Embryonic Hippocampal Neurons In Culture Research Articles

Ashwagandha and Its Active Ingredient, Withanolide A, Increase Phosphorylation of TrkB in Cultured Hippocampal Neurons

Aims: To primary rat embryonic hippocampal neurons in culture, ashwagandha or one of its active ingredients, withanolide A were added in the presence or absence of nutrient supplementation and then assayed for activity of the BDNF receptor, TrkB.
 Study Design: Primary hippocampal neurons were cultured and grown in nutrient-rich or nutrient-poor medium. Ashwagandha or withanolide A were then be added to both types of media with or without an inhibitor of TrkB or either the PI-3K or MAPK pathway.
 Place and Duration of Study: Department of Biological Sciences, California State University, Los Angeles, CA, USA, between July 2021 and August 2022.
 Methodology: Rat embryos were removed by cesarean section from mother rats at 18 days’ gestation and the hippocampi of the former dissected, plated into culture dishes, and treated with the appropriate drug(s) (see Study Design above). After 4 days, neurons were harvested for Western blotting. Optical density of Western blot bands were quantified and statistically analyzed in a 2-way ANOVA, using a level of statistical significance at P < .05.
 Results: Under normal conditions (with N2 supplement), ashwagandha, but not withanolide A, increased phospho-TrkB immunoreactivity when compared to the effects of vehicle (controls, F(11, 24) = 22.48, P < .001), although withanolide A did not quite reach statistical significance (P = .069) when compared to that of the controlled condition. Likewise, under nutrient-deprived conditions, both ashwagandha and withanolide A also increased phosphorylation of TrkB when compared to that of vehicle-nutrient-deprived conditions (P < .0001). The same results were obtained in the presence of inhibitors of TrkB itself and the PI-3K (ashwagandha, P < .001; withanolide A, P < .001) and MAPK (ashwagandha, P = .027; withanolide A, P = .045) pathways.
 Conclusion: Ashwagandha or withanolide A activates TrkB, in nutrient-deprived hippocampal neurons, underscoring its role in neuronal survival signaling.

Moringa oleifera with promising neuronal survival and neurite outgrowth promoting potentials

Ethnopharmacological relevanceMoringa oleifera Lam. (Moringaceae) by virtue of its high nutritional as well as ethnomedical values has been gaining profound interest both in nutrition and medicinal research. The leaf of this plant is used in ayurvedic medicine to treat paralysis, nervous debility and other nerve disorders. In addition, research evidence also suggests the nootropic as well as neuroprotective roles of Moringa oleifera leaf in animal models. The aim of the present study was to evaluate the effect of Moringa oleifera leaf in the primary hippocampal neurons regarding its neurotrophic and neuroprotective properties. Materials and methodsThe primary culture of embryonic hippocampal neurons was incubated with the ethanol extract of Moringa oleifera leaf (MOE). After an indicated time, cultures were either stained directly with a lipophilic dye, DiO, or fixed and immunolabeled to visualize the neuronal morphology. Morphometric analyses for neurite maturation and synaptogenesis were performed using Image J software. Neuronal viability was evaluated using trypan blue exclusion and lactate dehydrogenase assays. ResultsMOE promoted neurite outgrowth in a concentration-dependent manner with an optimal concentration of 30μg/mL. As a very initial effect, MOE significantly promoted the earlier stages of neuronal differentiation. Subsequently, MOE significantly increased the number and length of dendrites, the length of axon, and the number and length of both dendrite and axonal branches, and eventually facilitated synaptogenesis. The β-carotene, one major compound of MOE, promoted neuritogensis, but the increase was not comparable with the effect of MOE. In addition, MOE supported neuronal survival by protecting neurons from naturally occurring cell death in vitro. ConclusionsOur findings indicate that MOE promotes axodendritic maturation as well as provides neuroprotection suggesting a promising pharmacological importance of this nutritionally and ethnomedically important plant for the well-being of nervous system.

