Long-term culture of mouse cortical neurons as a model for neuronal development, aging, and death

A long-term cell culture system was used to study maturation, aging, and death of cortical neurons. Mouse cortical neurons were maintained in culture in serum-free medium (Neurobasal supplemented with B27) for 60 days in vitro (DIV). The levels of several proteins were evaluated by immunoblotting to demonstrate that these neurons matured by developing dendrites and synapses and remained continuously healthy for 60 DIV. During their maturation, cortical neurons showed increased or stable protein expression of glycolytic enzyme, synaptophysin, synapsin IIa, alpha and beta synucleins, and glutamate receptors. Synaptogenesis was prominent during the first 15 days and then synaptic markers remained stable through DIV60. Very early during dendritic development at DIV3, beta-synuclein (but not alpha-synuclein) was localized at the base of dendritic growth cones identified by MAP2 and alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor GluR1. In mature neurons, alpha and beta synucleins colocalized in presynaptic axon terminals. Expression of N-methyl-D-aspartate (NMDA) and AMPA receptors preceded the formation of synapses. Glutamate receptors continued to be expressed strongly through DIV60. Cortical neurons aging in vitro displayed a complex profile of protein damage as identified by protein nitration. During cortical neuron aging, some proteins showed increased nitration, while other proteins showed decreased nitration. After exposure to DNA damaging agent, young (DIV5) and old (DIV60) cortical neurons activated apoptosis mechanisms, including caspase-3 cleavage and poly(ADP)-ribose polymerase inactivation. We show that cultured mouse cortical neurons can be maintained for long term. Cortical neurons display compartmental changes in the localization of synucleins during maturation in vitro. These neurons sustain protein nitration during aging and exhibit age-related variations in the biochemistry of neuronal apoptosis.     

Neurabin/protein phosphatase-1 complex regulates dendritic spine morphogenesis and maturation

The majority of excitatory synapses in the mammalian brain form on filopodia and spines, actin-rich membrane protrusions present on neuronal dendrites. The biochemical events that induce filopodia and remodel these structures into dendritic spines remain poorly understood. Here, we show that the neuronal actin- and protein phosphatase-1-binding protein, neurabin-I, promotes filopodia in neurons and nonneuronal cells. Neurabin-I actin-binding domain bundled F-actin, promoted filopodia, and delayed the maturation of dendritic spines in cultured hippocampal neurons. In contrast, dimerization of neurabin-I via C-terminal coiled-coil domains and association of protein phosphatase-1 (PP1) with neurabin-I through a canonical KIXF motif inhibited filopodia. Furthermore, the expression of a neurabin-I polypeptide unable to bind PP1 delayed the maturation of neuronal filopodia into spines, reduced the synaptic targeting of AMPA-type glutamate (GluR1) receptors, and decreased AMPA receptor-mediated synaptic transmission. Reduction of endogenous neurabin levels by interference RNA (RNAi)-mediated knockdown also inhibited the surface expression of GluR1 receptors. Together, our studies suggested that disrupting the functions of a cytoskeletal neurabin/PP1 complex enhanced filopodia and impaired surface GluR1 expression in hippocampal neurons, thereby hindering the morphological and functional maturation of dendritic spines.     

Long‐term culture of mouse cortical neurons as a model for neuronal development,aging, and death

A long‐term cell culture system was used to study maturation, aging, and death of cortical neurons. Mouse cortical neurons were maintained in culture in serum‐free medium (Neurobasal supplemented with B27) for 60 days in vitro (DIV). The levels of several proteins were evaluated by immunoblotting to demonstrate that these neurons matured by developing dendrites and synapses and remained continuously healthy for 60 DIV. During their maturation, cortical neurons showed increased or stable protein expression of glycolytic enzyme, synaptophysin, synapsin IIa, α and β synucleins, and glutamate receptors. Synaptogenesis was prominent during the first 15 days and then synaptic markers remained stable through DIV60. Very early during dendritic development at DIV3, β‐synuclein (but not α‐synuclein) was localized at the base of dendritic growth cones identified by MAP2 and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole (AMPA) receptor GluR1. In mature neurons, α and β synucleins colocalized in presynaptic axon terminals. Expression of N‐methyl‐D ‐aspartate (NMDA) and AMPA receptors preceded the formation of synapses. Glutamate receptors continued to be expressed strongly through DIV60. Cortical neurons aging in vitro displayed a complex profile of protein damage as identified by protein nitration. During cortical neuron aging, some proteins showed increased nitration, while other proteins showed decreased nitration. After exposure to DNA damaging agent, young (DIV5) and old (DIV60) cortical neurons activated apoptosis mechanisms, including caspase‐3 cleavage and poly(ADP)‐ribose polymerase inactivation. We show that cultured mouse cortical neurons can be maintained for long term. Cortical neurons display compartmental changes in the localization of synucleins during maturation in vitro. These neurons sustain protein nitration during aging and exhibit age‐related variations in the biochemistry of neuronal apoptosis. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 9–23, 2002     

