Ischemia deteriorates the spike encoding of rat cerebellar Purkinje cells by raising intracellular Ca2+

Ischemia-induced excitotoxicity at cerebellar Purkinje cells is presumably due to a persistent glutamate action. To the fact that they are more vulnerable to ischemia than other glutamate-innervated neurons, we studied whether additional mechanisms are present and whether cytoplasm Ca²⁺ plays a key role in their ischemic excitotoxicity. Ischemic changes in the excitability of Purkinje cells were measured by whole-cell recording in cerebellar slices of rats with less glutamate action. The role of cytoplasm Ca²⁺ was examined by two-photon cellular imaging and BAPTA infusion in Purkinje cells. Lowering perfusion rate to cerebellar slices deteriorated spike timing and raised spike capacity of Purkinje cells. These changes were associated with the reduction of spike refractory periods and threshold potentials, as well as the loss of their control to spike encoding. Ischemia-induced functional deterioration at Purkinje neurons was accompanied by cytoplasm Ca²⁺ rise and prevented by BAPTA infusion. Therefore, the ischemia destabilizes the spike encoding of Purkinje cells via raising cytoplasm Ca²⁺ without a need for glutamate, which subsequently causes their excitotoxic death.     

Ca and acidosis synergistically lead to the dysfunction of cortical GABAergic neurons during ischemia

Cell death in cerebral ischemia is presumably initiated by neural excitotoxicity resulted from the dysfunction of inhibitory neurons in early stage. Molecular processes underlying the ischemic injury of inhibitory neurons remain to be elusive, which we investigated by biochemical manipulations with cellular imaging and patch clamp at GFP-labeled GABAergic cells in cortical slices. Ischemia induces Ca²⁺ elevation, acidosis and dysfunction in GABAergic cells. An elevation of cytoplasmic Ca²⁺ or H⁺ impairs the encoding of action potentials in these neurons. The effects of Ca²⁺ and H⁺ are additive in nature and occlude ischemic outcomes. Ischemia impairs spike production through prolonging spike refractory periods and raising threshold potentials. Therefore, calcium toxicity and acidosis during ischemia synergistically impair the dynamics of sodium channels and function of cortical GABAergic neurons, which lead to neural excitotoxicity. Our results also suggest that the cocktail therapeutics is needed to prevent neuronal death from ischemia.     

A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca²⁺-imaging using the superior green Ca²⁺-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type.     

Endothelial Piezo1: Life depends on it

Ryanodine receptors (RyRs), located in the sarcoplasmic/endoplasmic reticulum (SR/ER) membrane, are required for intracellular Ca²⁺ release that is involved in a wide range of cellular functions. In addition to Ca²⁺-induced Ca²⁺ release in cardiac cells and voltage-induced Ca²⁺ release in skeletal muscle cells, we recently identified another mode of intracellular Ca²⁺ mobilization mediated by RyR, i.e., nitric oxide-induced Ca²⁺ release (NICR), in cerebellar Purkinje cells. NICR is evoked by neuronal activity, is dependent on S-nitrosylation of type 1 RyR (RyR1) and is involved in the induction of long-term potentiation (LTP) of cerebellar synapses. In this addendum, we examined whether peroxynitrite, which is produced by the reaction of nitric oxide with superoxide, may also have an effect on the Ca²⁺ release via RyR1 and the cerebellar LTP. We found that scavengers of peroxynitrite have no significant effect either on the Ca²⁺ release via RyR1 or on the cerebellar LTP. We also found that an application of a high concentration of peroxynitrite does not reproduce neuronal activity-dependent Ca²⁺ release in Purkinje cells. These results support that NICR is induced by endogenous nitric oxide produced by neuronal activity through S-nitrosylation of RyR1.     

Quantal glutamate release is essential for reliable neuronal encodings in cerebral networks

Background

The neurons and synapses work coordinately to program the brain codes of controlling cognition and behaviors. Spike patterns at the presynaptic neurons regulate synaptic transmission. The quantitative regulations of synapse dynamics in spike encoding at the postsynaptic neurons remain unclear.