LKB1/STRAD Promotes Axon Initiation During Neuronal Polarization

Axon/dendrite differentiation is a critical step in neuronal development. In cultured hippocampal neurons, the accumulation of LKB1 and STRAD, two interacting proteins critical for establishing epithelial polarity, in an undifferentiated neurite correlates with its subsequent axon differentiation. Downregulation of either LKB1 or STRAD by siRNAs prevented axon differentiation, and overexpression of these proteins led to multiple axon formation. Furthermore, interaction of STRAD with LKB1 promoted LKB1 phosphorylation at a PKA site S431 and elevated the LKB1 level, and overexpressing LKB1 with a serine-to-alanine mutation at S431 (LKB1(S431A)) prevented axon differentiation. In developing cortical neurons in vivo, downregulation of LKB1 or overexpression of LKB1(S431A) also abolished axon formation. Finally, local exposure of the undifferentiated neurite to brain-derived neurotrophic factor or dibutyryl-cAMP promoted axon differentiation in a manner that depended on PKA-dependent LKB1 phosphorylation. Thus local LKB1/STRAD accumulation and PKA-dependent LKB1 phosphorylation represents an early signal for axon initiation.

Basic fibroblast growth factor: Lysine 134 is essential for its neuroprotective activity

Basic fibroblast growth factor (bFGF) is a heparin-binding growth factor known to cause cell proliferation, angiogenesis and neuroprotection. We have performed site-directed mutagenesis to identify the amino acids that are essential for heparin/growth factor interaction and for neuroprotection. Binding to heparin-acrylic beads was markedly reduced when lysine in position 134 of bFGF was replaced by alanine. Wildtype (wt)-bFGF was shown to protect rat primary cultures of embryonic hippocampal neurons against damage caused by staurosporine and to reduce the infarct size in mice after focal cerebral ischemia. These neuroprotective effects of wt-bFGF could not be shown for the mutant bFGF(K134A). Furthermore, phosphorylation of Akt and ERK1/2 was significantly reduced in cultured neurons treated with bFGF(K134A) indicating diminished intracellular signaling compared to neurons treated with wt-bFGF. In conclusion, lysine at position 134 of bFGF is essential for bFGF to bind heparin, then to interact with its receptor and, subsequently, to protect neurons against damage.

Molecular and Functional Characterization of the Electroneutral Na/HCO3 Cotransporter NBCn1 in Rat Hippocampal Neurons

We examined molecular and electrophysiological properties of the electroneutral sodium/bicarbonate cotransporter (NBCn1) that is present in rat hippocampal neurons. By PCR, a deletion variant (NBCn1-E) that lacks 123 amino acids in the cytoplasmic N-terminal domain was found in adult neurons. The previously characterized NBCn1-B, which does not have the deletion, was detected in embryonic neurons. In Xenopus oocytes, NBCn1-E raised the intracellular pH in the presence of HCO(3) without significantly affecting the membrane potential. Despite this electroneutral cotransport activity, the transporter mediated a steady-state current that positively shifted the resting potential by almost 30 mV. The mean reversal potential of the steady-state current was -21.2 mV, close to the resting potential of -21.4 mV. The reversal potential shifted 26 mV in response to a 10-fold increase of external Na(+) for concentrations above 10 mm. The current activity mediated by the transporter was unaffected by K(+), Mg(2+), Ca(2+), or Cl(-). Stable expression of NBCn1-E in human embryonic kidney cells also evoked an inward current that shifted the resting potentials more positive compared with the sham-transfected controls. In primary cultures of embryonic hippocampal neurons, the NBCn1 protein was localized in somatodendrites and synapses. NBCn1 protein was partially colocalized with the postsynaptic density protein PSD-95. Single-cell PCR showed that NBCn1 mRNA expression was present in both gamma-aminobutyric acid (GABA)ergic and non-GABAergic neurons. We propose that NBCn1 in hippocampal neurons may affect neuronal activity by regulating local pH as well as steady-state inward currents at synapses.

