Cultures Of Cerebellar Granule Cells Research Articles

Small Aβ1-42 oligomer-induced membrane depolarization of neuronal and microglial cells: role of N-methyl-D-aspartate receptors.

Although it is well documented that soluble beta amyloid (Aβ) oligomers are critical factors in the pathogenesis of Alzheimer's disease (AD) by causing synaptic dysfunction and neuronal death, the primary mechanisms by which Aβ oligomers trigger neurodegeneration are not entirely understood. We sought to investigate whether toxic small Aβ(1-42) oligomers induce changes in plasma membrane potential of cultured neurons and glial cells in rat cerebellar granule cell cultures leading to neuronal death and whether these effects are sensitive to the N-methyl-D-aspartate receptor (NMDA-R) antagonist MK801. We found that small Aβ(1-42) oligomers induced rapid, protracted membrane depolarization of both neurons and microglia, whereas there was no change in membrane potential of astrocytes. MK801 did not modulate Aβ-induced neuronal depolarization. In contrast, Aβ1(-42) oligomer-induced decrease in plasma membrane potential of microglia was prevented by MK801. Small Aβ(1-42) oligomers significantly elevated extracellular glutamate and caused neuronal necrosis, and both were prevented by MK801. Also, small Aβ(1-42) oligomers decreased resistance of isolated brain mitochondria to calcium-induced opening of mitochondrial permeability transition pore. In conclusion, the results suggest that the primary effect of toxic small Aβ oligomers on neurons is rapid, NMDA-R-independent plasma membrane depolarization, which leads to neuronal death. Aβ oligomers-induced depolarization of microglial cells is NMDA-R dependent.

The cellular state determines the effect of melatonin on the survival of mixed cerebellar cell culture.

The constitutive activation of nuclear factor-κB (NF-κB), a key transcription factor involved in neuroinflammation, is essential for the survival of neurons in situ and of cerebellar granule cells in culture. Melatonin is known to inhibit the activation of NF-κB and has a cytoprotective function. In this study, we evaluated whether the cytoprotective effect of melatonin depends on the state of activation of a mixed cerebellar culture that is composed predominantly of granule cells; we tested the effect of melatonin on cultured rat cerebellar cells stimulated or not with lipopolysaccharide (LPS). The addition of melatonin (0.1 nM–1 µM) reduced the survival of naïve cells while inhibiting LPS-induced cell death. Melatonin (100 nM) transiently (15 min) inhibited the nuclear translocation of both NF-κB dimers (p50/p50, p50/RelA) and, after 60 min, increased the activation of p50/RelA. Melatonin-induced p50/RelA activity in naïve cells resulted in the transcription of inducible nitric oxide synthase (iNOS) and the production of NO. Otherwise, in cultures treated with LPS, melatonin blocked the LPS-induced activation of p50/RelA and the reduction in p50/p50 levels and inhibited iNOS expression and NO synthesis. Therefore, melatonin in vehicle-treated cells induces cell death, while it protects against LPS-induced cytotoxicity. In summary, we confirmed that melatonin is a neuroprotective drug when cerebellar cells are challenged; however, melatonin can also lead to cell death when the normal balance of the NF-κB pathway is disturbed. Our data provide a mechanistic basis for understanding the influence of cell context on the final output response of melatonin.

Behavioral effect of dipeptide NGF mimetic GK-2 in an in vivo model of rat traumatic brain injury and its neuroprotective and regenerative properties in vitro

A protective behavioral effect of a nerve growth factor dipeptide mimetic GK-2 in the model of open focal trauma of rat brain sensorimotor cortex and its antioxidative and regenerative properties in cultures of rat cerebellar granule cells and mouse embryonal spinal ganglion, respectively, were studied. Intraperitoneal injections of GK-2 (1 mg/kg) for 5 days daily after traumatic brain injury improved significantly motor function of limbs. Moreover, supplementation the incubation medium with GK-2 (0.5–1.5 mg/l) decreased neuronal death induced by H2O2 in cerebellar granule cell cultures and stimulated neurite outgrowth from cultured mouse embryonal spinal ganglia. Our results suggest that GK-2 exhibits pronounced positive behavioral effect in vivo as well as neuroprotective and regenerative effects in vitro, and that these neuroprotective properties probably associated with cell survival but not with cell differentiation pathway.