罗格列酮对海马神经元树突发育的影响

罗格列酮(rosiglitazone,Rosig.)是噻唑烷二酮类(thiazolidinediones,TZDs)过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma,PPARγ)的激动剂,近年来,临床研究发现其具有神经保护作用,但对其作用机制目前仍没有完全研究清楚．利用活细胞成像的方法,观察罗格列酮对大鼠海马神经元树突丝和树突树发育的影响及其机制．结果显示,罗格列酮浓度依赖的增高神经元树突丝密度,对树突丝长度、运动速度并没有影响．此外,罗格列酮也不影响树突树的总分支、总长度以及各级分支的数目和长度．PPARγ 特异性拮抗剂GW9662完全阻断了罗格列酮介导的树突丝密度增高．结果表明罗格列酮可能通过PPARγ途径影响神经元的早期发育,这可能是罗格列酮发挥神经保护作用的潜在机制．     

Cell Surface Heparan Sulfate Proteoglycan Syndecan-2 Induces the Maturation of Dendritic Spines in Rat Hippocampal Neurons

Dendritic spines are small protrusions that receive synapses, and changes in spine morphology are thought to be the structural basis for learning and memory. We demonstrate that the cell surface heparan sulfate proteoglycan syndecan-2 plays a critical role in spine development. Syndecan-2 is concentrated at the synapses, specifically on the dendritic spines of cultured hippocampal neurons, and its accumulation occurs concomitant with the morphological maturation of spines from long thin protrusions to stubby and headed shapes. Early introduction of syndecan-2 cDNA into immature hippocampal neurons, by transient transfection, accelerates spine formation from dendritic protrusions. Deletion of the COOH-terminal EFYA motif of syndecan-2, the binding site for PDZ domain proteins, abrogates the spine-promoting activity of syndecan-2. Syndecan-2 clustering on dendritic protrusions does not require the PDZ domain-binding motif, but another portion of the cytoplasmic domain which includes a protein kinase C phosphorylation site. Our results indicate that syndecan-2 plays a direct role in the development of postsynaptic specialization through its interactions with PDZ domain proteins.     

Time-lapse imaging of dendritic spines in vitro

Dendritic spines are small protrusions present postsynaptically at approximately 90% of excitatory synapses in the brain. Spines undergo rapid spontaneous changes in shape that are thought to be important for alterations in synaptic connectivity underlying learning and memory. Visualization of these dynamic changes in spine morphology are especially challenging because of the small size of spines (approximately 1 microm). Here we describe a microscope system, based on a spinning-disk confocal microscope, suitable for imaging mature dendritic spines in brain slice preparations, with a time resolution of seconds. We discuss two commonly used in vitro brain slice preparations and methods for transfecting them. Preparation and transfection require approximately 1 d, after which slices must be cultured for at least 21 d to obtain spines of mature morphology. We also describe imaging and computer analysis routines for studying spine motility. These procedures require in the order of 2 to 4 h.     

Developmental downregulation of GABAergic drive parallels formation of functional synapses in cultured mouse neocortical networks

Networks of cortical neurons in vitro spontaneously develop synchronous oscillatory electrical activity at around the second week in culture. However, the underlying mechanisms and in particular the role of GABAergic interneurons in initiation and synchronization of oscillatory activity in developing cortical networks remain elusive. Here, we examined the intrinsic properties and the development of GABAergic and glutamatergic input onto presumed projection neurons (PNs) and large interneurons (L-INs) in cortical cultures of GAD67-GFP mice. Cultures developed spontaneous synchronous activity already at 5-7 days in vitro (DIV), as revealed by imaging transient changes in Fluo-3 fluorescence. Concurrently, spontaneous glutamate-mediated and GABA(A)-mediated postsynaptic currents (sPSCs) occured at 5 DIV. For both types of neurons the frequency of glutamatergic and GABAergic sPSCs increased with DIV, whereas the charge transfer of glutamatergic sPSCs increased and the charge transfer of GABAergic sPSCs decreased with cultivation time. The ratio between GABAergic and the overall charge transfer was significantly reduced with DIV for L-INs and PNs, indicating an overall reduction in GABAergic synaptic drive with maturation of the network. In contrast, analysis of miniature PSCs (mPSCs) revealed no significant changes of charge transfer with DIV for both types of neurons, indicating that the reduction in GABAergic drive was not due to a decreased number of functional synapses. Our data suggest that the global reduction in GABAergic synaptic drive together with more synaptic input to PNs and L-INs during maturation may enhance rhythmogenesis of the network and increase the synchronization at the level of population bursts.     