Differential regulation of GABAA receptor and gephyrin postsynaptic clustering in immature hippocampal neuronal cultures

Gephyrin is a postsynaptic scaffolding protein involved in clustering of glycine- and GABA(A) receptors at inhibitory synapses. The role of gephyrin in GABAergic synapses, the nature of its interactions with GABA(A) receptors, and the mechanisms of targeting to GABAergic synapses are largely unknown. To gain further insights into these questions, the formation of GABA(A) receptor and gephyrin clusters and their distribution relative to presynaptic terminals were investigated in immature cultures of embryonic hippocampal neurons using triple immunofluorescence staining. GABA(A) receptor clusters, labeled for the alpha2 subunit, formed independently of gephyrin clusters, and were distributed on neurites at constant densities, either extrasynaptically or, to a lesser extent, postsynaptically, apposed to synapsin-I-positive axon terminals. In contrast, gephyrin clusters were always associated with GABA(A) receptors and were preferentially localized postsynaptically. Their density increased linearly with the extent of innervation, which developed rapidly during the first week in vitro. These results suggested that GABA(A) receptor clustering is mediated by cell-autonomous mechanisms independent of synapse formation. Their association with gephyrin is dynamically regulated and may contribute to stabilization at postsynaptic sites. Labeling for vesicular glutamate transporters revealed that most synapses in these immature cultures were presumably glutamatergic, implying that postsynaptic GABA(A) receptor and gephyrin clusters initially were located in "mismatched" synapses. However, clusters appropriately localized in GABAergic synapses were distinctly larger and more intensely stained. Altogether, these results demonstrate that the targeting of GABA(A) receptor and gephyrin clusters to GABAergic synapses occurs secondarily and is regulated by presynaptic factors that are not essential for clustering.

Enhancement of dendritic branching in cultured hippocampal neurons by 17β-estradiol is mediated by nitric oxide

Both 17β-estradiol (E2) and nitric oxide (NO) are important in neuronal development, learning and memory, and age-related memory changes. There is growing evidence that a number of estrogen receptor-mediated effects of estradiol utilize nitric oxide as an intermediary. The role of estradiol in hippocampal neuronal differentiation and function has particular implications for learning and memory.Low levels of estradiol (10nM) significantly increase dendritic branching in cultured embryonic rat hippocampal neurons (158% of control). This study investigates the hypothesis that the estrogen-stimulated increase in dendritic branching is mediated by nitric oxide. We found that nitric oxide donors also produce significantly increased dendritic branching S-nitroso-N-acetylpenicillamine (SNAP: 119%; 2,2′-(hydroxynitrosohydrazino)bis-ethanamine (NOC-18): 128% of control). We then determined that the increases in dendritic branching stimulated by estradiol or by a nitric oxide donor were both blocked by an inhibitor of guanylyl cyclase. Dendritic branching was also stimulated by a cell permeable analog of cyclic guanosine monophosphate (dibutyryl-cGMP: 173% of control). Estradiol-stimulated dendritic branching was reversed by the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (carboxy-PTIO). This study provides evidence that estradiol influences the development of embryonic hippocampal neurons in culture by increasing the production of nitric oxide or by increasing the sensitivity of the neurons to nitric oxide. Nitric oxide in turn stimulates dendritic branching via activation of guanylyl cyclase.

Generation of reactive oxygen species, release of l-glutamate and activation of caspases are required for oxygen-induced apoptosis of embryonic hippocampal neurons in culture

Oxygen-induced cell death in embryonic neurons is a useful in vitro model of neuronal apoptosis to study the molecular mechanisms underlying the cell death induced by oxidative stress. In the present study, we examined the involvement of reactive oxygen species and glutamate in the high (50%) oxygen-induced death of cultured hippocampal neurons. During the course of cell death, increases in O 2 − and hydrogen peroxide (H 2O 2) levels were observed. On the other hand, superoxide dismutase (SOD), catalase and deferoxamine (DFX), which have inhibitory effects on the generation of O 2 −, H 2O 2 and hydroxyl radicals, respectively, protected the neurons. These results suggested that both O 2 − and H 2O 2 play important roles in this apoptosis. Antagonists of NMDA and AMPA/kinate (AMPA/KA) receptors and an inhibitor of glutamate release partially prevented the apoptosis, suggesting that exposure to high oxygen enhances glutamate release, which results in activation of NMDA receptor and AMPA/KA receptor. In addition, specific nitric oxide (NO) scavenger and NO synthetase inhibitors blocked the apoptosis, indicating that NO and/or peroxynitrite are involved in this mechanism of cell death. Caspase inhibitors also blocked the neuronal apoptosis. These results suggested that multiple effectors including generation of reactive oxygen species, release of l-glutamate and activation of caspases are activated during the death induced by high oxygen.

