synapse.

Transient incubation of cultured hippocampal neurons in the absence of magnesium induces rhythmic and synchronized epileptiform-like activity.

Cell culture models are important tools to study epileptogenesis mechanisms. The aim of this work was to characterize the spontaneous and synchronized rhythmic activity developed by cultured hippocampal neurons after transient incubation in zero Mg2+ to model Status Epilepticus. Cultured hippocampal neurons were transiently incubated with a Mg2+-free solution and the activity of neuronal networks was evaluated using single cell calcium imaging and whole-cell current clamp recordings. Here we report the development of synchronized and spontaneous [Ca2+]i transients in cultured hippocampal neurons immediately after transient incubation in a Mg2+-free solution. Spontaneous and synchronous [Ca2+]i oscillations were observed when the cells were then incubated in the presence of Mg2+. Functional studies also showed that transient incubation in Mg2+-free medium induces neuronal rhythmic burst activity that was prevented by antagonists of glutamate receptors. In conclusion, we report the development of epileptiform-like activity, characterized by spontaneous and synchronized discharges, in cultured hippocampal neurons transiently incubated in the absence of Mg2+. This model will allow studying synaptic alterations contributing to the hyperexcitability that underlies the development of seizures and will be useful in pharmacological studies for testing new drugs for the treatment of epilepsy.

Synaptogenesis Stimulates a Proteasome-Mediated Ribosome Reduction in Axons.

Ribosomes and a subset of cellular mRNAs are trafficked into axons of developing neurons. The axonal localization of translational machinery allows new proteins to be rapidly and locally synthesized during axonal growth and pathfinding. However, in mature neurons, axonal ribosomes are significantly reduced or even absent. The mechanism that elicits this removal is currently unknown. Here, we demonstrate that synapse formation is the trigger for ribosome reduction in mature axons. In vivo analysis shows that axonal ribosome levels decrease in rat brain at a developmental stage coincident with synapse formation. Next, we observe in vitro that different synaptogenic inducers trigger an overall decrease of ribosomal proteins and rRNA in the axons of spinal motor neurons. We further observe that this process is dependent on the ubiquitin-proteasome system but not on autophagy. Together, these data identify synaptogenesis as the long missing biological trigger that leads to ribosome disappearance during axonal maturation.

BDNF increases synaptic NMDA receptor abundance by enhancing the local translation of Pyk2 in cultured hippocampal neurons.

The effects of brain-derived neurotrophic factor (BDNF) in long-term synaptic potentiation (LTP) are thought to underlie learning and memory formation and are partly mediated by local protein synthesis. Here, we investigated the mechanisms that mediate BDNF-induced alterations in the synaptic proteome that are coupled to synaptic strengthening. BDNF induced the synaptic accumulation of GluN2B-containing NMDA receptors (NMDARs) and increased the amplitude of NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) in cultured rat hippocampal neurons by a mechanism requiring activation of the protein tyrosine kinase Pyk2 and dependent on cellular protein synthesis. Single-particle tracking using quantum dot imaging revealed that the increase in the abundance of synaptic NMDAR currents correlated with their enhanced stability in the synaptic compartment. Furthermore, BDNF increased the local synthesis of Pyk2 at the synapse, and the observed increase in Pyk2 protein abundance along dendrites of cultured hippocampal neurons was mediated by a mechanism dependent on the ribonucleoprotein hnRNP K, which bound to Pyk2 mRNA and dissociated from it upon BDNF application. Knocking down hnRNP K reduced the BDNF-induced synaptic synthesis of Pyk2 protein, whereas its overexpression enhanced it. Together, these findings indicate that hnRNP K mediates the synaptic distribution of Pyk2 synthesis, and hence the synaptic incorporation of GluN2B-containing NMDARs, induced by BDNF, which may affect LTP and synaptic plasticity.

Alterations in GABAA-Receptor Trafficking and Synaptic Dysfunction in Brain Disorders.