Methodology/Principal Findings

With dual whole-cell recordings at synapse-paired cells in mouse cortical slices, we have investigated the regulation of synapse dynamics to neuronal spike encoding at cerebral circuits assembled by pyramidal neurons and GABAergic ones. Our studies at unitary synapses show that postsynaptic responses are constant over time, such as glutamate receptor-channel currents at GABAergic neurons and glutamate transport currents at astrocytes, indicating quantal glutamate release. In terms of its physiological impact, our results demonstrate that the signals integrated from quantal glutamatergic synapses drive spike encoding at GABAergic neurons reliably, which in turn precisely set spike encoding at pyramidal neurons through feedback inhibition.

Conclusion/Significance

Our studies provide the evidences for the quantal glutamate release to drive the spike encodings precisely in cortical circuits, which may be essential for programming the reliable codes in the brain to manage well-organized behaviors.     

Neurons respond to hyposmotic conditions by an increase in intracellular free calcium

The effect of hyposmotic conditions on the concentration of intracellular free calcium ([Ca²⁺]_i) was studied in cultured cerebellar granule cells and cerebral cortical neurons after loading of the cells with the fluorescent Ca²⁺ chelator Fluo-3. It was found that in both types of neurons exposure to media with a decrease in osmolarity of 20 to 50% of the osmolarity in the isosmotic medium (320 mOsm) led to a dose dependent increase in [Ca²⁺]_i with a time course showing the highest value at the earliest measured time point, i.e. 40 s after exposure to the hyposmotic media and a subsequent decline towards the basal level during the following 320 s. The response in the cortical neurons was larger than in the granule cells but both types of neurons exhibited a similar increase in [Ca²⁺]_i after expoxure to 50 mM K⁺ which was of the same magnitude as the increase in [Ca²⁺]_i observed in the cortical neurons exposed for 40 s to a medium with a 50% reduction in osmolarity. In both types of neurons the blocker of voltage gated Ca²⁺ channels verapamil had no effect on the hyposmolarity induced increase in [Ca²⁺]_i. On the contrary, this increase in [Ca²⁺]_i was dependent upon external calcium and could be inhibited partly or completely by the inorganic blockers of Ca²⁺ channels Mg²⁺ and La³⁺. Dantrolene which prevents release of Ca²⁺ from internal stores had no effect. The results show that exposure of neurons to hyposmotic conditions leading to swelling results in a large increase in free intracellular Ca²⁺ which represents an influx of Ca²⁺ rather than a release of Ca²⁺ from internal, dantrolene sensitive stores.     

Co-cultures of inferior olive and cerebellum: Electrophysiological evidence for multiple innervation of Purkinje cells by olivary axons

Slices of inferior olive (IO) and cerebellum were co-cultured for several weeks by means of the roller tube technique. Recordings were carried out intracellularly from Purkinje cells (PCs) which were identified morphologically by intracellular injection of the fluorescent dye Lucifer yellow, or by immunohistochemical stainings with antibodies raised against the 28 kD Ca²⁺-binding protein calbindin. Following stimulation of olivary tissue, an all-or-none full complex spike response was recorded in some PCs consisting of a fast rising spike followed by a depolarizing potential. In other PCs, graded stimulation of the olivary explant induced synaptic potentials which were characterized by step-wise variation in their amplitude and resembled the ones occurring spontaneously. In contrast, only smoothly graded synaptic potentials were observed in cerebellar mono-cultures. These results indicate that some of the PCs in olivo-cerebellar co-cultures are innervated by several olivary neurons.     