Expression of a dominant negative TrkB receptor, T1, reveals a requirement for presynaptic signaling in BDNF-induced synaptic potentiation in cultured hippocampal neurons.

We have developed a method to analyze the relative contributions of pre- and postsynaptic actions of a particular gene product in neurons in culture and potentially in slices using adenovirus-mediated gene transfer. A recombinant virus directed the expression of both a GFP reporter protein and TrkB.T1, a C-terminal truncated dominant negative TrkB neurotrophin receptor. When expressed in the presynaptic cell at synapses between embryonic hippocampal neurons in culture, the dominant negative TrkB.T1 inhibited two forms of synaptic potentiation induced by the neurotrophin brain-derived neurotrophic factor (BDNF): (i) greater evoked synaptic transmission and (ii) higher frequency of spontaneous miniature synaptic currents. These inhibition effects are not seen if the transgene is expressed only in the postsynaptic cell. We conclude that BDNF-TrkB signal transduction in the presynaptic terminal leads to both types of potentiation and is therefore the primary cause of synaptic enhancement by BDNF in these neurons.

Reduction of voltage-dependent calcium currents in mumps virus-infected cultures of rat hippocampal neurons

The effects of a mumps virus infection on functional properties of embryonic hippocampal neurons in culture were analysed with special emphasis on voltage-dependent Ca2+ channels. Cultures with higher or lower density of glial cells (not treated or treated with mitotic inhibitor, respectively) were infected with the relatively non-cytolytic RW strain of mumps virus and currents were recorded from neurons using whole cell voltage clamp. More than 65% of neurons and glial cells contained viral antigens 1-2 days post infection (p.i.). Glial cells remained infected 6-7 days p.i., while the ratio of infected versus uninfected neurons, especially in cultures with higher glial cell density, was reduced. In both infected and uninfected cultures the somal voltage-dependent Ca2+ currents were stronger in cultures with a higher glial cell density, which indicates that these currents are influenced by glial cells. Introduction of the virus into cultures caused a selective decrease in inward Ca2+ currents, which was most marked at days 6-7 p.i., and which included both infected and unifected neurons. Spontaneous synaptic currents and other ion channel conductances appeared normal in the infected cultures. Dantrolene, which inhibits release of Ca2+ from intracellular stores, decreased the neurons that died during the infection. Taken together the results show that a mumps viral infection can selectively alter the number of function of somal voltage dependent Ca2+ channels in immature hippocampal neurons and that this may reflect a disturbed glia-nerve cell interaction.

Microtubule fragmentation and partitioning in the axon during collateral branch formation

Axons within the brain branch principally by the formation of collaterals rather than by bifurcation of the terminal growth cone (O'Leary and Terashima, 1988). This same behavior is recapitulated in cultures of embryonic hippocampal neurons (Dotti et al., 1988), rendering them ideal for studies on the cell biological mechanisms underlying collateral branch formation. In the present study, we focused on changes in the microtubule (MT) array that occur as these axons branch. In particular, we explored the mechanism by which MT number is locally increased to accommodate the need for more MTs during collateral branch formation. Serial reconstruction analyses indicate that MT number increases by severalfold and that MT length decreases correspondingly within the parent axon in the discrete region giving rise to the branch. These observations strongly suggest that MTs within the parent axon undergo a local fragmentation in this region, and hence raise the possibility that a portion of these new MTs might be destined for transport into the branch. To address this latter issue, we used quantitative immunofluorescence to compare the proportion of newly assembled to total MT polymer in different regions of the axon. As previously reported (Brown et al., 1992), the region of the axon contiguous with the terminal growth cone is particularly rich in newly assembled polymer. In contrast, there was no distinguishable difference in the proportion of newly assembled polymer in the newly formed collateral branches compared to the shaft region of the parent axon. These results indicate that the MTs within the newly formed collateral branches are on average assembled at the same time as those within the parent axon, and thus strongly suggest that the MTs in the collateral branch were assembled in the parent axon and then translocated into the branch. We conclude on the basis of these observations that collateral branch formation requires a local fragmentation of MTs within the parent axon, followed by the partitioning of a portion of the MT fragments into the branch. These short MTs presumably then resume their movement and elongation down the collateral branch as well as down the parent axon for the steady and orderly increase of both MT arrays.

