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.     

Zinc-induced neuronal death in cortical neurons.

Although Zn2+ is normally stored and released in the brain, excessive exposure to extracellular Zn2+ can be neurotoxic. The purpose of the present study was to determine the type of neuronal cell death, necrosis versus apoptosis, induced by Zn2+ exposure. Addition of 10-50 microM ZnCl2 to the bathing medium of murine neuronal and glial cell cultures induced, over the next 24 hrs., Zn2+-concentration-dependent neuronal death; some glial death also occurred with Zn2+ concentrations above 30 microM. The neuronal death induced by 20 microM Zn2+ was characterized by coarse chromatin condensation, the formation of apoptotic bodies, and internucleosomal DNA fragmentation. It was attenuated in cortical cell cultures prepared from mice null for the bax gene, and by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-CH2F (ZVAD, 100 microM), but not by the NMDA receptor antagonist, D-2-amino-5-phosphonovalerate (D-APV, 200 microM ). In contrast, the neuronal death induced by 50 microM Zn2+ was characterized by plasma membrane disruption and random DNA fragmentation; this death was attenuated by D-APV, but exhibited little sensitivity to ZVAD or deletion of bax. These results suggest that Zn2+ can induce cell death with characteristics of either apoptosis or necrosis, depending on the intensity of the Zn2+ exposure.     

The effect of iron on mammalian cortical neurons in culture

We added iron in the ferric form to predominantly neuronal, cortical cell cultures, and determined clonazepam-displaceable [³H]diazepam binding, choline acetyltransferase activity, high-affinity [³H]GABA uptake, and glutamic acid decarboxylase activity. Chronic exposure (14 days) to low concentrations (0.01, 0.04, and 0.1 g/ml) of added ferric iron resulted in a significant decrease in each of the measures studied.     

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     

Effects of simulated microgravity on the development and maturation of dissociated cortical neurons

Although a wealth of evidence supports the hypothesis that some functions of the nervous system may be altered during exposure to microgravity, the possible changes in basic neuronal physiology are not easy to assess. Indeed, few studies have examined whether microgravity affects the development of neurons in culture. In the present study, a suspension of dissociated cortical cells from rat embryos were exposed to 24 h of simulated microgravity before plating in a normal adherent culture system. Both preexposed and control cells were used after a period of 7-10 d in vitro. The vitality and the level of reactive oxygen species of cultures previously exposed did not differ from those of normal cultures. Cellular characterization by immunostaining with a specific antibody displayed normal neuronal phenotype in control cells, whereas pretreatment in simulated microgravity revealed an increase of glial fibrillary acidic protein fluorescence in the elongated stellate glial cells. Electrophysiological recording indicated that the electrical properties of neurons preexposed were comparable with those of controls. Overall, our results indicate that a short time of simulated microgravity preexposure does not affect dramatically the ability of dissociated neural cells to develop and differentiate in an adherent culture system.     

Acetylcholine synthesis by chick spinal cord neurons in dissociated cell culture

Acetylcholine (ACh) synthesis was examined in cultures of chick spinal cord cells to follow the development of the cholinergic neurons. The cells, prepared from 4-day-old embryonic chick spinal cords, were grown either alone in dissociated cell cultures (SC cultures) or with chick myotubes (SC-M cultures). ACh synthesis was measured by incubating the cultures in [³Hcholine and using high-voltage paper electrophoresis to quantitate the amount of [³H]ACh present in cell extracts prepared from the labeled cultures. The amount of [³H]ACh synthesized in SC-M cultures was strictly proportional to the number of spinal cord cells used to prepare the cultures, and was linear with the time of incubation in [³H]choline for periods up to 1 hr. Maximal rates of synthesis were observed with [³H]choline concentrations in excess of 100 μM. Such rates for 1-week-old SC-M cultures were approximately 10–20 pmoles of [³H]ACh/hr/10⁵ spinal cord cells. Studies on the stability of the intracellular [³H]ACh revealed the presence of a major pool with a half-time of 20–30 min. A second, small pool decayed more rapidly. No detectable [³H]ACh was spontaneously released from the cells, suggesting that most of the decay represented intracellular degradation. Development of cholinergic neurons as monitored by [³H]ACh synthesis continued over a 2-week period in SC-M cultures and paralleled general cell growth. When examined at 1 week, SC-M cultures had about a 50% greater capacity for [³H]ACh synthesis and 60% more choline acetyltransferase activity than did SC cultures. No difference was observed in the stability of the [³H]ACh formed for the two types of cultures at 1 week, and no further difference was observed in the rates of [³H]ACh synthesis at 2 weeks. Growth of SC cultures in medium containing different amounts of chick embryo extract (2–10%) or in medium with fetal calf serum (10%) instead of extract produced only small differences in the measured rates of [³H]ACh synthesis. Thus chick spinal cord cells can undergo some of the early stages of cholinergic development in cell culture without sustained contact with skeletal myotubes, one of the normal postsynaptic target cells for the cholinergic neuron population. No absolute requirement for muscle factors was revealed under these conditions, although such factors may have been provided by other cell types in the spinal cord population or may have been present in other additions to the culture medium.     