Neurotoxic effects of indocyanine green -cerebellar granule cell culture viability study

The aim of this study was to examine neurotoxicity indocyanine green (ICG). We assessed viability of primary cerebellar granule cell culture (CGC) exposed to ICG to test two mechanisms that could be the first triggers causing neuronal toxicity: imbalance in calcium homeostasis and the degree of oligomerization of ICG molecules. We have observed this imbalance in CGC after exposure to 75-125μΜ ICG and dose and application sequence dependent protective effect of Gadovist on surviving neurons in vitro when used with ICG. Spectroscopic studies suggest the major cause of toxicity of the ICG is connected with oligomers formation. ICG at concentration of 25 μM (which is about 4 times higher than the highest concentration of ICG in the brain applied in in-vivo human studies) is not neurotoxic in the cell culture.

Differential Neuronal Targeting of a New and Two Known Calcium Channel β4Subunit Splice Variants Correlates with Their Regulation of Gene Expression

The β subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 α1 subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain β subunits are targeted into the nucleus, where they interact directly with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of β isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual β variants in specific neuronal functions. In the present study, an alternatively spliced β4 subunit lacking the variable N terminus (β4e) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGCs) and modulates P/Q-type calcium currents in tsA201 cells and CaV2.1 surface expression in neurons. Compared with the other two known full-length β4 variants (β4a and β4b), β4e is most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To determine the importance of nuclear targeting of β4 subunits for transcriptional regulation, we performed whole-genome expression profiling of CGCs from lethargic (β4-null) mice individually reconstituted with β4a, β4b, and β4e. Notably, the number of genes regulated by each β4 splice variant correlated with the rank order of their nuclear-targeting properties (β4b > β4a > β4e). Together, these findings support isoform-specific functions of β4 splice variants in neurons, with β4b playing a dual role in channel modulation and gene regulation, whereas the newly detected β4e variant serves exclusively in calcium-channel-dependent functions.

Three Splice Variants of the Calcium Channel Beta4 Subunit Display Differential Targeting and Gene Regulation in Neurons

The β subunits of voltage-gated calcium channels regulate surface expression and gating properties of CaV1 and CaV2 α1 subunits. All four CaVβ genes are expressed in the brain, but only mutation/lack of β4 causes a neurological phenotype (epilepsy, ataxia) in humans and mice. The β4 isoform is also targeted into the nucleus. There it directly interacts with the epigenetic machinery, suggesting a calcium channel-independent role of β4 in transcriptional regulation. CaVβ subunits are subject to abundant alternative splicing. However, little is known about the specific functions of individual β splice variants in excitable cells. Here we identified a new alternatively spliced β4 transcript, β4e. mRNA and protein of this splice are highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGC). Overexpression of β4e modulates P/Q-type calcium currents in tsA cells and promotes surface expression of native synaptic CaV2.1 channels in hippocampal neurons. Compared to the other two known full-length β4 variants (β4a, β4b) β4e is most abundantly expressed in the distal axon. Consistent with the described role of N-terminal sequences in nuclear import, β4e, which lacks these sequences, does not show nuclear targeting. The importance of nuclear targeting for the putative role of β4 in transcriptional regulation was examined by whole genome expression profiling of CGCs from β4-null mice individually reconstituted with β4a, β4b, or β4e. Strikingly, the capacity of β4 splice variants to regulate neuronal genes depended on their nuclear targeting with a rank order β4b>β4a>β4e. Together these findings indicate that in neurons the three β4 splice variants serve distinct functions. Whereas β4b plays a dual role in channel modulation and gene regulation, the newly detected β4e variant functions primarily as calcium channel subunit. Support: FWF P23479, P24079, W1101, F4406.

The role of the glutamatergic NMDA receptor in nanosilver-evoked neurotoxicity in primary cultures of cerebellar granule cells

Nanoparticles are known to enter the vertebrate brain, but little is known about their neurotoxicity. The aim of this study is to investigate mechanisms of the contribution of AgNPs to neuronal cell death using primary cultures of rat cerebellar granule cells (CGCs). We tested the role of glutamatergic N-methyl-d-aspartate receptors (NMDA) in AgNP-evoked neurotoxicity using MK-801, a noncompetitive inhibitor of NMDAR. We used commercially available 0.2% PVP-coated AgNPs <100nm in a concentration range of 2.5–75μg/ml sonicated with fetal calf serum. After a 10min incubation period, a dose-dependent increase in the uptake of 45Ca2+ into neurons was observed in the presence of 25–75μg/mL AgNPs which was completely abolished by addition of MK-801. Using the fluorescent dye fluo3 AM we observed an increase in the intracellular calcium level by 87% compared to control. ROS production was found to increase by about 30% over control after a 30-min incubation with 75μg/mL AgNPs. Further, we observed a significant decrease in the mitochondrial potential during a 30-min incubation with AgNPs. Administration of MK-801 was found to provide a protective effect. Our results show that excitotoxicity via activation of NMDA receptor, followed by calcium imbalance, destabilization of mitochondrial function and ROS production, indicate an important mechanism involved in neurotoxicity evoked by AgNPs in cultured neurons.