Purification and characterization of neurotrophic factor for retinal cholinergic neurons derived from cultured hippocampal neurons.

A neurotrophic factor that supports the development of cholinergic retinal neurons was purified from media conditioned by a primary culture of embryonic hippocampal neurons. Retinal choline acetyltransferase (ChAT), which is located exclusively in amacrine cells, served as a marker for the development of retinal cholinergic neurons. In a serum-free control culture, retinal neurons from 17-day-old rat fetus displayed little increase in the enzyme activity and a low proportion of neurite-bearing cells (15-20%) within 7 days. The conditioned media, when added to the retinal neuron culture, dose-dependently increased ChAT activity and the number of neurite-bearing cells (40-60%), the maximum ChAT activity being approximately sixfold higher than that in the control. The fraction with these stimulatory activities was purified by Sephadex G-15 column chromatography and two times reverse-phase HPLC. The final fraction showed approximately 3,000-fold higher purification as compared with that in the Sephadex G-15 fraction. Gas-phase protein sequencing analysis of the final fraction yielded a peptide sequence: Tyr-Leu-Leu-Pro-Ala-Gln-Val-Asn-Ile-Asp. A synthetic peptide with this sequence dose-dependently stimulated ChAT activity in the retinal cell culture and dissociated cell culture of the septal nucleus. These findings suggest that the developing hippocampal neurons produce a neurotrophic peptide that stimulates the development of cholinergic neurons.

Involvement of GABA A receptors in the outgrowth of cultured hippocampal neurons

Whereas GABA is a major inhibitory neurotransmitter in the adult central nervous system, recent experiments performed in our laboratory have shown that the activation of GABA A receptors in the hippocampus leads to excitatory effects during the early post-natal period. The possible consequence of a depolarizing effect of GABA was assessed on the neuritic outgrowth of embryonic hippocampal neurons in culture. No morphological alterations were observed when hippocampal neurons were cultured for three days in the presence of muscimol, a GABA A receptor agonist. In contrast, the neuritic outgrowth of cultured hippocampal neurons was profoundly affected by the presence of bicuculline in the culture medium. In the presence of this GABA A receptor antagonist neurons displayed a reduction in the number of primary neurites and branching points, resulting in a concomitant decrease of the total neuritic length. Thus, this study suggests that GABA, acting on GABA A subtype of receptors, is able to affect the development of the hippocampus.

Systemic interleukin-1β decreases brain-derived neurotrophic factor messenger RNA expression in the rat hippocampal formation

Brain-derived neurotrophic factor is selectively expressed at relatively high levels in the rat hippocampal formation (for review, see Ref. 12; see also Refs 8, 13, 19, 20, 27) where it is thought to be involved in mechanisms of neurodegeneration and/or neural protection related to the plasticity of hippocampal neurons. Functional responses to brain-derived neurotrophic factor appear to be mediated by a tyrosine receptor kinase B with the possible involvement of the p75 low-affinity nerve growth factor receptor protein. Among the many characteristics of Alzheimer's disease is an upregulation of immune mediators in and around senile plaques in Alzheimer's disease. Recently, interleukin-1 has been shown to be detrimental to the long-term survival of embryonic hippocampal neurons in culture. Thus, if the same occurs in vivo, it is possible that the accumulation of interleukin-1 in Alzheimer's disease hippocampus may be responsible for altered hippocampal neuron synaptic plasticity. This may occur either by a direct action of interleukin-1 on hippocampal neurons or possibly indirectly by stimulating beta-amyloid production. Other indirect mechanisms may involve growth or survival factors such as the neurotrophin brain-derived neurotrophic factor which is thought to play an important role in the plastic responses of hippocampal neurons. A recent study showed that brain-derived neurotrophic factor mRNA is selectively decreased in the dentate gyrus in Alzheimer's disease. The reason(s) for the decrease of brain-derived neurotrophic factor mRNA is not known, but one possibility may be associated with the enhanced expression of interleukin-1 in the hippocampus of Alzheimer's disease patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane currents induced by l-homocysteic acid in mouse cultured hippocampal neurons