Synchronized dynamics of cortical neurons with time-delay feedback

The dynamics of three mutually coupled cortical neurons with time delays in the coupling are explored numerically and analytically. The neurons are coupled in a line, with the middle neuron sending a somewhat stronger projection to the outer neurons than the feedback it receives, to model for instance the relay of a signal from primary to higher cortical areas. For a given coupling architecture, the delays introduce correlations in the time series at the time-scale of the delay. It was found that the middle neuron leads the outer ones by the delay time, while the outer neurons are synchronized with zero lag times. Synchronization is found to be highly dependent on the synaptic time constant, with faster synapses increasing both the degree of synchronization and the firing rate. Analysis shows that pre-synaptic input during the inter-spike interval stabilizes the synchronous state, even for arbitrarily weak coupling, and independent of the initial phase. The finding may be of significance to synchronization of large groups of cells in the cortex that are spatially distanced from each other.     

The effects of cholinergic neuromodulation on neuronal phase-response curves of modeled cortical neurons

The response of an oscillator to perturbations is described by its phase-response curve (PRC), which is related to the type of bifurcation leading from rest to tonic spiking. In a recent experimental study, we have shown that the type of PRC in cortical pyramidal neurons can be switched by cholinergic neuromodulation from type II (biphasic) to type I (monophasic). We explored how intrinsic mechanisms affected by acetylcholine influence the PRC using three different types of neuronal models: a theta neuron, single-compartment neurons and a multi-compartment neuron. In all of these models a decrease in the amount of a spike-frequency adaptation current was a necessary and sufficient condition for the shape of the PRC to change from biphasic (type II) to purely positive (type I).     

Survival and development in culture of dissociated parasympathetic neurons from ciliary ganglia

Parasympathetic neurons from avian embryonic ciliary ganglia survive in low density culture when neurons are free from contact with other cells. A charged substratum, polyornithine, and a conditioned medium permit cell survival and vigorous neurite formation. The heart-conditioned medium must be present continuously and is active after dialysis. Neurites elongate rapidly, branch extensively, and follow patterns of charged substratum provided in the culture dish.     

Characterization of cortical neuronal and glial alterations during culture of organotypic whole brain slices from neonatal and mature mice

Background

Organotypic brain slice culturing techniques are extensively used in a wide range of experimental procedures and are particularly useful in providing mechanistic insights into neurological disorders or injury. The cellular and morphological alterations associated with hippocampal brain slice cultures has been well established, however, the neuronal response of mouse cortical neurons to culture is not well documented.

Methods

In the current study, we compared the cell viability, as well as phenotypic and protein expression changes in cortical neurons, in whole brain slice cultures from mouse neonates (P4–6), adolescent animals (P25–28) and mature adults (P50+). Cultures were prepared using the membrane interface method.

Results

Propidium iodide labeling of nuclei (due to compromised cell membrane) and AlamarBlue™ (cell respiration) analysis demonstrated that neonatal tissue was significantly less vulnerable to long-term culture in comparison to the more mature brain tissues. Cultures from P6 animals showed a significant increase in the expression of synaptic markers and a decrease in growth-associated proteins over the entire culture period. However, morphological analysis of organotypic brain slices cultured from neonatal tissue demonstrated that there were substantial changes to neuronal and glial organization within the neocortex, with a distinct loss of cytoarchitectural stratification and increased GFAP expression (p<0.05). Additionally, cultures from neonatal tissue had no glial limitans and, after 14 DIV, displayed substantial cellular protrusions from slice edges, including cells that expressed both glial and neuronal markers.