Studying synaptic efficiency by post-hoc immunolabelling

BackgroundIn terms of vesicular recycling, synaptic efficiency is a key determinant of the fidelity of synaptic transmission. The ability of a presynaptic terminal to reuse its vesicular content is thought to be a signature of synaptic maturity and this process depends on the activity of several proteins that govern exo/endocytosis. Upon stimulation, individual terminals in networks of cultured cerebellar granule neurons exhibit heterogeneous exocytic responses, which reflect the distinct states of maturity and plasticity intrinsic to individual synaptic terminals. This dynamic scenario serves as the substrate for processes such as scaling, plasticity and synaptic weight redistribution. Presynaptic strength has been associated with the activity of several types of proteins, including the scaffolding proteins that form the active zone cytomatrix and the proteins involved in presynaptic exocytosis.MethodsWe have combined fluorescence imaging techniques using the styryl dye FM1-43 in primary cultures of cerebellar granule cells with subsequent post-hoc immunocytochemistry in order to study synaptic efficiency in terms of vesicular release. We describe a protocol to easily quantify these results with minimal user intervention.ResultsIn this study we describe a technique that specifically correlates presynaptic activity with the levels of presynaptic markers. This method involves the use of the styryl dye FM1-43 to estimate the release capacity of a synaptic terminal, and the subsequent post-hoc immunolabelling of thousands of individual nerve terminals. We observed a strong correlation between the release capacity of the nerve terminal and the levels of the RIM1α but not the Munc13-1 protein in the active zone.ConclusionsOur findings support those of previous studies and point out to RIM1α as a crucial factor in determining synaptic efficiency. These results also demonstrate that this technique is a useful tool to analyse the molecular differences underlying the heterogeneous responses exhibited by neuronal networks.

GABAA Receptors of Cerebellar Granule Cells in Culture: Interaction with Benzodiazepines.

GABAA receptor mediated inhibition plays an important role in modulating the input/output dynamics of cerebellum. A characteristic of cerebellar GABAA receptors is the presence in cerebellar granule cells of subunits such as α6 and δ which give insensitivity to classical benzodiazepines. In fact, cerebellar GABAA receptors have generally been considered a poor model for testing drugs which potentially are active at the benzodiazepine site. In this overview we show how rat cerebellar granule cells in culture may be a useful model for studying new benzodiazepine site agonists. This is based on the pharmacological separation of diazepam-sensitive α1 β2/3 γ2 receptors from those which are diazepam-insensitive and contain the α6 subunit. This is achieved by utilizing furosemide/Zn2+ which block α6 containing and incomplete receptors.

Small Nogo-66-binding peptide promotes neurite outgrowth through RhoA inhibition after spinal cord injury

Abortive regeneration in the adult mammalian central nervous system (CNS) is partially mediated through CNS myelin proteins, among which Nogo-A plays an important role. Nogo-66, which is located at the C-terminus of Nogo-A, inhibits axonal regrowth through the Nogo-66/NgR signalling pathway. In this study, two small peptides were tested in a neurite outgrowth assay and spinal cord injury (SCI) model to examine the effects of these molecules on the inhibition of Nogo-66/NgR signalling. PepIV was selected from a phage display peptide library as a Nogo-66 binding molecule. And PepII was synthesized as a potential NgR antagonist. The results indicated that PepIV and PepII decrease the mRNA levels of the small GTPase RhoA and partially neutralize CNS myelin inhibition to cultured cerebellar granule cells (CGCs). Moreover, treatment with both peptides was propitious to maintaining residual axons after SCI, thereby promoting regeneration and locomotion recovery. Because RhoA plays a role in stabilizing the cytoskeleton in growth cones and axons, enhanced neurite outgrowth might reflect a decrease in RhoA expression through PepIV and PepII treatment. Moreover, PepIV induced lower RhoA mRNA expression compared with PepII. Therefore, PepIV could block Nogo-66/NgR signalling and reduce RhoA mRNA level, and then contribute to neuronal survival and axonal regrowth after SCI, showing its ability to reverse CNS myelin inhibition to regeneration. Furthermore, selected small peptide might cover some unknown active sites on CNS myelin proteins, which could be potential targets for improving neurite outgrowth after injury.

