Primary Hippocampal Cell Cultures Research Articles

Quinic acid contributes to neurogenesis: Targeting Notch pathway a key player in hippocampus

Coordinated proliferation and differentiation of neural stem cells (NSCs) results in continuous neurogenesis. The present study provides novel insights into the Notch intracellular signaling in neuronal cell proliferation, maintenance, migration, and differentiation regulated by naturally based Quinic acid (QA) in primary hippocampal cell culture. Further, this study might help in the discovery and development of lead molecules that can overcome the challenges in the treatment of neurodegenerative diseases. The growth supporting effect of QA was studied using Alamar Blue assay. The migratory potential of QA was evaluated using scratch assay. The in vitro H2O2-induced oxidative stress model was used to upregulate neuronal survival after QA treatment. The RT-qPCR and immunocytochemical analysis were performed for selected markers of Notch signaling to determine the proliferation, differentiation, and maintenance of NSCs at gene and molecular levels. The Mash1 and Ngn2 are the upstream proneural genes of the Notch pathway which were included to evaluate the differentiation of NSCs into mature neurons after treatment with QA.Furthermore, regarding the role of QA in maintaining the pool of NPCs, we used Notch1 and Hes1 markers for proliferation analysis. Also, secondary neuronal markers i.e. Pax6, PCNA, and Mcm2 were included in this study and their gene expression analysis was analyzed following treatment with QA. Based on the study’s results, we suggest that naturally based QA can promote the growth and differentiation of neonatal NSCs residing in hippocampal regions into neuronal lineage. Therefore, we propose that the neurogenic potential of QA can be employed to prevent and treat neurodegenerative diseases.

SARS-CoV-2 Viroporin E Induces Ca2+ Release and Neuron Cell Death in Primary Cultures of Rat Hippocampal Cells Aged In Vitro.

The COVID-19 pandemic was caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which may lead to serious respiratory, vascular and neurological dysfunctions. The SARS-CoV-2 envelope protein (E protein) is a structural viroporin able to form ion channels in cell membranes, which is critical for viral replication. However, its effects in primary neurons have not been addressed. Here we used fluorescence microscopy and calcium imaging to study SARS-CoV-2 viroporin E localization and the effects on neuron damage and intracellular Ca2+ homeostasis in a model of rat hippocampal neurons aged in vitro. We found that the E protein quickly enters hippocampal neurons and colocalizes with the endoplasmic reticulum (ER) in both short-term (6-8 days in vitro, DIV) and long-term (20-22 DIV) cultures resembling young and aged neurons, respectively. Strikingly, E protein treatment induces apoptosis in aged neurons but not in young neurons. The E protein induces variable increases in cytosolic Ca2+ concentration in hippocampal neurons. Ca2+ responses to the E protein are due to Ca2+ release from intracellular stores at the ER. Moreover, E protein-induced Ca2+ release is very small in young neurons and increases dramatically in aged neurons, consistent with the enhanced Ca2+ store content in aged neurons. We conclude that the SARS-CoV-2 E protein quickly translocates to ER endomembranes of rat hippocampal neurons where it releases Ca2+, probably acting like a viroporin, thus producing Ca2+ store depletion and neuron apoptosis in aged neurons and likely contributing to neurological damage in COVID-19 patients.

Prolactin-induced neuroprotection against excitotoxicity is mediated via PI3K/AKT and GSK3β/NF-κB in primary cultures of hippocampal neurons

Prolactin (PRL) is a polypeptide hormone that has been reported to play a significant role in neuroprotection against neuronal excitotoxicity produced by glutamate (Glu) or kainic acid (KA) in both, in vitro and in vivo models. However, the molecular mechanisms involved in PRL’s neuroprotective effects in the hippocampus have not been completely elucidated. The aim of the present study was to assess the signaling pathways involved in PRL neuroprotection against excitotoxicity. Primary rat hippocampal neuronal cell cultures were used to assess PRL-induced signaling pathway activation. The effects of PRL on neuronal viability, as well as its effects on activation of key regulatory pathways, phosphoinositide 3-kinases/Protein Kinase B (PI3K/AKT) and glycogen synthase kinase 3β / nuclear factor kappa B (GSK3β/NF-κB), were evaluated under conditions of Glutamate-induced excitotoxicity. Additionally, the effect on downstream regulated genes such as Bcl-2 and Nrf2, was assessed. Here, we show that the PI3K/AKT signaling pathway is activated by PRL treatment during excitotoxicity, promoting neuronal survival through upregulation of active AKT and GSK3β/NF-κB, resulting in induction of Bcl-2 and Nrf2 gene expression. Inhibition of the PI3K/AKT signaling pathway abrogated the protective effect of PRL against Glu-induced neuronal death. Overall, results indicate that the neuroprotective actions of PRL are mediated in part, by the activation of the AKT pathway and survival genes. Our data support the idea that PRL could be useful as a potential neuroprotective agent in different neurological and neurodegenerative diseases.