GABAA receptors (GABAAR) are the major players in fast inhibitory neurotransmission in the central nervous system (CNS). Regulation of GABAAR trafficking and the control of their surface expression play important roles in the modulation of the strength of synaptic inhibition. Different pieces of evidence show that alterations in the surface distribution of GABAAR and dysregulation of their turnover impair the activity of inhibitory synapses. A diminished efficacy of inhibitory neurotransmission affects the excitatory/inhibitory balance and is a common feature of various disorders of the CNS characterized by an increased excitability of neuronal networks. The synaptic pool of GABAAR is mainly controlled through regulation of internalization, recycling and lateral diffusion of the receptors. Under physiological condition these mechanisms are finely coordinated to define the strength of GABAergic synapses. In this review article, we focus on the alteration in GABAAR trafficking with an impact on the function of inhibitory synapses in various disorders of the CNS. In particular we discuss how similar molecular mechanisms affecting the synaptic distribution of GABAAR and consequently the excitatory/inhibitory balance may be associated with a wide diversity of pathologies of the CNS, from psychiatric disorders to acute alterations leading to neuronal death. A better understanding of the cellular and molecular mechanisms that contribute to the impairment of GABAergic neurotransmission in these disorders, in particular the alterations in GABAAR trafficking and surface distribution, may lead to the identification of new pharmacological targets and to the development of novel therapeutic strategies.

The RNA-Binding Protein hnRNP K Mediates the Effect of BDNF on Dendritic mRNA Metabolism and Regulates Synaptic NMDA Receptors in Hippocampal Neurons.

Brain-derived neurotrophic factor (BDNF) is an important mediator of long-term synaptic potentiation (LTP) in the hippocampus. The local effects of BDNF depend on the activation of translation activity, which requires the delivery of transcripts to the synapse. In this work, we found that neuronal activity regulates the dendritic localization of the RNA-binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K) in cultured rat hippocampal neurons by stimulating BDNF-Trk signaling. Microarray experiments identified a large number of transcripts that are coimmunoprecipitated with hnRNP K, and about 60% of these transcripts are dissociated from the protein upon stimulation of rat hippocampal neurons with BDNF. In vivo studies also showed a role for TrkB signaling in the dissociation of transcripts from hnRNP K upon high-frequency stimulation (HFS) of medial perforant path-granule cell synapses of male rat dentate gyrus (DG). Furthermore, treatment of rat hippocampal synaptoneurosomes with BDNF decreased the coimmunoprecipitation of hnRNP K with mRNAs coding for glutamate receptor subunits, Ca2+- and calmodulin-dependent protein kinase IIß (CaMKIIß) and BDNF. Downregulation of hnRNP K impaired the BDNF-induced enhancement of NMDA receptor (NMDAR)-mediated mEPSC, and similar results were obtained upon inhibition of protein synthesis with cycloheximide. The results demonstrate that BDNF regulates specific populations of hnRNP-associated mRNAs in neuronal dendrites and suggests an important role of hnRNP K in BDNF-dependent forms of synaptic plasticity.

Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons.

This protocol aims at standardizing the procedure to obtain primary cultures of hippocampal and cerebrocortical neurons for in vitro experiments. Cultures should be prepared from cells isolated during embryonic development when neuronal precursor cells are not yet fully differentiated. This helps increasing the quality and quantity of cells, while offering minimal cell death that often occurs during dissociation of differentiated neurons. Cells plated under the appropriate conditions, either in Petri-dishes or in multi-well plates, will develop and establish synaptic contacts over time since the neuronal culture medium provides the nutrients and trophic factors required for differentiation. In this protocol we describe the methodology for the preparation of both cortical and hippocampal neuronal cultures.

Downregulation of GABAA Receptor Recycling Mediated by HAP1 Contributes to Neuronal Death in In Vitro Brain Ischemia.