Prenatal exposure to bisphenol A interferes with the development of cerebellar granule neurons in mice and chicken

In mice, prenatal exposure to low doses of bisphenol A has been shown to affect neurogenesis and neuronal migration in cortex, resulting in disturbance of both neuronal positioning and the network formation between thalamus and cortex in the offspring brain. In the present study we investigated whether prenatal exposure to bisphenol A disturbs the neurodevelopment of the cerebellum. Two different model systems were used; offspring from two strains of mice from mothers receiving bisphenol A in the drinking water before mating, during gestation and lactation, and chicken embryos exposed to bisphenol A (in the egg) on embryonic day 16 for 24h before preparation of cerebellar granule cell cultures. In the cerebellum, tight regulation of the level of transcription factor Pax6 is critical for correct development of granule neurons. During the development, the Pax6 level in granule neurons is high when these cells are located in the external granule layer and during their migration to the internal granule layer, and it is then reduced. We report that bisphenol A induced an increase in the thickness of the external granule layer and also an increase in the total cerebellar Pax6 level in 11 days old mice offspring. In cultured chicken cerebellar granule neurons from bisphenol A injected eggs the Pax6 level was increased day 6 in vitro. Together, these findings indicate that bisphenol A may affect the granule neurons in the developing cerebellum and thereby may disturb the correct development of the cerebellum.

Role of Ca2+ imbalance mediated by NMDA and ryanodine receptors in acute TBBPA toxicity in primary cultures of rat cerebellar granule cells

Role of Ca2+ imbalance mediated by NMDA and ryanodine receptors in acute TBBPA toxicity in primary cultures of rat cerebellar granule cells

PI3 k/akt inhibition induces apoptosis through p38 activation in neurons

Accumulating evidence suggests that the PI3K/AKT pathway is a pro-survival signalling system in neurons. Therefore, the inhibition of this pathway may be implicated in the degeneration of neurons in Parkinson's disease (PD), Alzheimer's disease (AD), and other neurological disorders. Here we study the participation of the mitogen-activated protein kinase (MAPK) pathway on apoptosis induced by PI3K/AKT inhibition in cultured cerebellar granule cells (CGCs).LY294002, a specific PI3K/AKT inhibitor, selectively activated the p38 MAPK kinase pathway and enhanced c-Jun phosphorylation, but did not activate JNK. The pharmacological inhibitors SB203580 (p38 inhibitor) and SP600125 (a JNK inhibitor) protected primary cultures of rat CGCs from LY294002-induced apoptosis. Furthermore, both compounds decreased the phosphorylation of c-Jun and lowered mRNA levels of the pro-apoptotic gene dp5, a direct target of c-Jun. Taken together, our data demonstrate that PI3K/AKT inhibition induces neuronal apoptosis, a process that is mediated by the activation of p38 MAPK/c-Jun/dp5.

Heroin activates Bim via c-Jun N-terminal kinase/c-Jun pathway to mediate neuronal apoptosis

Heroin is reported to cause spongiform leukoencephalopathy (SLE) in heroin addicts and the exact mechanism has not yet been identified. In the present study, we found that heroin could induce apoptosis of primary cultured cerebellar granule cells (CGCs) and Bim was upregulated both transcriptionally and post transcriptionally during CGCs apoptosis. Upregulated Bim translocated to mitochondria and Bax was activated under heroin treatment. Genetic knockdown of Bim using lentiviruses significantly prevented neuronal apoptosis induced by heroin. Meanwhile, c-Jun N-terminal kinase (JNK)/c-Jun pathway was activated in heroin-induced apoptosis. Bim was demonstrated as a downstream target of JNK/c-Jun pathway in this process because pharmacological inhibition of JNK reduced the levels of Bim mRNA and protein. These results indicate that Bim plays a critical role in the neurotoxic process by heroin and JNK/c-Jun pathway acts upstream of Bim in regulating heroin-induced neuronal death. This represents a detailed mechanism of heroin-induced neuronal apoptosis and may provide a new and effective strategy to treat heroin-induced addiction and SLE.