Increased number of nitric oxide synthase immunoreactive Purkinje cells and dentate nucleus neurons in schizophrenia

There is growing interest in the cerebellum as a site of neuropathological changes in schizophrenia. Reports showing that schizophrenics have higher nitric oxide synthase (NOS) activity and MAPKinase levels in the vermis, point to possible aberrations in the cerebellar signal transduction of schizophrenics. It has been speculated that Ca²⁺-dependent extracellular to intracellular signal transduction may be disrupted in the cerebellum of schizophrenics. We decided to test this hypothesis by studying the nitrergic system and markers of the Ca²⁺-triggered signal cascade in the cerebellum of schizophrenics, depressives and controls. The cellular distribution of two calcium sensor proteins (VILIP-1 and VILIP-3) and of neuronal NOS immunoreactivity was studied morphometrically in the flocculonodulus, the inferior vermis and the dentate nucleus of 9 schizophrenics, 7 depressive patients and 9 matched controls. In comparison to controls and depressed patients there were fewer Nissl-stained neurons in the dentate nucleus of schizophrenics. The number of NOS-expressing Purkinje neurons was however strongly increased. In the flocculonodulus and the vermis no differences between the groups were found with regard to the density of Nissl-stained Purkinje cells. The number of NOS-expressing Purkinje neurons was increased in schizophrenics, however. No differences between schizophrenics, depressives and controls were found in the number of VILIP-1 immunoreactive dentate nucleus neurons and VILIP-3 immunoreactive vermal and flocculonodular Purkinje cells. Our data provide further histochemical evidence in favor of structural abnormalities in discrete cerebellar regions of schizophrenics. They confirm and extend earlier reports of increased cerebellar NOS immunoreactivity in schizophrenia and point to possible neurodevelopmental disturbances. Our failure to show an altered expression of two calcium sensor proteins possibly points to a less important role of calcium signaling in cerebellar pathology of the disease.     

Short-term hypoxia induces a selective death of GABAergic neurons

It is known that brief episodes of hypoxia protect neurons from death caused by global ischemia and hypoxia (hypoxic preconditioning). At the same time, brief hypoxia may cause a phenomenon of posthypoxic hyperexcitability during reoxygenation, which can lead to the death of separate neurons due to their individual differences. In this work we compare the effects of short-term hypoxia on the initiation of preconditioning and posthypoxic hyperexcitability in two populations of neurons: inhibitory GABAergic neurons and excitatory glutamatergic neurons. Preconditioning effect was evaluated according to the suppression of the NMDA-receptor activity. The phenomenon of posthypoxic hyperexcitability was estimated by the appearance of spontaneous synchronized Ca²⁺ spikes in the neuronal network during reoxygenation after each episode of hypoxia. It is shown that the preconditioning effect occurs only in glutamatergic neurons. In the GABAergic neurons the effect of preconditioning was not observed. The activity of NMDA receptors in these neurons was not suppressed but increased after each episode of hypoxia. At the moment of posthypoxic synchronous Ca²⁺-spike generation, a global increase of the cytoplasmic Ca²⁺ concentration occurred in a few of GABAergic neurons, followed by the apoptotic death of these cells. The anti-inflammatory cytokine, interleukin-10 (IL-10) prevented the development of posthypoxic hyperexcitability, inhibiting spontaneous synchronous Ca²⁺ spike, and protected GABAergic neurons from the death, restoring the preconditioning effect in them. PI3-kinase inhibitors wortmannin and LY294002 prevented the IL-10 protective effect abolishing the inhibiting effect of IL-10 on the generation of the Ca²⁺ synchronous spike. These findings point out to the leading role of GABAergic neurons in the development of posthypoxic hyperexcitability. We suggest that the reason for posthypoxic hyperexcitability in the network is a weakening of the inhibiting effect of GABAergic neurons. Activation of different signaling pathways leading to activation of PKB- and PKG-dependent phosphorylation in the neurons of this type represents a possible strategy to protect neurons from death during hypoxia.     