EphB receptors regulate dendritic spine morphogenesis through the recruitment/phosphorylation of focal adhesion kinase and RhoA activation

Dendritic filopodia are small protrusions on the surface of neuronal dendrites that transform into dendritic spines upon synaptic contact with axon terminals. The formation of dendritic spines is a critical aspect of synaptic development. Dendritic spine morphogenesis is characterized by filopodia shortening followed by the formation of mature mushroom-shaped spines. Here we show that activation of the EphB receptor tyrosine kinases in cultured hippocampal neurons by their ephrinB ligands induces morphogenesis of dendritic filopodia into dendritic spines. This appears to occur through assembly of an EphB-associated protein complex that includes focal adhesion kinase (FAK), Src, Grb2, and paxillin and the subsequent activations of FAK, Src, paxillin, and RhoA. Furthermore, Cre-mediated knock-out of loxP-flanked fak or RhoA inhibition blocks EphB-mediated morphogenesis of dendritic filopodia. Finally, EphB-mediated RhoA activation is disrupted by FAK knock-down. These data suggest that EphB receptors are upstream regulators of FAK in dendritic filopodia and that FAK-mediated RhoA activation contributes to assembly of actin filaments in dendritic spines.     

Protection by exogenous pyruvate through a mechanism related to monocarboxylate transporters against cell death induced by hydrogen peroxide in cultured rat cortical neurons

In cortical neurons cultured for 3 or 9 days in vitro (DIV), exposure to hydrogen peroxide (H(2)O(2)) led to a marked decrease in cell viability in a concentration-dependent manner at a concentration range of 10 microm to 1 mm irrespective of the duration between 6 and 24 h. However, H(2)O(2) was more potent in decreasing cellular viability in cortical neurons cultured for 9 DIV than in those for 3 DIV. Pyruvate was effective in preventing the neuronal cell death at 1 mm even when added 1-3 h after the addition of H(2)O(2). Semi-quantitative RT-PCR and western blotting analyses revealed significantly higher expression of both mRNA and protein for a particular monocarboxylate transporter (MCT) in neurons cultured for 9 DIV than in those for 3 DIV. A specific inhibitor of MCT significantly attenuated the neuroprotection by pyruvate in neurons cultured for 9 DIV, without markedly affecting that in neurons cultured for 3 DIV. These results suggest that vulnerability to H(2)O(2) may at least in part involve expression of particular MCT isoforms responsible for the bi-directional transport of pyruvate across cell surfaces in cultured rat cortical neurons.     

Sleep contributes to dendritic spine formation and elimination in the developing mouse somatosensory cortex

Sleep is maximal during early postnatal life when rapid and extensive synapse remodeling occurs. It remains unknown whether and how sleep affects synapse development and plasticity. Using transcranial two‐photon microscopy, we examined the formation and elimination of fluorescently labeled dendritic spines and filopodia of Layer 5 pyramidal neurons in the barrel cortex of 3‐week‐old mice during wakefulness and sleep. We observed high turnover of dendritic protrusions over 2 h in both wake and sleep states. The formation rate of dendritic spines or filopodia over 2 h was comparable between the two states. The elimination rate of dendritic spines or filopodia was lower during 2‐h wakefulness than during 2‐h sleep. Similar results were observed on dendritic protrusion dynamics over 12‐h light/dark cycle when mice spent more time asleep or awake. The substantial remodeling of dendritic protrusions during the sleep state supports the notion that sleep plays an important role in the development and plasticity of synaptic connections in the mouse cortex. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