ErbB1 epidermal growth factor receptor is a valid target for reducing the effects of multiple inhibitors of axonal regeneration

Pharmacological inhibitors of epidermal growth factor receptor (ErbB1) attenuate the ability of CNS myelin to inhibit axonal regeneration. However, it has been claimed that such effects are mediated by off-target interactions. We have tested the role of ErbB1 in axonal regeneration by culturing neurons from ErbB1 knockout mice in the presence of various inhibitors of axonal regeneration: CNS myelin, chondroitin sulfate proteoglycans (CSPG), fibrinogen or polyinosinic:polycytidylic acid (poly I:C). We confirmed that ErbB1 was activated in cultures of cerebellar granule cells exposed to inhibitors of axonal regeneration and that ErbB1 kinase inhibitors promoted neurite outgrowth under these conditions. In the presence of myelin, fibrinogen, CSPG and poly I:C ErbB1 −/− neurons grew longer neurites than neurons expressing ErbB1. Furthermore, inhibitors of ErbB1 kinase did not improve neurite outgrowth from ErbB1 −/− neurons, ruling out an off-target mechanism of action. ErbB1 kinase activity is therefore a valid target for promoting axonal elongation in the presence of many of the molecules believed to contribute to the failure of axonal regeneration in the injured CNS.

Synergistic neurotoxicity of oxygen-glucose deprivation and tetrabromobisphenol A in vitro: role of oxidative stress

BackgroundTetrabromobisphenol A (TBBPA) is a toxic brominated flame retardant. Previous studies have demonstrated that exposure of primary cultures of rat cerebellar granule cells (CGC) to ≥10μM TBBPA induces toxicity and excitotoxicity, and the underlying mechanism may involve calcium imbalance and oxidative stress. Here we examined whether the application of TBBPA at subtoxic concentrations may exacerbate acute damage of CGC challenged with oxygen-glucose deprivation (OGD), and evaluated with fluorescent indicators the involvement of calcium imbalance, mitochondrial depolarization and oxidative stress. MethodsSurvival of CGC was assessed 24h after OGD/TBBPA using fluorescent dyes. An OGD challenge lasting for 45, 60 or 75min induced a duration-dependent injury to the neurons. ResultsApplication of 2.5, 5 or 7.5μM TBBPA for 45min to normoxic and glucose-containing incubation medium did not reduce the viability of cultured CGC, but this compound exacerbated the toxic effects of OGD in a concentration-dependent way. Moreover, TBBPA had a slight effect on calcium homeostasis and mitochondrial membrane potential, but significantly activated the production of reactive oxygen species in CGC. The application of H2O2 at 5, 10 and 25μM mimicked the effects of TBBPA on OGD toxicity, while 0.1mM ascorbic acid or 1mM glutathione ameliorated this toxicity. ConclusionThese results suggest the involvement of oxidative stress in the synergistic neurotoxic effects of TBBPA and OGD.

Culturing of Cerebellar Granule Cells to Study Neuronal Migration: Gradient and Local Perfusion Assays

Cultures of cerebellar granule cells are a suitable model to analyze the mechanisms governing neuronal migration. In this unit, we describe a protocol to obtain cultures of dissociated granule cells at a low density, where individual cells can be easily observed. In addition, we include a protocol for studying neuronal migration in these cultures, using single, actively migrating cerebellar granule cells. Following this protocol, a factor of interest can be applied either in a gradient concentration by means of a micropipet located near the neuron, or in a homogeneous concentration by locally perfusing a certain region of the neuron. Time-lapse images are taken to analyze changes in the speed and/or directionality of the observed neuron. Overall, the two protocols take more or less a day and a half to perform, and are a useful way to evaluate a certain factor/drug for its chemotactic activity or its capacity to alter migration speed.