Calcium signalling in cortical and thalamic neurons of rats with streptozotocin-induced diabetes

Intracellular Ca²⁺ transients were measured with the use of a Ca²⁺-sensitive fluorescent indicator, fura-2, in neocortical and thalamic neurons in brain slices from control rats and rats with uncompensated streptozotocin-induced diabetes. The transients were evoked by high-potassium (50 mM)-induced membrane depolarization. The amplitude of depolarization-induced Ca²⁺ transients demonstrated a tendency to increase under diabetic conditions, beeing more expressed in cortical neurons compared with thalamic ones. The transients in cortical neurons from diabetic animals became also more susceptible to the blocking action of nifedipine (100μM) and less sensitive to Ni²⁺ (50μM), indicating that diabetic changes affect mostly Ca²⁺ transients triggered by high-voltage activated (L-type) calcium channels. The duration of a statistically significant increase was observed in the residual elevation of intracellular Ca²⁺ changes. However, a statistically significant increase was observed in the residual elevation of intracellular Ca²⁺ measured 60 sec after termination of membrane depolarization in both cortical and thalamic neurons, indicating alterations in the mechanisms that restore the resting level of Ca²⁺ in the cytosol. It is concluded that uncomensated insulin-dependent diabetes, which according to earlier data substantially alters calcium signalling in primary sensory neurons, also affects such signalling in the neurons of higher brain structures including the thalamus and cortex.     

Dendritic differentiation of cerebellar Purkinje cells is promoted by ryanodine receptors expressed by Purkinje and granule cells

Cerebellar Purkinje cells have the most elaborate dendritic trees among neurons in the brain. We examined the roles of ryanodine receptor (RyR), an intracellular Ca²⁺ release channel, in the dendrite formation of Purkinje cells using cerebellar cell cultures. In the cerebellum, Purkinje cells express RyR1 and RyR2, whereas granule cells express RyR2. When ryanodine (10 µM), a blocker of RyR, was added to the culture medium, the elongation and branching of Purkinje cell dendrites were markedly inhibited. When we transferred small interfering RNA (siRNA) against RyR1 into Purkinje cells using single‐cell electroporation, dendritic branching but not elongation of the electroporated Purkinje cells was inhibited. On the other hand, transfection of RyR2 siRNA into granule cells also inhibited dendritic branching of Purkinje cells. Furthermore, ryanodine reduced the levels of brain‐derived neurotrophic factor (BDNF) in the culture medium. The ryanodine‐induced inhibition of dendritic differentiation was partially rescued when BDNF was exogenously added to the culture medium in addition to ryanodine. Overall, these results suggest that RyRs expressed by both Purkinje and granule cells play important roles in promoting the dendritic differentiation of Purkinje cells and that RyR2 expressed by granule cells is involved in the secretion of BDNF from granule cells. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 467–480, 2014     

Distribution of different types of calcium channels in the brain structures

This study demonstrates that low voltage-activated (LVA) Ca²⁺ channels and at least five pharmacologically distinct high voltage-activated (HVA) Ca²⁺ channels exist in most rat CNS neurons, with the exception of cerebellar Purkinje cells. These LVA and HVA Ca²⁺ channels (including their subtypes) are differentially distributed among various CNS regions. The pharmacological diversity of these channels and their heterogeneous distribution on the nerve cell somata and nerve endings appear relevant in the development of new and region-specific pharmacological agents for clinical use.     

Anatomy of zebrafish cerebellum and screen for mutations affecting its development

The cerebellum is important for the integration of sensory perception and motor control, but its structure has mostly been studied in mammals. Here, we describe the cell types and neural tracts of the adult zebrafish cerebellum using molecular markers and transgenic lines. Cerebellar neurons are categorized to two major groups: GABAergic and glutamatergic neurons. The Purkinje cells, which are GABAergic neurons, express parvalbumin7, carbonic anhydrase 8, and aldolase C like (zebrin II). The glutamatergic neurons are vglut1⁺ granule cells and vglut2^high cells, which receive Purkinje cell inputs; some vglut2^high cells are eurydendroid cells, which are equivalent to the mammalian deep cerebellar nuclei. We found olig2⁺ neurons in the adult cerebellum and ascertained that at least some of them are eurydendroid cells. We identified markers for climbing and mossy afferent fibers, efferent fibers, and parallel fibers from granule cells. Furthermore, we found that the cerebellum-like structures in the optic tectum and antero-dorsal hindbrain show similar Parvalbumin7 and Vglut1 expression profiles as the cerebellum. The differentiation of GABAergic and glutamatergic neurons begins 3 days post-fertilization (dpf), and layers are first detectable 5 dpf. Using anti-Parvalbumin7 and Vglut1 antibodies to label Purkinje cells and granule cell axons, respectively, we screened for mutations affecting cerebellar neuronal development and the formation of neural tracts. Our data provide a platform for future studies of zebrafish cerebellar development.     