Downregulation of GABAergic synaptic transmission contributes to the increase in overall excitatory activity in the ischemic brain. A reduction of GABAA receptor (GABAAR) surface expression partly accounts for this decrease in inhibitory activity, but the mechanisms involved are not fully elucidated. In this work, we investigated the alterations in GABAAR trafficking in cultured rat hippocampal neurons subjected to oxygen/glucose deprivation (OGD), an in vitro model of global brain ischemia, and their impact in neuronal death. The traffic of GABAAR was evaluated after transfection of hippocampal neurons with myc-tagged GABAAR ß3 subunits. OGD decreased the rate of GABAAR ß3 subunit recycling and reduced the interaction of the receptors with HAP1, a protein involved in the recycling of the receptors. Furthermore, OGD induced a calpain-mediated cleavage of HAP1. Transfection of hippocampal neurons with HAP1A or HAP1B isoforms reduced the OGD-induced decrease in surface expression of GABAAR ß3 subunits, and HAP1A maintained the rate of receptor recycling. Furthermore, transfection of hippocampal neurons with HAP1 significantly decreased OGD-induced cell death. These results show a key role for HAP1 protein in the downmodulation of GABAergic neurotransmission during cerebral ischemia, which contributes to neuronal demise.

Role of GABAA R trafficking in the plasticity of inhibitory synapses.

Neuronal excitability depends on the balance between inhibitory and excitatory neurotransmission, which in the CNS are mainly mediated by GABA and glutamate respectively. The plasticity of glutamatergic synapses and the underlying molecular mechanisms have been characterized to a large extent. In comparison, much less is known regarding the plasticity of GABAergic synapses, which is also important in the maintenance of the excitatory/inhibitory balance. GABAergic synapses, similarly to the glutamatergic synapses, adjust their strength depending on the pattern of neuronal activity. These alterations take place in the pre- and postsynaptic compartments, and short- and long-term alterations have been described. At the postsynaptic level the plasticity of inhibitory synapses is largely mediated by modulation of the expression, localization and function of GABAA receptors, by mechanisms involving the participation of scaffold proteins and structural molecules. This review is focused on the key mechanisms that regulate GABAA receptor trafficking in response to alterations in neuronal activity or to stimulation of plasma membrane receptors. These alterations in GABAergic neurotransmission are important in the refinement of the pattern of activity of neuronal networks. In this work, we review some of the mechanisms contributing to the plasticity of inhibitory synapses in the CNS, focusing on the regulation of GABAA receptor (GABAA R) trafficking in response to alterations in neuronal activity or to stimulation of different classes of plasma membrane-associated receptors. Alterations in these mechanisms are important in the refinement of neuronal network activity. This article is part of a mini review series: "Synaptic Function and Dysfunction in Brain Diseases".

Calpains and neuronal damage in the ischemic brain: The swiss knife in synaptic injury.

The excessive extracellular accumulation of glutamate in the ischemic brain leads to an overactivation of glutamate receptors with consequent excitotoxic neuronal death. Neuronal demise is largely due to a sustained activation of NMDA receptors for glutamate, with a consequent increase in the intracellular Ca(2+) concentration and activation of calcium- dependent mechanisms. Calpains are a group of Ca(2+)-dependent proteases that truncate specific proteins, and some of the cleavage products remain in the cell, although with a distinct function. Numerous studies have shown pre- and post-synaptic effects of calpains on glutamatergic and GABAergic synapses, targeting membrane- associated proteins as well as intracellular proteins. The resulting changes in the presynaptic proteome alter neurotransmitter release, while the cleavage of postsynaptic proteins affects directly or indirectly the activity of neurotransmitter receptors and downstream mechanisms. These alterations also disturb the balance between excitatory and inhibitory neurotransmission in the brain, with an impact in neuronal demise. In this review we discuss the evidence pointing to a role for calpains in the dysregulation of excitatory and inhibitory synapses in brain ischemia, at the pre- and post-synaptic levels, as well as the functional consequences. Although targeting calpain-dependent mechanisms may constitute a good therapeutic approach for stroke, specific strategies should be developed to avoid non-specific effects given the important regulatory role played by these proteases under normal physiological conditions.