Conclusion

In summary, we present a substantial evaluation of the viability and morphological changes that occur in the neocortex of whole brain tissue cultures, from different ages, over an extended period of culture.     

High affinity choline uptake by spinal cord neurons in dissociated cell culture

Spinal cord-myotube cultures prepared with dissociated embryonic chick spinal cord cells and myoblasts exhibit a high affinity mechanism for accumulating choline. The uptake mechanism has a K_m of 3.4 ± 0.5 μM (7) and a V_m of 40.0 ± 0.1 (7) pmoles/min/mg of protein (mean ± SEM; number of determinations in parentheses). It is inhibited 90–95% by 10 μM hemicholinium-3 or by replacement of Na⁺ in the incubation solution with Li⁺. Part of the choline (10–20%) accumulated by the high affinity system is converted to acetylcholine (ACh). Uptake studies on spinal cord cells and myotubes grown separately demonstrate that the spinal cord cells can account for virtually all of the choline uptake observed in the mixed cultures. Myotubes are unnecessary under these conditions for the expression of the high affinity uptake mechanism by spinal cord cells. Neurons are not the only cell type in culture to exhibit high affinity choline uptake. Chick fibroblasts in both rapidly growing and stationary phase can accumulate choline with kinetics similar to those observed for the high affinity uptake by spinal cord cells. Little if any of the choline accumulated by fibroblasts, however, is converted to ACh. In most uptake studies with spinal cord cells, contributions from fibroblasts were minimized by carrying out the analysis at a time when few non-neuronal cells were present in the spinal cord cultures. These observations suggest that a population of chick central nervous system (CNS) neurons develop a high affinity choline uptake mechanism in cell culture that has many of the properties described for uptake by cholinergic neurons in vivo and that at least part of the choline accumulated by the system can be used for neurotransmitter synthesis.     

Synaptic connections in a dissociated mixed culture of rat dorsal root ganglion and spinal cord neurons

Postsynaptic currents and action potentials recorded from neurons in a mixed culture of rat dorsal root ganglion and spinal cord cells are described. The existence of mutual synaptic connections between the above two types of neurons is demonstrated. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 358–360, July–August, 2006.     

Dendritic spines of developing rat cortical neurons in culture

The formation of spines and their association with synapses were examined in developing cultured rat cortical neurons using fluorescence labeling techniques. Small protrusions were found on the processes of cultured cortical neurons after seven days in vitro (DIV), and the density of protrusions almost halved during the second week in vitro, after which it remained unchanged throughout the third week in vitro. The proportion of protrusions associated with the accumulation of the presynaptic marker, synaptophysin, increased steadily from <5% at 7 DIV to approximately 50% at 21 DIV. Based on the absence or presence of an enlargement at the end, protrusions on processes were further divided into filopodia and spines, respectively. The percentage of protrusions that were classified as spines increased steadily from approximately 5% at 3-4 DIV to approximately 80% at 18-20 DIV. The percentage of spines associated with synaptophysin accumulation increased gradually as the cortical neurons developed in vitro, reaching a plateau of approximately 40% after two weeks. However, the percentage of filopodia associated with synaptophysin accumulation never exceeded 5% during the first three weeks in vitro. Double-label staining the microfilaments and beta-tubulin or phosphorylated neurofilament H of cultured neurons further revealed many spines without any nearby axon-like processes. These findings suggest that spines are the dominant form of protrusion on the processes of more mature cortical neurons, that spines are the preferential sites where synapses reside, and that maintaining constant contact with axons is not essential for the formation of spines in cultured cortical neurons.     