The postnatal development of intrinsic properties and spike encoding at cortical GABAergic neurons

GABAergic neurons play a critical role in maintaining the homeostasis of brain functions for well-organized behaviors. It is not known about the dynamical change in signal encoding at these neurons during postnatal development. We investigated this issue at GFP-labeled GABAergic neurons by whole-cell recording in cortical slices of mice. Our results show that the ability of spike encoding at GABAergic neurons is improved during postnatal development. This change is associated with the reduction of refractory periods and threshold potentials of sequential spikes, as well as the improvement of linear correlations between intrinsic properties and spike capacity. Therefore, the postnatal maturation of the spike encoding capacity at GABAergic neurons will stabilize the excitatory state of cerebral cortex.     

Cerebellar Hypoplasia in Mice Lacking Selenoprotein Biosynthesis in Neurons

Selenium exerts many, if not most, of its physiological functions as a selenocysteine moiety in proteins. Selenoproteins are involved in many biochemical processes including regulation of cellular redox state, calcium homeostasis, protein biosynthesis, and degradation. A neurodevelopmental syndrome called progressive cerebello-cortical atrophy (PCCA) is caused by mutations in the selenocysteine synthase gene, SEPSECS, demonstrating that selenoproteins are essential for human brain development. While we have shown that selenoproteins are required for correct hippocampal and cortical interneuron development, little is known about the functions of selenoproteins in the cerebellum. Therefore, we have abrogated neuronal selenoprotein biosynthesis by conditional deletion of the gene encoding selenocysteyl tRNA^[Ser]Sec (gene symbol Trsp). Enzymatic activity of cellular glutathione peroxidase and cytosolic thioredoxin reductase is reduced in cerebellar extracts from Trsp-mutant mice. These mice grow slowly and fail to gain postural control or to coordinate their movements. Histological analysis reveals marked cerebellar hypoplasia, associated with Purkinje cell death and decreased granule cell proliferation. Purkinje cell death occurs along parasagittal stripes as observed in other models of Purkinje cell loss. Neuron-specific inactivation of glutathione peroxidase 4 (Gpx4) used the same Cre driver phenocopies tRNA^[Ser]Sec mutants in several aspects: cerebellar hypoplasia, stripe-like Purkinje cell loss, and reduced granule cell proliferation. Parvalbumin-expressing GABAergic interneurons (stellate and/or basket cells) are virtually absent in tRNA^[Ser]Sec-mutant mice, while some remained in Gpx4-mutant mice. Our data show that selenoproteins are specifically required in postmitotic neurons of the developing cerebellum, thus providing a rational explanation for cerebellar hypoplasia as occurring in PCCA patients.     

Inhibition of spontaneous synchronous activity of hippocampal neurons by excitation of GABAergic neurons