A synaptic strategy for consolidation of convergent visuotopic maps

The mechanisms by which experience guides refinement of converging afferent pathways are poorly understood. We describe a vision-driven refinement of corticocollicular inputs that determines the consolidation of retinal and visual cortical (VC) synapses on individual neurons in the superficial superior colliculus (sSC). Highly refined corticocollicular terminals form 1-2 days after eye-opening (EO), accompanied by VC-dependent filopodia sprouting on proximal dendrites, and PSD-95 and VC-dependent quadrupling of functional synapses. Delayed EO eliminates synapses, corticocollicular terminals, and spines on VC-recipient dendrites. Awake recordings after EO show that VC and retina cooperate to activate sSC neurons, and VC light responses precede sSC responses within intervals promoting potentiation. Eyelid closure is associated with more protracted cortical visual responses, causing the majority of VC spikes to follow those of the colliculus. These data implicate spike-timing plasticity as a mechanism for cortical input survival, and support a cooperative strategy for retinal and cortical coinnervation of the sSC.     

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis ¹. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.     

Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis

Dendritic spines are small protrusions along dendrites where the postsynaptic components of most excitatory synapses reside in the mature brain. Morphological changes in these actin-rich structures are associated with learning and memory formation. Despite the pivotal role of the actin cytoskeleton in spine morphogenesis, little is known about the mechanisms regulating actin filament polymerization and depolymerization in dendritic spines. We show that the filopodia-like precursors of dendritic spines elongate through actin polymerization at both the filopodia tip and root. The small GTPase Rif and its effector mDia2 formin play a central role in regulating actin dynamics during filopodia elongation. Actin filament nucleation through the Arp2/3 complex subsequently promotes spine head expansion, and ADF/cofilin-induced actin filament disassembly is required to maintain proper spine length and morphology. Finally, we show that perturbation of these key steps in actin dynamics results in altered synaptic transmission.     

Proteins that promote filopodia stability, but not number, lead to more axonal-dendritic contacts

Dendritic filopodia are dynamic protrusions that are thought to play an active role in synaptogenesis and serve as precursors to spine synapses. However, this hypothesis is largely based on a temporal correlation between filopodia formation and synaptogenesis. We investigated the role of filopodia in synapse formation by contrasting the roles of molecules that affect filopodia elaboration and motility, versus those that impact synapse induction and maturation. We used a filopodia inducing motif that is found in GAP-43, as a molecular tool, and found this palmitoylated motif enhanced filopodia number and motility, but reduced the probability of forming a stable axon-dendrite contact. Conversely, expression of neuroligin-1 (NLG-1), a synapse inducing cell adhesion molecule, resulted in a decrease in filopodia motility, but an increase in the number of stable axonal contacts. Moreover, RNAi knockdown of NLG-1 reduced the number of presynaptic contacts formed. Postsynaptic scaffolding proteins such as Shank1b, a protein that induces the maturation of spine synapses, increased the rate at which filopodia transformed into spines by stabilization of the initial contact with axons. Taken together, these results suggest that increased filopodia stability and not density, may be the rate-limiting step for synapse formation.     

Synaptically Released Matrix Metalloproteinase Activity in Control of Structural Plasticity and the Cell Surface Distribution of GluA1-AMPA Receptors

Synapses are particularly prone to dynamic alterations and thus play a major role in neuronal plasticity. Dynamic excitatory synapses are located at the membranous neuronal protrusions called dendritic spines. The ability to change synaptic connections involves both alterations at the morphological level and changes in postsynaptic receptor composition. We report that endogenous matrix metalloproteinase (MMP) activity promotes the structural and functional plasticity of local synapses by its effect on glutamate receptor mobility and content. We used live imaging of cultured hippocampal neurons and quantitative morphological analysis to show that chemical long-term potentiation (cLTP) induces the permanent enlargement of a subset of small dendritic spines in an MMP-dependent manner. We also used a superresolution microscopy approach and found that spine expansion induced by cLTP was accompanied by MMP-dependent immobilization and synaptic accumulation as well as the clustering of GluA1-containing AMPA receptors. Altogether, our results reveal novel molecular and cellular mechanisms of synaptic plasticity.     

Filopodia: molecular architecture and cellular functions

Filopodia are thin, actin-rich plasma-membrane protrusions that function as antennae for cells to probe their environment. Consequently, filopodia have an important role in cell migration, neurite outgrowth and wound healing and serve as precursors for dendritic spines in neurons. The initiation and elongation of filopodia depend on the precisely regulated polymerization, convergence and crosslinking of actin filaments. The increased understanding of the functions of various actin-associated proteins during the initiation and elongation of filopodia has provided new information on the mechanisms of filopodia formation in distinct cell types.     

