Embryonic Hippocampal Cultures Research Articles

Methods for shipping live primary cortical and hippocampal neuron cultures from postnatal mice

Primary neuronal cultures have proven to be a powerful tool for studying mechanisms in neuroscience. It is technically challenging and expensive to reproduce high quality viable neuronal cultures. Laboratories that are not experienced or equipped to prepare primary neuron cultures may have difficulty producing consistent cultures for experiments. It has previously been shown that live rat embryonic hippocampal cultures can be shipped from laboratories that produce them. Here, we show that variations to this procedure allow for shipping postnatal mouse cultures of hippocampal and cortical primary neurons using standard commercial couriers. We also show that after shipping, primary neurons are viable, express synaptic markers, and demonstrate physiological activity, making them relevant models over immortalized cell lines. Among the many applications of this technique would be the preparation of cultured neurons from transgenic mouse lines in one laboratory and sharing them with distant collaborators, reducing variability.

Cep215 is essential for morphological differentiation of astrocytes

Cep215 (also known as Cdk5rap2) is a centrosome protein which is involved in microtubule organization. Cep215 is also placed at specific subcellular locations and organizes microtubules outside the centrosome. Here, we report that Cep215 is involved in morphological differentiation of astrocytes. Cep215 was specifically localized at the glial processes as well as centrosomes in developing astrocytes. Morphological differentiation of astrocytes was suppressed in the Cep215-deleted P19 cells and in the Cep215-depleted embryonic hippocampal culture. We confirm that the microtubule organizing function of Cep215 is critical for the glial process formation. However, Cep215 is not involved in the regulation of cell proliferation nor cell specification. Based on the results, we propose that Cep215 organizes microtubules for glial process formation during astrocyte differentiation.

Sex-specific features of spine densities in the hippocampus

Previously, we found that in dissociated hippocampal cultures the proportion of large spines (head diameter ≥ 0.6 μm) was larger in cultures from female than from male animals. In order to rule out that this result is an in vitro phenomenon, we analyzed the density of large spines in fixed hippocampal vibratome sections of Thy1-GFP mice, in which GFP is expressed only in subpopulations of neurons. We compared spine numbers of the four estrus cycle stages in females with those of male mice. Remarkably, total spine numbers did not vary during the estrus cycle, while estrus cyclicity was evident regarding the number of large spines and was highest during diestrus, when estradiol levels start to rise. The average total spine number in females was identical with the spine number in male animals. The density of large spines, however, was significantly lower in male than in female animals in each stage of the estrus cycle. Interestingly, the number of spine apparatuses, a typical feature of large spines, did not differ between the sexes. Accordingly, NMDA-R1 and NMDA-R2A/B expression were lower in the hippocampus and in postsynaptic density fractions of adult male animals than in those of female animals. This difference could already be observed at birth for NMDA-R1, but not for NMDA-R2A/B expression. In dissociated embryonic hippocampal cultures, no difference was seen after 21 days in culture, while the difference was evident in postnatal cultures. Our data indicate that hippocampal neurons are differentiated in a sex-dependent manner, this differentiation being likely to develop during the perinatal period.

Análise da evolução de culturas neurais

This paper analyses the electrical activity recorded in an experiment involving the maturation of dissociated embryonic hippocampal cultures, using Multielectrode Array (MEA) devices, starting from the moment of the culture’s placement in the device until its death, a period of 0 to 88 days in vitro (DIV). Classical “spike” analysis is applied to three different cultures, showing the progression of “spikes” and “bursts”. Particular attention is devoted to the last step associated with cellular death, as an unexpected rise in electrical activity amplitude is observed.

Neurite Outgrowth of Neural Progenitors in Presence of Inhibitory Proteoglycans

Attempts to promote host regeneration after spinal cord injury (SCI) have often resulted in poor axon extension due to formation of a glial scar, which creates a dense physical barrier around the injury and contains molecules that inhibit regeneration and repair of adult injured axons. Previous studies have shown that, while transplants of multipotent neural stem cells (NSC) integrate poorly in the injury site, the use of neuronal-restricted precursor cells (NRP) together with glial-restricted precursor cells (GRP) allow differentiation and integration of neurons, possibly because NRP are able to overcome chondroitin sulfate proteoglycan (CSPG) inhibition. To investigate this possibility, we grew mixed cultures of NRP/GRP on CSPG at inhibitory concentrations, using embryonic hippocampal cultures as controls. We found that NRP/GRP grown on CSPG survive and differentiate into neurons with no significant changes in neurite length, relative to growth on control polylysine substrate, and in contrast to a significant inhibition of axon growth in hippocampal cultures grown on CSPG-coated substrate. There was, however, a significant decrease in neurite number and branching in both cultures, indicating that CSPG also has important effects on neuronal morphology. These data suggest that embryonic neurons supported by glial cells derived from NRP/GRP transplants are less sensitive to inhibitory effects of CSPG in the glial scar, and are thus an appropriate source for neuronal cell replacement and reconnection of damaged circuits after SCI.