Use of liposome-encapsulated estetrol for treatment of neonatal hypoxic-ischemic encephalopathy

Estetrol (E4) is a natural estrogen synthesized only during pregnancy. It has strong neuroprotective and antioxidative activities. The aim of the present study was to define the neuroprotective potency of E4 encapsulated either in liposome (Lipo-E4) or in drug-in cyclodextrin (HP-β-CD) in liposome (DCL) system, and compare them with a single use of E4. In vitro studies were performed in an oxidative stress model of primary hippocampal neuronal cell cultures, followed by the lactate dehydrogenase activity and cell proliferation assays. In vivo studies were conducted by using a model of neonatal hypoxic-ischemic encephalopathy in immature rat pups. Brain samples were studied by (immuno)histochemistry for the detection of survived cells, expression of microtubule-associated protein-2, myelin basic protein, doublecortin and vascular-endothelial growth factor. Concentrations of glial fibrillary acidic protein in blood serum were studied by ELISA. In vitro, cell proliferation was significantly up-regulated in cultures treated either by DCL-E4 or E4 compared to the control cells, whereas DCL-E4 treated cells had significantly higher survival rate than the cells treated by E4 alone. Evaluation of brain samples showed that DCL-E4 and a high dose of E4 alone significantly preserve the grey and the white matter loses, and diminish GFAP expression in blood. Although DCL-E4 and E4 have similar effect on neurogenesis in the hippocampus and the cortex, DCL-E4 treatment significantly up-regulates angiogenesis in the hippocampus compared to a single use of E4. Present work reveals for the first time that liposome-encapsulated E4 might be a better alternative to a single use of E4.

Characterisation of Primary Human Hippocampal Astrocyte Cell Culture Following Exposure to Hypoxia.

The present study aimed to understand the characterisation of human hippocampal astrocyte following hypoxia exposure. Based on the preliminary screening, 15 min was chosen as the time point and the cells were exposed to different oxygen percentages. The Trypan blue viability assay used to examine cell death. Immunofluorescence assay, glial fibrillary acidic protein (GFAP) was used to portray the morphology of astrocytes. The hypoxia-inducible factor 1 (HIF-1) staining was performed to confirm hypoxia induced cell death and there was a dramatic expression of HIF-1α displayed in exposed astrocyte cells compared to the control. In molecular level, genes were chosen, such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), GFAP, HIF-1α and B-cell lymphoma 2 (Bcl-2) and ran the reverse transcription-polymerase chain reaction (RT-PCR). Microscope revealed a filamentous and clear nucleus appearance in a control whereas the rupture nuclei with no rigid structure of the cell were found in the 3% oxygen. The control and hypoxia cells were also stained with the annexin V-fluorescein isothiocyanate (annexin V-FITC). Fluorescence microscope reveals astrocyte cells after hypoxia showed higher expression of nuclei but not in control. Merging PI and FITC showed the differences of nuclei expression between the control and hypoxia. In the molecular analysis, there were significant changes of GFAP, HIF-1α and Bcl-2 in hypoxia exposed cells when compared to the control group. Cells that were exposed to hypoxia (3% oxygen for 15 min) clearly showed damage. General view of human hippocampal astrocyte genomic response to hypoxia was obtained.

Astrocyte-mediated Transduction of Muscle Fiber Contractions Synchronizes Hippocampal Neuronal Network Development

Exercise supports brain health in part by enhancing hippocampal function. The leading hypothesis is that muscles release factors when they contract (e.g., lactate, myokines, growth factors) that enter circulation and reach the brain where they enhance plasticity (e.g., increase neurogenesis and synaptogenesis). However, it remains unknown how the muscle signals are transduced by the hippocampal cells to modulate network activity and synaptic development. Thus, we established an in vitro model in which the media from contracting primary muscle cells (CM) is applied to developing primary hippocampal cell cultures on a microelectrode array. We found that the hippocampal neuronal network matures more rapidly (as indicated by synapse development and synchronous neuronal activity) when exposed to CM than regular media (RM). This was accompanied by a 4.4- and 1.4-fold increase in the proliferation of astrocytes and neurons, respectively. Further, experiments established that factors released by astrocytes inhibit neuronal hyper-excitability induced by muscle media, and facilitate network development. Results provide new insight into how exercise may support hippocampal function by regulating astrocyte proliferation and subsequent taming of neuronal activity into an integrated network.

Hydrogen Peroxide Induced Toxicity Is Reversed by the Macrocyclic IRAP-Inhibitor HA08 in Primary Hippocampal Cell Cultures.

Angiotensin IV (Ang IV), a metabolite of Angiotensin II, is a bioactive hexapeptide that inhibits the insulin-regulated aminopeptidase (IRAP). This transmembrane zinc metallopeptidase with many biological functions has in recent years emerged as a new pharmacological target. IRAP is expressed in a variety of tissues and can be found in high density in the hippocampus and neocortex, brain regions associated with cognition. Ang IV is known to improve memory tasks in experimental animals. One of the most potent IRAP inhibitors known today is the macrocyclic compound HA08 that is significantly more stable than the endogenous Ang IV. HA08 combines structural elements from Ang IV and the physiological substrates oxytocin and vasopressin, and binds to the catalytic site of IRAP. In the present study we evaluate whether HA08 can restore cell viability in rat primary cells submitted to hydrogen peroxide damage. After damaging the cells with hydrogen peroxide and subsequently treating them with HA08, the conceivable restoring effects of the IRAP inhibitor were assessed. The cellular viability was determined by measuring mitochondrial activity and lactate dehydrogenase (LDH) release. The mitochondrial activity was significantly higher in primary hippocampal cells, whereas the amount of LDH was unaffected. We conclude that the cell viability can be restored in this cell type by blocking IRAP with the potent macrocyclic inhibitor HA08, although the mechanism by which HA08 exerts its effects remains unclear.