Filopodia are required for cortical neurite initiation

Extension of neurites from a cell body is essential to form a functional nervous system; however, the mechanisms underlying neuritogenesis are poorly understood. Ena/VASP proteins regulate actin dynamics and modulate elaboration of cellular protrusions. We recently reported that cortical axon-tract formation is lost in Ena/VASP-null mice and Ena/VASP-null cortical neurons lack filopodia and fail to elaborate neurites. Here, we report that neuritogenesis in Ena/VASP-null neurons can be rescued by restoring filopodia formation through ectopic expression of the actin nucleating protein mDia2. Conversely, wild-type neurons in which filopodia formation is blocked fail to elaborate neurites. We also report that laminin, which promotes the formation of filopodia-like actin-rich protrusions, rescues neuritogenesis in Ena/VASP-deficient neurons. Therefore, filopodia formation is a key prerequisite for neuritogenesis in cortical neurons. Neurite initiation also requires microtubule extension into filopodia, suggesting that interactions between actin-filament bundles and dynamic microtubules within filopodia are crucial for neuritogenesis.     

Effect of HDAC Inhibitors on Neuroprotection and Neurite Outgrowth in Primary Rat Cortical Neurons Following Ischemic Insult

Histone deacetylase inhibitors (HDACi)—valproic acid (VPA) and trichostatin A (TSA) promote neurogenesis, neurite outgrowth, synaptic plasticity and neuroprotection. In this study, we investigated whether VPA and TSA promote post-ischemic neuroprotection and neuronal restoration in rat primary cortical neurons. On 6 days in vitro (DIV), cortical neurons were exposed to oxygen-glucose deprivation for 90 min. Cells were returned to normoxic conditions and cultured for 1, 3, or 7 days with or without VPA and TSA. Control cells were cultured in normoxic conditions only. On 7, 9, and 13 DIV, cells were measured neurite outgrowth using the Axiovision program and stained with Tunel staining kit. Microtubule associated protein-2 immunostaining and tunel staining showed significant recovery of neurite outgrowth and post-ischemic neuronal death by VPA or TSA treatment. We also determined levels of acetylated histone H3, PSD95, GAP 43 and synaptophysin. Significant increases in all three synaptic markers and acetylated histone H3 were observed relative to non-treated cells. Post-ischemic HDACi treatment also significantly raised levels of brain derived neurotrophic factor (BDNF) expression and secreted BDNF. Enhanced BDNF expression by HDACi treatment might have been involved in the post-ischemic neuroprotection and neuronal restorative effects. Our findings suggest that both VPA and TSA treatment during reoxygenation after ischemia may help post-ischemic neuroprotection and neuronal regeneration via increased BDNF expression and activation.     

Maturation-dependent reduced responsiveness of intracellular free Ca2+ ions to repeated stimulation by N-methyl-D-aspartate in cultured rat cortical neurons

In contrast to other ionotropic glutamate receptors, N-methyl-d-aspartate (NMDA) receptor channels are rather stable after the simulation. Brief exposure to NMDA at 50 microM rapidly increased the fluorescence intensity for increased intracellular free Ca(2+) levels in a reversible- and concentration-dependent manner in rat cortical neurons cultured for 3-15 days in vitro (DIV), while EC(50) values were significantly decreased in proportion to cellular maturation from 3 to 15 DIV. Although a constant increase was persistently seen in the fluorescence throughout the sustained exposure to NMDA for 60 min irrespective of the cell maturation from 3 to 15 DIV, the second brief exposure for 5 min resulted in a less efficient increase in the fluorescence than that found after the first brief exposure for 5 min in a manner dependent on intervals between the two repetitive brief exposures. In vitro maturation significantly shortened the interval required for the reduced responsiveness to the second brief exposure, while in immature neurons prolonged intervals were required for the reduced responsiveness to the second brief exposure to NMDA. Moreover, brief exposure to NMDA led to a marked decrease in immunoreactivity to extracellular loop of NR1 subunit in cultured neurons not permeabilized in proportion to the time after washing. These results suggest that cellular maturation would facilitate the desensitization process to repeated stimulation by NMDA, without markedly affecting that to sustained stimulation, through a mechanism related to the decreased number of NMDA receptors expressed at cell surfaces in cultured rat cortical neurons.