Potential antiviral drug pueraria crude extract and puerarin protect against ethanol-induced cytotoxicity in embryonic mouse hippocampal cultures

To examine whether Chinese medical herb Pueraria crude extract (CP) and standard of pure puerarin (SP) possess the same neuroprotective effects during concomitant ethanol (EtOH) treatment. Hippocampus cultures were prepared from mice at gestational age of 18 day. Cell viability was measured by MTT assay. RT-PCR was employed to determine mRNA expression of superoxide dismutase (SOD). As measured by MTT assay, supplementation with 15 mg/L CP or 10 mg/L SP afforded neuroprotection against all EtOH concentrations (50, 200 and 350 mmol/L, respectively) in embryonic hippocampal culture system. In addition, both 15 mg/L CP and 10 mg/L SP could decrease expression of SOD at mRNA level. This study suggests that CP and SP could decrease oxidative stress induced by ethanol treatment by the decreased expression of SOD at mRNA level, and demonstrates antioxidative neuroprotective effect of CP and SP against developmental ethanol exposure in vitro.

Analysis of SNAP-25 immunoreactivity in hippocampal inhibitory neurons during development in culture and in situ

Synaptosomal associated protein of 25 kDa (SNAP-25) is a component of the soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex which plays a central role in synaptic vesicle exocytosis. We have previously demonstrated that adult rat hippocampal GABAergic synapses, both in culture and in brain, are virtually devoid of SNAP-25 immunoreactivity and are less sensitive to the action of botulinum toxin type A, which cleaves this SNARE protein [Neuron 41 (2004) 599]. In the present study, we extend our findings to the adult mouse hippocampus and we also provide demonstration that hippocampal inhibitory synapses lacking SNAP-25 labeling belong to parvalbumin-, calretinin- and cholecystokinin-positive interneurons. A partial colocalization between SNAP-25 and glutamic acid decarboxylase is instead detectable in developing mouse hippocampus at P0 and, at a lesser extent, at P5. In rat embryonic hippocampal cultures at early developmental stages, SNAP-25 immunoreactivity is detectable in a percentage of GABAergic neurons, which progressively reduces with time in culture. Consistent with the presence of the substrate, botulinum toxin type A is partially effective in inhibiting synaptic vesicle recycling in immature GABAergic neurons. Since SNAP-25, beside its role as a SNARE protein, is involved in additional processes, such as neurite outgrowth and regulation of calcium dynamics, the presence of higher levels of the protein at specific stages of neuronal differentiation may have implications for the construction and for the functional properties of brain circuits.

Expression and Function of Neurotrophins and Their Receptors in Cultured Human Keratinocytes

Whereas nerve growth factor has been extensively studied in human keratinocytes, little is known on the role of other members of the neurotrophin family. We investigated the expression and function of neurotrophins and neurotrophin receptors in cultured human keratinocytes. We demonstrated by reverse transcription-polymerase chain reaction that keratinocytes synthesize neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5. These cells also express tyrosinase kinase A and C, the nerve growth factor and neuro-trophin-3 high-affinity receptors, respectively. On the other hand, only the truncated extracellular isoform of tyrosinase kinase B, the high-affinity brain-derived neurotrophic factor and neurotrophin-4/5 receptor, is detected in keratinocytes. Moreover, neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5 proteins are secreted by human keratinocytes at low levels. Keratinocyte stem cells synthesize the highest amounts of nerve growth factor, while they secrete higher levels of nerve growth factor as compared with transit amplifying cells. Neurotrophin-3 stimulates keratinocyte proliferation, where brain-derived neurotrophic factor or neurotrophin-4/5 does not exert any effect on keratinocyte proliferation. Addition of neurotrophin-3 slightly upregulates the secretion of nerve growth factor, whereas nerve growth factor strongly augments neurotrophin-3 release. Ultraviolet B irradiation downregulates nerve growth factor, whereas it augments neurotrophin-3 and neurotrophin-4/5 protein levels. Ultraviolet A irradiation increases the level of neurotrophin-3, whereas it does not exert any effect on the other neurotrophins. Finally, neurotrophins other than nerve growth factor fail to protect human keratinocytes from ultraviolet B-induced apoptosis. This work delineates a functional neurotrophin network, which may contribute to epidermal homeostasis.

Inhibition of neuronal maturation in primary hippocampal neurons from tau deficient mice.