Multiple domains in the C-terminus of NMDA receptor GluN2B subunit contribute to neuronal death following in vitro ischemia.

Global cerebral ischemia induces selective degeneration of specific subsets of neurons throughout the brain, particularly in the hippocampus and cortex. One of the major hallmarks of cerebral ischemia is excitotoxicity, characterized by overactivation of glutamate receptors leading to intracellular Ca(2+) overload and ultimately neuronal demise. N-methyl-d-aspartate receptors (NMDARs) are considered to be largely responsible for excitotoxic injury due to their high Ca(2+) permeability. In the hippocampus and cortex, these receptors are most prominently composed of combinations of two GluN1 subunits and two GluN2A and/or GluN2B subunits. Due to the controversy regarding the differential role of GluN2A and GluN2B subunits in excitotoxic cell death, we investigated the role of GluN2B in the activation of pro-death signaling following an in vitro model of global ischemia, oxygen and glucose deprivation (OGD). For this purpose, we used GluN2B(-/-) mouse cortical cultures and observed that OGD-induced damage was reduced in these neurons, and partially prevented in wild-type rat neurons by a selective GluN2B antagonist. Notably, we found a crucial role of the C-terminal domain of the GluN2B subunit in triggering excitotoxic signaling. Indeed, expression of YFP-GluN2B C-terminus mutants for the binding sites to post-synaptic density protein 95 (PSD95), Ca(2+)-calmodulin kinase IIα (CaMKIIα) or clathrin adaptor protein 2 (AP2) failed to mediate neuronal death in OGD conditions. We focused on the GluN2B-CaMKIIα interaction and found a determinant role of this interaction in OGD-induced death. Inhibition or knock-down of CaMKIIα exerted a neuroprotective effect against OGD-induced death, whereas overexpression of this kinase had a detrimental effect. Importantly, in comparison with neurons overexpressing wild-type CaMKIIα, neurons overexpressing a mutant form of the kinase (CaMKII-I205K), unable to interact with GluN2B, were partially protected against OGD-induced damage. Taken together, our results identify crucial determinants in the C-terminal domain of GluN2B subunits in promoting neuronal death in ischemic conditions. These mechanisms underlie the divergent roles of the GluN2A- and GluN2B-NMDARs in determining neuronal fate in cerebral ischemia.

Gephyrin Cleavage in In Vitro Brain Ischemia Decreases GABAA Receptor Clustering and Contributes to Neuronal Death.

GABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the central nervous system, and changes in GABAergic neurotransmission modulate the activity of neuronal networks. Gephyrin is a scaffold protein responsible for the traffic and synaptic anchoring of GABAA receptors (GABAAR); therefore, changes in gephyrin expression and oligomerization may affect the activity of GABAergic synapses. In this work, we investigated the changes in gephyrin protein levels during brain ischemia and in excitotoxic conditions, which may affect synaptic clustering of GABAAR. We found that gephyrin is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with glutamate, as well as after intrahippocampal injection of kainate, giving rise to a stable cleavage product. Gephyrin cleavage was also observed in cultured hippocampal neurons subjected to transient oxygen-glucose deprivation (OGD), an in vitro model of brain ischemia, and after transient middle cerebral artery occlusion (MCAO) in mice, a model of focal brain ischemia. Furthermore, a truncated form of gephyrin decreased the synaptic clustering of the protein, reduced the synaptic pool of GABAAR containing γ2 subunits and upregulated OGD-induced cell death in hippocampal cultures. Our results show that excitotoxicity and brain ischemia downregulate full-length gephyrin with a concomitant generation of truncated products, which affect synaptic clustering of GABAAR and cell death.