The concentration-response relationship of membrane currents induced by l-homocysteic acid was studied on mouse embryonic hippocampal neurons in culture ( n = 56). In the majority of neurons two phases in the dose-response relationship could be distinguished. The first was characterized by responses to 3–100 μM l-homocysteic acid which desensitized with a time-constant > 1 s in a concentration-dependent manner and were antagonized by 30 μM d- l-2-amino-5-phosphonovaleric acid indicating activation of the N-methyl- d-aspartate receptors. At higher concentrations of l-homocysteic acid this component was strongly depressed. The second phase was characterized by sustained responses that were concentration-dependent (1 mM l-homocysteic acid maximum concentration tested) and were not blocked by d- l-2-amino-5-phosphonovaleric acid indicating activation of non-N-methyl- d-aspartate receptors. Eight neurons did not exhibit these two-phase characteristics in the concentration-response relationship at the beginning of the recording. The magnitude of responses to l-homocysteic acid was positively related to concentration and the responses were partially blocked by d- l-2-amino-5-phosphonovaleric acid. In these neurons, however, repeated applications of l-homocysteic acid at concentrations 30 μM up to 300 μM resulted in a long-lasting, three- to four-fold increase of the membrane current. This increase was completely blocked by d- l-2-amino-5-phosphonovaleric acid (50–100 μM) suggesting that it was produced by activation of receptors. It is suggested that the long-lasting increase of the membrane current after repeated l-homocysteic acid application reflects long-term changes in the mechanisms involved in controlling N-methyl- d-aspartate receptors from an intracellular site in which Ca 2+ plays an important but not an exclusive role.

Structure-activity relationships for amino acid transmitter candidates acting at N-methyl-D-aspartate and quisqualate receptors

Dose-response curves for activation of excitatory amino acid receptors on mouse embryonic hippocampal neurons in culture were recorded for 15 excitatory amino acids, including the L-isomers of glutamate, aspartate, and a family of endogenous sulfur amino acids. In the presence of 3 microM glycine, with no extracellular Mg, micromolar concentrations of 11 of these amino acids produced selective activation of N-methyl-D-aspartate (NMDA) receptors. L-Glutamate was the most potent NMDA agonist (EC50 2.3 microM) and quinolinic acid the least potent (EC50 2.3 mM). Dose-response curves were well fit by the logistic equation, or by a model with 2 independent agonist binding sites. The mean limiting slope of log-log plots of NMDA receptor current versus agonist concentration (1.93) suggests that a 2-site model is appropriate. There was excellent correlation between agonist EC50S determined in voltage clamp experiments and KdS determined for NMDA receptor binding (Olverman et al., 1988). With no added glycine, and 1 mM extracellular Mg, responses to NMDA were completely blocked; responses to kainate and quisqualate were unchanged. Under these conditions, glutamate and the sulfur amino acids activated a rapidly desensitizing response, similar to that evoked by micromolar concentrations of quisqualate and AMPA, but mM concentrations of L-aspartate, homoquinolinic acid, and quinolinic acid failed to elicit a non-NMDA receptor-mediated response. Except for L-glutamate (EC50 480 microM), the low potency of the sulfur amino acids prevented the study of complete dose-response curves for the rapidly desensitizing response at quisqualate receptors. Small-amplitude nondesensitizing quisqualate receptor responses were activated by much lower concentrations of all quisqualate receptor agonists. Full dose-response curves for the nondesensitizing response were obtained for 9 amino acids; L-glutamate was the most potent endogenous agonist (EC50 19 microM). Domoate (EC50 13 microM) and kainate (EC50 143 microM) activated large-amplitude, nondesensitizing responses.