Chlorination of ortho-position on Polychlorinated Biphenyls Increases Protein Kinase C Activity in Neuronal Cells

Polychlorinated biphenyls (PCBs) are persistent and bioaccumulative environmental pollutants. Recently, it is suggested that neurotoxic effects such as motor dysfunction and impairment in memory and learning have been associated with PCB exposure. However, structure relationship of PCB congeners with neurotoxic effects remains unknown. Since PKC signaling pathway is implicated in the modulation of motor behavior as well as learning and memory and the role of PKC are subspecies-specific, we attempted to study the effects of structurally distinct PCBs on the total PKC activity as well as subspecies of PKC in cerebellar granule cell culture model. Cells were exposed to 0, 25 and 50 μM of PCB-126, PCB-169, PCB-114, PCB-157, PCB-52 and PCB-4 for 15 min. Cells were subsequently analyzed by [3H] phorbol ester binding assay or immunoblotted against PKC-α and -ε monoclonal antibodies. While non-dioxin-like-PCB (PCB-52 and PCB-4) induced a translocation of PKC-α and -ε from cytosol to membrane fraction, dioxin-like PCBs (PCB-126, -169, -114, -157) had no effects. [3H] Phorbol ester binding assay also revealed structure-dependent increase similar to translocation of PKC isozymes. While PCB-4 induced translocation of PKC-α and -ε was inhibited by ROS inhibitor, the pattern of translocation was not affected in presence of AhR inhibitor. It is suggested that PCB-4-induced PKC activity may not be mediated via AhR-dependent pathway. Taken together, our findings suggest that chlorination of ortho-position in PCB may be a critical structural moiety associated with neurotoxic effects, which may be preferentially mediated via non-AhR-dependent pathway. Therefore, the present study may contribute to understanding the neurotoxic mechanism of PCBs as well as providing a basis for establishing a better neurotoxic assessment.

Ligand-induced changes in Oestrogen and thyroid hormone receptor expression in the developing rat cerebellum: A comparative quantitative PCR and Western blot study

Oestrogen (E2) and thyroid hormones (THs) are key regulators of cerebellar development. Recent reports implicate a complex mechanism through which E2 and THs influence the expression levels of each other's receptors (ERs and TRs) to precisely mediate developmental signals and modulate signal strength. We examined the modulating effects of E2 and THs on the expression levels of their receptor mRNAs and proteins in cultured cerebellar cells obtained from 7-day-old rat pups. Cerebellar granule cell cultures were treated with either E2, THs or a combination of these hormones, and resulting receptor expression levels were determined by quantitative PCR and Western blot techniques. The results were compared to non-treated controls and to samples obtained from 14-day-old in situ cerebella. Additionally, we determined the glial effects on the regulation of ER-TR expression levels. The results show that (i) ER and TR expression depends on the combined presence of E2 and THs; (ii) glial cells mediate the hormonal regulation of neuronal ER-TR expression and (iii) loss of tissue integrity results in characteristic changes in ER-TR expression levels. These observations suggest that both E2 and THs, in adequate amounts, are required for the precise orchestration of cerebellar development and that alterations in the ratio of E2/THs may influence signalling mechanisms involved in neurodevelopment. Comparison of data from in vitro and in situ samples revealed a shift in receptor expression levels after loss of tissue integrity, suggesting that such adjusting/regenerative mechanisms may function after cerebellar tissue injury as well.

The Therapeutic effect of Memantine through the Stimulation of Synapse Formation and Dendritic Spine Maturation in Autism and Fragile X Syndrome

Although the pathogenic mechanisms that underlie autism are not well understood, there is evidence showing that metabotropic and ionotropic glutamate receptors are hyper-stimulated and the GABAergic system is hypo-stimulated in autism. Memantine is an uncompetitive antagonist of NMDA receptors and is widely prescribed for treatment of Alzheimer's disease treatment. Recently, it has been shown to improve language function, social behavior, and self-stimulatory behaviors of some autistic subjects. However the mechanism by which memantine exerts its effect remains to be elucidated. In this study, we used cultured cerebellar granule cells (CGCs) from Fmr1 knockout (KO) mice, a mouse model for fragile X syndrome (FXS) and syndromic autism, to examine the effects of memantine on dendritic spine development and synapse formation. Our results show that the maturation of dendritic spines is delayed in Fmr1-KO CGCs. We also detected reduced excitatory synapse formation in Fmr1-KO CGCs. Memantine treatment of Fmr1-KO CGCs promoted cell adhesion properties. Memantine also stimulated the development of mushroom-shaped mature dendritic spines and restored dendritic spine to normal levels in Fmr1-KO CGCs. Furthermore, we demonstrated that memantine treatment promoted synapse formation and restored the excitatory synapses to a normal range in Fmr1-KO CGCs. These findings suggest that memantine may exert its therapeutic capacity through a stimulatory effect on dendritic spine maturation and excitatory synapse formation, as well as promoting adhesion of CGCs.