Differential role of the proteasome in the early and late phases of BDNF-induced facilitation of LTP.

The neurotrophin brain-derived neurotrophic factor (BDNF) mediates activity-dependent long-term changes of synaptic strength in the CNS. The effects of BDNF are partly mediated by stimulation of local translation, with consequent alterations in the synaptic proteome. The ubiquitin-proteasome system (UPS) also plays an important role in protein homeostasis at the synapse by regulating synaptic activity. However, whether BDNF acts on the UPS to mediate the effects on long-term synaptic potentiation (LTP) has not been investigated. In the present study, we show similar and nonadditive effects of BDNF and proteasome inhibition on the early phase of synaptic potentiation (E-LTP) induced by theta-burst stimulation of rat hippocampal CA1 synapses. The effects of BDNF were blocked by the proteasome activator IU1, suggesting that the neurotrophin acts by decreasing proteasome activity. Accordingly, BDNF downregulated the proteasome activity in cultured hippocampal neurons and in hippocampal synaptoneurosomes. Furthermore, BDNF increased the activity of the deubiquitinating enzyme UchL1 in synaptoneurosomes and upregulated free ubiquitin. In contrast to the effects on posttetanic potentiation, proteasome activity was required for BDNF-mediated LTP. These results show a novel role for BDNF in UPS regulation at the synapse, which is likely to act together with the increased translation activity in the regulation of the synaptic proteome during E-LTP.

GABA(A) receptor dephosphorylation followed by internalization is coupled to neuronal death in in vitro ischemia.

Cerebral ischemia is characterized by an early disruption of GABAergic neurotransmission contributing to an imbalance of the excitatory/inhibitory equilibrium and neuronal death, but the molecular mechanisms involved are not fully understood. Here we report a downregulation of GABA(A) receptor (GABA(A)R) expression, affecting both mRNA and protein levels of GABA(A)R subunits, in hippocampal neurons subjected to oxygen-glucose deprivation (OGD), an in vitro model of ischemia. Similar alterations in the abundance of GABA(A)R subunits were observed in in vivo brain ischemia. OGD reduced the interaction of surface GABA(A)R with the scaffold protein gephyrin, followed by clathrin-dependent receptor internalization. Internalization of GABA(A)R was dependent on glutamate receptor activation and mediated by dephosphorylation of the ß3 subunit at serine 408/409. Expression of phospho-mimetic mutant GABA(A)R ß3 subunits prevented receptor internalization and protected hippocampal neurons from ischemic cell death. The results show a key role for ß3 GABA(A)R subunit dephosphorylation in the downregulation of GABAergic synaptic transmission in brain ischemia, contributing to neuronal death. GABA(A)R phosphorylation might be a therapeutic target to preserve synaptic inhibition in brain ischemia.

BDNF regulates the expression and distribution of vesicular glutamate transporters in cultured hippocampal neurons.

BDNF is a pro-survival protein involved in neuronal development and synaptic plasticity. BDNF strengthens excitatory synapses and contributes to LTP, presynaptically, through enhancement of glutamate release, and postsynaptically, via phosphorylation of neurotransmitter receptors, modulation of receptor traffic and activation of the translation machinery. We examined whether BDNF upregulated vesicular glutamate receptor (VGLUT) 1 and 2 expression, which would partly account for the increased glutamate release in LTP. Cultured rat hippocampal neurons were incubated with 100 ng/ml BDNF, for different periods of time, and VGLUT gene and protein expression were assessed by real-time PCR and immunoblotting, respectively. At DIV7, exogenous application of BDNF rapidly increased VGLUT2 mRNA and protein levels, in a dose-dependent manner. VGLUT1 expression also increased but only transiently. However, at DIV14, BDNF stably increased VGLUT1 expression, whilst VGLUT2 levels remained low. Transcription inhibition with actinomycin-D or α-amanitine, and translation inhibition with emetine or anisomycin, fully blocked BDNF-induced VGLUT upregulation. Fluorescence microscopy imaging showed that BDNF stimulation upregulates the number, integrated density and intensity of VGLUT1 and VGLUT2 puncta in neurites of cultured hippocampal neurons (DIV7), indicating that the neurotrophin also affects the subcellular distribution of the transporter in developing neurons. Increased VGLUT1 somatic signals were also found 3 h after stimulation with BDNF, further suggesting an increased de novo transcription and translation. BDNF regulation of VGLUT expression was specifically mediated by BDNF, as no effect was found upon application of IGF-1 or bFGF, which activate other receptor tyrosine kinases. Moreover, inhibition of TrkB receptors with K252a and PLCγ signaling with U-73122 precluded BDNF-induced VGLUT upregulation. Hippocampal neurons express both isoforms during embryonic and neonatal development in contrast to adult tissue expressing only VGLUT1. These results suggest that BDNF regulates VGLUT expression during development and its effect on VGLUT1 may contribute to enhance glutamate release in LTP.