Conflicting evidence supports a role for tau as an essential neuronal cytoskeletal protein or as a redundant protein whose function can be fulfilled by other microtubule-associated proteins. To investigate the function of tau in axonogenesis, we created tau deficient mice by disrupting the TAU gene. The engineered mice do not express the tau protein, appear physically normal and are able to reproduce. In contrast to a previously reported tau knockout mouse, embryonic hippocampal cultures from tau deficient mice show a significant delay in maturation as measured by axonal and neuritic extensions. The classic technique of selectively enhancing axonal growth by growth on laminin substrates failed to restore normal neuronal maturation of tau knockout neurons. By mating human TAU-gene transgenic and tau knockout mice, we reconstituted tau-deficient neurons with human tau proteins and restored a normal pattern of axonal growth and neuronal maturation. The ability of human tau proteins to rescue tau-deficient mouse neurons confirms that tau expression affects the rate of neurite extension.

Vitamin E and β-carotene protect against ethanol combined with ischemia in an embryonic rat hippocampal culture model of fetal alcohol syndrome

Neurodevelopmental damage can occur as a result of in utero exposure to alcohol. Oxidative stress processes are one of many proposed mechanisms thought to contribute to nervous system dysfunction characterized in fetal alcohol syndrome (FAS). Therefore, this study examined neuroprotective effects of antioxidant supplementation during ethanol (EtOH) treatment (0, 200, 400, 800 or 1600 mg/dl) combined with concomitants of EtOH exposure: acute (2-h) ischemia (aISCH) and chronic (16-h) hypoglycemia (cHG). The antioxidants vitamin E and β-carotene protected embryonic hippocampal cultures against 0–1600 mg/dl EtOH/aISCH/cHG treatments. In addition, neuronal viability, as measured by MTT ((3,4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; 5 mg/ml)), was equal to untreated cultures when supplemented with vitamin E or β-carotene at 0–800 mg/dl or 0–200 mg/dl EtOH/aISCH/cHG, respectively. These in vitro studies mirror potential in utero ethanol-exposed CNS conditions and may lead to therapeutic strategies targeted at attenuating neurodevelopmental FAS-related deficits.

Neurotrophin-3 promotes the survival of oligodendrocyte precursors in embryonic hippocampal cultures under chemically defined conditions

Embryonic rat hippocampal cells were cultured in basal medium with or without addition of the neurotrophin NT-3. After culturing in these extreme conditions, the effects of NT-3 on the neuronal and on the glial components were assessed. Neurons survived even in the absence of NT-3 but failed to reach terminal differentiation. On the other hand, NT-3 promoted the survival but not the proliferation and/or the differentiation of oligodendrocytes precursors present in the same culture, an effect that was reversed by the addition of neutralizing antibodies against NT-3. Type I or II astrocytes were not affected by NT-3. These results reinforce the role for NT-3 in oligodendrocyte lineage development and allow to dissect the roles of this neurotrophin in survival and in proliferation/differentiation of oligodendrocytes.

Short exposure to methylazoxymethanol causes a long‐term inhibition of axonal outgrowth from cultured embryonic rat hippocampal neurons

Methylazoxymethanol (MAM) is an alkylating agent that is used to induce microencephaly by killing mitotically active neuroblasts. We found that at later developmental times, MAM exposure can result in abnormal fiber growth in vivo. However, there have not been any previous studies on the effects of MAM on differentiating neurons. We examined the outcome of short exposure to MAM on postmitotic embryonic hippocampal cultures during the establishment of axonal polarity. At 0, 1, or 2 days in vitro (DIV), neurons were treated with 0.1 nM-1 μM MAM for 3 hr and then transferred to glial conditioned media. At 3 DIV, the cells were fixed and analyzed by immuno-fluorescent staining for neuron viability and differentiation. Control cells initiate several minor processes; one process elongates rapidly at about 1 DIV eventually becoming an axon, while extensive dendritic growth occurs after 3–4 DIV. Neurons treated with 1 μM MAM at 0 or 1 DIV showed a marked inhibition of neurite growth and withdrawal of axons without affecting cell viability. These cells continued to show minimal neurite outgrowth at 7 DIV, even when transferred to a glial coculture. In contrast, cells treated initially with MAM, after neuronal polarity is established at 2 DIV, showed no effect on axonal growth. To determine the effects of MAM on the neuronal cytoskeleton, we examined the in vitro assembly of brain microtubules in a one cycle assay. Exposure to MAM depleted the soluble pool of proteins, including microtubule-associated protein 1B (MAP1B) and MAP2, which are required for neurite outgrowth, through a nonspecific process. Under non-saturating conditions, there were no changes in the total amount of microtubules assembled or the coassembly of MAP1B and MAP2 in the presence of MAM. These results demonstrate that MAM can directly affect differentiating neurons, indicating that an early disruption of axonal outgrowth may have long-term effects. © 1996 Wiley-Liss, Inc.