Neurotrophic and neurotoxic effects of zinc on neonatal cortical neurons

Although zinc exerts direct neurotoxic action, this metal is also essential for the activity of numerous biological systems and zinc deficiency has been associated with various pathologies. We investigated the cellular responses and neuronal viability following exposure to different concentrations of zinc in primary cultures of neonatal rat cortical neurons. Higher concentrations of zinc (0.15 and 0.2 mM) triggered excessive zinc influx, glutathione depletion and ATP loss leading to necrotic neuronal death. In contrast, lower concentrations of zinc (0.05 and 0.1 mM) attenuated serum-deprivation induced apoptotic neuronal death. The antiapoptotic action of low amounts of zinc was found both in mixed cultures and neuron-enriched cultures indicating the independence of glial mediator. Neurotrophic action was not accompanied by significant alteration in those cellular responses but required chelatable zinc. The N-methyl-D-aspartate (NMDA) antagonist, MK-801, mimicked the beneficial effect of zinc in protecting neuronal death. Moreover, both MK-801 and zinc eliminated NMDA-induced neuronal injury. The results suggest that zinc is an intrinsic factor for neuron survival and exogenous zinc, in low amounts, is an active neuroprotectant against serum deprivation in part through the antagonism of NMDA receptor activation.     

The Kv4.2 mediates excitatory activity-dependent regulation of neuronal excitability in rat cortical neurons

Neuronal excitability can cooperate with synaptic transmission to control the information storage. This regulation of neuronal plasticity can be affected by alterations in neuronal inputs and accomplished by modulation of voltage-dependent ion channels. In this study, we report that enhanced excitatory input negatively regulated neuronal excitability. Enhanced excitatory input by glutamate, electric field stimulation or high K⁺ increased transient outward K⁺ current, whereas did not affect the delayed rectifier K⁺ current in rat cultured cortical neurons. Both the voltage-dependent K⁺ channel 4.2 and 4.3 subunits contributed to the increase. The increase in the K⁺ current density by Kv4.2 was ascribed to its cytoplasmic membrane translocation, which was mediated by NMDA type of glutamate receptor. Furthermore, enhanced excitatory input inhibited neuronal excitability. Taken together, our results suggest that excitatory neurotransmission affects neuronal excitability via the regulation of the K⁺ channel membrane translocation.     

Nerve growth factor-independent development of embryonic mouse sympathetic neurons in dissociated cell culture

Although ganglia from neonatal mouse sympathetic ganglia require nerve growth factor (NGF) for survival in culture, explanted sympathetic ganglia from early embryonic stages do not require added NGF for survival and growth. To determine whether the change in growth factor requirement is due to changes in the neurons themselves, to variations in neuronal populations, or to changes in nonneuronal cells, we examined the response to growth factors by dissociated sympathetic neurons at various stages of development. Results indicate that neurons from the 14-day gestational (E14) superior cervical ganglion (SCG) do not require NGF for initial survival and neurite extension, but do require the conditioned medium neurite extension factor, CMF. By 2 to 3 days thereafter, whether in vivo or in culture, most neurons have developed a requirement for NGF for survival in culture. During the same period, there is a concomitant increase in responsiveness to NGF alone as a trophic agent. Changes in response to NGF are not due to changes in NGF content of ganglia, to interactions in culture with nonneuronal cells, or to age-related differences in NGF requirements for maximum survival. The changes in growth factor requirements may be related to mechanisms regulating specificity of nerve-target connections.     

Identification and characterization of a population of motile neurons in long-term cortical culture

The specific phenotypes and progression to maturity of primary cortical neurons in long-term culture correlate well with neurons in vivo. Utilizing a model of neuronal injury in long-term cultures at 21 days in vitro (DIV), we have identified a distinct population of neurons that translocate into the injury site. 5-bromo-2'-deoxyUridine (BrdU) incorporation studies demonstrated that neurons with the capacity to translocate were 21 days old. However, this motile ability is not consistent with the traditional view of the maturation and structural stability of neurons in long-term culture. Therefore, we examined the neurons' cytoskeletal profile using immunocytochemistry, to establish relative stage of maturation and phenotype. Expression of marker proteins including beta-III-tubulin, alpha-internexin, NF-L and NF-M, tau and L1 indicated the neurons were differentiated, and in some cases polarized. The neurons did not immunolabel with NF-H or MAP2, which might suggest they had not reached the level of maturity of other neurons in culture. They did not express the microtubule-associated migration marker doublecortin (DCX). Cytoskeletal disrupting agents were used to further investigate the role of the microtubule cytoskeleton in translocation, and microtubule destabilization significantly enhanced aspects of their motility. Finally, molecular guidance cues affected their motility in a similar manner to that reported for both axon guidance and early neuron migration. Therefore, this study has identified and characterized a population of motile neurons in vitro that have the capacity to migrate into a site of injury. These studies provide new information on the structurally dynamic features of subsets of neurons.     