The molecular mechanisms of the neuronal spontaneous synchronous activity (SSA) regulation by population of GABAergic neurons have been investigated in rat hippocampal culture. The neurons from this population contain Ca²⁺-permeable KA receptors on the presynaptic membrane. Using image analysis, confocal microscopy and immunocytochemistry, we identified by the shape of Ca²⁺ signal the population of GABAergic neurons with unique charachteristics allowing these neurons to control SSA. The SSA in a neuronal network was suppressed by the KA-receptor mediated [Ca²⁺]i increase in neurons of this population. Agonists of GluR5/GluK1-containing KA receptors (domoic acid (DA), SYM2081, and ATPA) evoked a fast high-amplitude Ca²⁺ signal without desensitization only in this population of neurons. This fact points to Ca²⁺ permeability of KA receptors in these neurons. The GABA(A) receptor antagonist bicuculline increased the activity of AMPA but not KA receptors of these neurons, indicating presynaptical localization of KA receptors. Depolarization of cells induced by KCl (unlike bicuculline-induced depolarization) increased the activity of AMPA and KA receptors twofold, which points to the dependence of the activity on depolarization. A tenfold increase of the SSA frequency in neurons of this population caused an increase in the basal [Ca²⁺]i level, which was accompanied by inhibition of SSA in another numerous population of neurons, suggesting that an increased GABAergic inhibition takes place. Prolonged high-frequency oscillations causes a global [Ca²⁺]i increase in the neurons of this population and their subsequent death. Thus, KA receptors in the population of fast GABAergic neurons may implement a negative feedback under hyperexcitation by glutamate enhancing GABA release due to the fast and prolonged [Ca²⁺]i increase. It has been shown that this mechanism can be used to suppress hyperactivation of a certain population of neurons under high-frequency SSA and ischemia. It is obvious that selective death of inhibitory neurons from this population may lead to hyperexcitability of certain brain regions.     

AMPA Receptor-Mediated Alterations of Intracellular Calcium Homeostasis in Rat Cerebellar Purkinje Cells In Vitro: Correlates to Dark Cell Degeneration

In the rat cerebellar slice preparation in vitro, excessive DL-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)-receptor activation elicits a characteristic type of excitotoxicity of Purkinje cells (PCs) known as dark cell degeneration (DCD). DCD models neurotoxicity of PCs and hippocampal pyramidal neurons in vivo following hyperexcitable states. The intent of this study was to: a) determine whether AMPA-induced neurotoxicity of PCs is correlated with temporally and spatially restricted rises in intracellular Ca²⁺ and b) whether GYKI 52466 and nominal external Ca²⁺, conditions that reduced expression of AMPA-elicited DCD, altered the induced Ca²⁺ patterns. Employing the Ca²⁺-sensitive dye Fluo-3 and a confocal laser scanning microscope, we evaluated changes in intracellular Ca²⁺ within PCs in a cerebellar slice preparation. AMPA application alone (30 M for 30 min) caused a significant initial rise in perinuclear and cytoplasmic Ca²⁺ that returned to control levels during the latter part of the AMPA exposure period. Following removal of AMPA (expression period), perinuclear and cytoplasmic Ca²⁺ displayed a significant delayed rise peaking transiently 60 min after AMPA removal. The efficacy of GYKI 52466 and nominal external Ca²⁺ conditions to attenuate AMPA-induced DCD was correlated to reductions in AMPA-induced transient elevations in perinuclear and cytoplasmic Ca²⁺ levels during the expression phase and to a lesser extent during the exposure period. The present data suggest that during the expression phase, the delayed perinuclear and cytoplasmic Ca²⁺ transient may be the harbinger of impending loss of Ca²⁺ homeostasis and cell damage.     

Glutamate triggers intracellular Ca2+ oscillations and nitric oxide release by inducing NAADP- and InsP3-dependent Ca2+ release in mouse brain endothelial cells

The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the target cells, which activates the Ca²⁺/Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca²⁺]_i and NO production. The current study assessed whether and how glutamate drives Ca²⁺-dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca²⁺]_i, which was maximally activated at 200 μM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca²⁺ oscillations were triggered by rhythmic endogenous Ca²⁺ mobilization and maintained over time by extracellular Ca²⁺ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca²⁺ release was mediated by InsP₃-sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca²⁺ entry mediated Ca²⁺ entry during ongoing Ca²⁺ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca²⁺ signals. Of note, glutamate induced Ca²⁺-dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca²⁺ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.