Excitotoxic stimulation downregulates the ubiquitin-proteasome system through activation of NMDA receptors in cultured hippocampal neurons.

Overactivation of glutamate receptors contributes to neuronal damage (excitotoxicity) in ischemic stroke but the detailed mechanisms are not fully elucidated. Brain ischemia is also characterized by an impairment of the activity of the proteasome, one of the major proteolytic systems in neurons. We found that excitotoxic stimulation with glutamate rapidly decreases ATP levels and the proteasome activity, and induces the disassembly of the 26S proteasome in cultured rat hippocampal neurons. Downregulation of the proteasome activity, leading to an accumulation of ubiquitinated proteins, was mediated by calcium entry through NMDA receptors and was only observed in the nuclear fraction. Furthermore, excitotoxicity-induced proteasome inhibition was partially sensitive to cathepsin-L inhibition and was specifically induced by activation of extrasynaptic NMDA receptors. Oxygen and glucose deprivation induced neuronal death and downregulated the activity of the proteasome by a mechanism dependent on the activation of NMDA receptors. Since deubiquitinating enzymes may regulate proteins half-life by counteracting ubiquitination, we also analyzed how their activity is regulated under excitotoxic conditions. Glutamate stimulation decreased the total deubiquitinase activity in hippocampal neurons, but was without effect on the activity of Uch-L1, showing that not all deubiquitinases are affected. These results indicate that excitotoxic stimulation with glutamate has multiple effects on the ubiquitin-proteasome system which may contribute to the demise process in brain ischemia and in other neurological disorders.

Cleavage of the vesicular glutamate transporters under excitotoxic conditions.

Glutamate is loaded into synaptic vesicles by vesicular glutamate transporters (VGLUTs), and alterations in the transporters expression directly regulate neurotransmitter release. We investigated changes in VGLUT1 and VGLUT2 protein levels after ischemic and excitotoxic insults. The results show that VGLUT2 is cleaved by calpains after excitotoxic stimulation of hippocampal neurons with glutamate, whereas VGLUT1 is downregulated to a lower extent. VGLUT2 was also cleaved by calpains after oxygen/glucose deprivation (OGD), and downregulated after middle cerebral artery occlusion (MCAO) and intrahippocampal injection of kainate. In contrast, VGLUT1 was not affected after OGD. Incubation of isolated synaptic vesicles with recombinant calpain also induced VGLUT2 cleavage, with a little effect observed for VGLUT1. N-terminal sequencing analysis showed that calpain cleaves VGLUT2 in the C-terminus, at Asn(534) and Lys(542). The truncated GFP-VGLUT2 forms were found to a great extent in non-synaptic regions along neurites, when compared to GFP-VGLUT2. These findings show that excitotoxic and ischemic insults downregulate VGLUT2, which is likely to affect glutamatergic transmission and cell death, especially in the neonatal period when the transporter is expressed at higher levels.