Short exposure to methylazoxymethanol causes a long-term inhibition of axonal outgrowth from cultured embryonic rat hippocampal neurons.

Methylazoxymethanol (MAM) is an alkylating agent that is used to induce microencephaly by killing mitotically active neuroblasts. We found that at later developmental times, MAM exposure can result in abnormal fiber growth in vivo. However, there have not been any previous studies on the effects of MAM on differentiating neurons. We examined the outcome of short exposure to MAM on postmitotic embryonic hippocampal cultures during the establishment of axonal polarity. At 0, 1, or 2 days in vitro (DIV), neurons were treated with 0.1 nM-1 microM MAM for 3 hr and then transferred to glial conditioned media. At 3 DIV, the cells were fixed and analyzed by immunofluorescent staining for neuron viability and differentiation. Control cells initiate several minor processes; one process elongates rapidly at about 1 DIV eventually becoming an axon, while extensive dendritic growth occurs after 3-4 DIV. Neurons treated with 1 microM MAM at 0 or 1 DIV showed a marked inhibition of neurite growth and withdrawal of axons without affecting cell viability. These cells continued to show minimal neurite outgrowth at 7 DIV, even when transferred to a glial coculture. In contrast, cells treated initially with MAM, after neuronal polarity is established at 2 DIV, showed no effect on axonal growth. To determine the effects of MAM on the neuronal cytoskeleton, we examined the in vitro assembly of brain microtubules in a one cycle assay. Exposure to MAM depleted the soluble pool of proteins, including microtubule-associated protein 1B (MAP1B) and MAP2, which are required for neurite outgrowth, through a nonspecific process. Under non-saturating conditions, there were no changes in the total amount of microtubules assembled or the coassembly of MAP1B and MAP2 in the presence of MAM. These results demonstrate that MAM can directly affect differentiating neurons, indicating that an early disruption of axonal outgrowth may have long-term effects.

Statin, a Marker of Cell Cycle Arrest, Is Overexpressed during the Early Phase of Delayed NMDA Toxicity in Hippocampal Cell Cultures

Statin is a 57-kDa protein exclusively expressed in nuclei of nonproliferating mammalian cells. Recent studies have suggested that statin may play a role in the maintenance of growth arrest. Several lines of evidence also support the notion that a variety of genes and gene products are modulated during cell proliferation and cell death. The present study examined the possibility that statin expression could be modulated during neuronal injury using N-methyl-Daspartate (NMDA)-induced toxicity to rat embryonic hippocampal cultures as a model. Immunocytochemical studies using a monoclonal antibody to statin revealed a prominent nuclear localization of statin in cultured hippocampal cells. Western blot analysis showed that this antibody recognizes a 57-kDa protein band, indicative of the presence of statin in this preparation. Brief exposure of hippocampal neurons to NMDA (500 μM produced severe neuronal degeneration over the subsequent hours. NMDA-treated neurons markedly overexpressed statin. Both NMDA-induced neuronal toxicity and statin overexpression were prevented by the NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohept-5,10-imine hydrogen maleate (MK-801). Interestingly, time course studies indicate that the increased expression of statin observed following NMDA exposure clearly preceded the appearance of the first signs of neuronal death us determined by vital staining. In addition, exposure of hippocampal neurons to the Ca2+ ionophore, A23187, produced a marked increase in statin immunodetection, indicating that statin expression is likely regulated in a Ca2+-dependent manner. Thus, these results show that statin, which is expressed at low levels in embryonic rat cultured hippocampal neurons, is rapidly overexpressed following a toxic insult produced by the activation of the NMDA receptor. The observation that statin overexpression occurs prior to neuronal death raises the possibility that the up-regulation of statin could be used as an early index of neuronal injury.

Cellular targets and trophic functions of neurotrophin-3 in the developing rat hippocampus

The expression of the neurotrophins and trk receptors in the hippocampus has directed attention toward their roles in the development and maintenance of this region. We have examined the effects of the neurotrophins NT-3, BDNF, and NGF in cultures of developing rat hippocmpal cells by two criteria: rapid induction of c- fos and neurotrophic responses. The selective induction of c- fos mRNA suggests the presence of functional receptors for NT-3 and BDNF, but not NGF, in embryonic hippocampal cultures. The NT-3-responsive cells were localized in pyramidal neurons of areas CAI through CA3 and dentate granular and hilar cells of postnatal organotypic slices, as detected by c-Fos immunocytochemistry. In addition to immediate early responses, NT-3 caused a 10-fold increase in the number of cells expressing the neuronal antigen calbindin-D 28k. This increase was dose dependent, with maximal stimulation at 10 ng/ml. In contrast, BDNF elicited small but significant calbindin responses. These results indicate biological responses to NT-3 in the CNS and suggest roles for for this neurotrophin during hippocampal neurogenesis.