Epidermal growth factor induces oxidative neuronal injury in cortical culture

Recently, we have demonstrated that certain neurotrophic factors can induce oxidative neuronal necrosis by acting at the cognate tyrosine kinase-linked receptors. Epidermal growth factor (EGF) has neurotrophic effects via the tyrosine kinase-linked EGF receptor (EGFR), but its neurotoxic potential has not been studied. Here, we examined this possibility in mouse cortical culture. Exposure of cortical cultures to 1-100 ng/ml EGF induced gradually developing neuronal death, which was complete in 48-72 h; no injury to astrocytes was noted. Electron microscopic findings of EGF-induced neuronal death were consistent with necrosis; severe mitochondrial swelling and disruption of cytoplasmic membrane occurred, whereas nuclei appeared relatively intact. The EGF-induced neuronal death was accompanied by increased free radical generation and blocked by the anti-oxidant Trolox. Suggesting mediation by the EGFR, an EGFR tyrosine kinase-specific inhibitor, C56, attenuated EGF-induced neuronal death. In addition, inhibitors of extracellular signal-regulated protein kinase 1/2 (Erk-1/2) (PD98056), protein kinase A (H89), and protein kinase C (GF109203X) blocked EGF-induced neuronal death. A p38 mitogen-activated protein kinase inhibitor (SB203580) or glutamate antagonists (MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione) showed no protective effect. The present results suggest that prolonged activation of the EGFR may trigger oxidative neuronal injury in central neurons.     

Network dynamics and synchronous activity in cultured cortical neurons

Neurons extracted from specific areas of the Central Nervous System (CNS), such as the hippocampus, the cortex and the spinal cord, can be cultured in vitro and coupled with a micro-electrode array (MEA) for months. After a few days, neurons connect each other with functionally active synapses, forming a random network and displaying spontaneous electrophysiological activity. In spite of their simplified level of organization, they represent an useful framework to study general information processing properties and specific basic learning mechanisms in the nervous system. These experimental preparations show patterns of collective rhythmic activity characterized by burst and spike firing. The patterns of electrophysiological activity may change as a consequence of external stimulation (i.e., chemical and/or electrical inputs) and by partly modifying the "randomness" of the network architecture (i.e., confining neuronal sub-populations in clusters with micro-machined barriers). In particular we investigated how the spontaneous rhythmic and synchronous activity can be modulated or drastically changed by focal electrical stimulation, pharmacological manipulation and network segregation. Our results show that burst firing and global synchronization can be enhanced or reduced; and that the degree of synchronous activity in the network can be characterized by simple parameters such as cross-correlation on burst events.     

Zinc toxicity on neonatal cortical neurons: involvement of glutathione chelation

Several mechanisms have been implicated in pathological neuronal death including zinc neurotoxicity, calcium excitotoxicity and oxidative injury. Glutathione (GSH) serves to provide reducing equivalents for the maintenance of oxidant homeostasis, and also plays roles in intracellular and intercellular signaling in the brain. We investigated the role of GSH homeostasis in the neurotoxic action of zinc using both mixed cortical cultures containing neurons and glia, and cortical neurons prepared from 1-day-old rats. Zinc caused neuronal cell death in a concentration-dependent manner. In parallel, a high concentration of zinc depleted GSH, in a time-dependent manner, preceding the onset of neuronal damage. Depletion of GSH by diethylmaleate injured neurons and exacerbated zinc-induced death. In contrast, replenishment of GSH attenuated zinc neurotoxicity. The thiol-containing compounds N-acetylcysteine and GSH chemically chelated zinc leading to decreases in the influx of zinc, the fall in GSH level and neuronal death. Interestingly, the glycolytic substrate pyruvate, but not lactate, chelated zinc concentration dependently and prevented its toxicity. On the other hand, pyrrolidine dithiocarbamate, serving as a zinc chaperon, enhanced its entry and toxicity. The results suggest that zinc non-enzymatically depleted GSH, an intrinsic factor for neuron survival, leading to activation of the cellular death signal and eventually neuronal death.