Dysregulation of miRNAs Levels in Glycogen Synthase Kinase-3β Overexpressing Mice and the Role of miR-221-5p in Synaptic Function

Glycogen synthase kinase-3β (GSK-3β) is a highly expressed kinase in the brain, where it has an important role in synaptic plasticity. Aberrant activity of GSK-3β leads to synaptic dysfunction which results in the development of several neuropsychiatric and neurological diseases. Notably, overexpression of constitutively active form of GSK-3β (GSK-3β[S9A]) in mice recapitulates the cognitive and structural defects characteristic for neurological and psychiatric disorders. However, the mechanisms by which GSK-3β regulates synaptic functions are not clearly known. Here, we investigate the effects of GSK-3β overactivity on neuronal miRNA expression in the mouse hippocampus. We found that GSK-3β overactivity downregulates miRNA network with a potent effect on miR-221-5p (miR-221*). Next, characterization of miR-221* function in primary hippocampal cell culture transfected by miR-221* inhibitor, showed no structural changes in dendritic spine shape and density. Using electrophysiological methods, we found that downregulation of miR-221* increases excitatory synaptic transmission in hippocampal neurons, probably via postsynaptic mechanisms. Thus, our data reveal potential mechanism by which GSK-3β and miRNAs might regulate synaptic function and therefore also synaptic plasticity.

Toxic Effect of Streptozotocin on Cultured Mouse Hippocampal Neurons.

In primary dissociated hippocampal cell cultures from 18-day-old mouse embryos, streptozotocin in concentrations of 2-5 mM produced a dose-dependent cytotoxic effect on day 3 in vitro, whereas on day 11 of culturing, the neurons were resistant to streptozotocin. The neurons in the 3-day cultures were functionally immature, which was seen from their weak spontaneous bioelectric activity in the form of rare single action potentials; by day 11 of culturing, the neurons reached a high level of differentiation and their functional properties acquired a character of network burst activity. Thus, streptozotocin had the most pronounced cytotoxic effect on immature hippocampal neurons in vitro.

Microarray profiling emphasizes transcriptomic differences between hippocampal in vivo tissue and in vitro cultures.

Primary hippocampal cell cultures are routinely used as an experimentally accessible model platform for the hippocampus and brain tissue in general. Containing multiple cell types including neurons, astrocytes and microglia in a state that can be readily analysed optically, biochemically and electrophysiologically, such cultures have been used in many in vitro studies. To what extent the in vivo environment is recapitulated in primary cultures is an on-going question. Here, we compare the transcriptomic profiles of primary hippocampal cell cultures and intact hippocampal tissue. In addition, by comparing profiles from wild type and the PrP 101LL transgenic model of prion disease, we also demonstrate that gene conservation is predominantly conserved across genetically altered lines.

Interleukin-1\u03b2 mediates alterations in mitochondrial fusion/fission proteins and memory impairment induced by amyloid-\u03b2 oligomers

BackgroundThe lack of effective treatments for Alzheimer’s disease (AD) reflects an incomplete understanding of disease mechanisms. Alterations in proteins involved in mitochondrial dynamics, an essential process for mitochondrial integrity and function, have been reported in AD brains. Impaired mitochondrial dynamics causes mitochondrial dysfunction and has been associated with cognitive impairment in AD. Here, we investigated a possible link between pro-inflammatory interleukin-1 (IL-1), mitochondrial dysfunction, and cognitive impairment in AD models.MethodsWe exposed primary hippocampal cell cultures to amyloid-β oligomers (AβOs) and carried out AβO infusions into the lateral cerebral ventricle of cynomolgus macaques to assess the impact of AβOs on proteins that regulate mitochondrial dynamics. Where indicated, primary cultures were pre-treated with mitochondrial division inhibitor 1 (mdivi-1), or with anakinra, a recombinant interleukin-1 receptor (IL-1R) antagonist used in the treatment of rheumatoid arthritis. Cognitive impairment was investigated in C57BL/6 mice that received an intracerebroventricular (i.c.v.) infusion of AβOs in the presence or absence of mdivi-1. To assess the role of interleukin-1 beta (IL-1β) in AβO-induced alterations in mitochondrial proteins and memory impairment, interleukin receptor-1 knockout (Il1r1−/−) mice received an i.c.v. infusion of AβOs.ResultsWe report that anakinra prevented AβO-induced alteration in mitochondrial dynamics proteins in primary hippocampal cultures. Altered levels of proteins involved in mitochondrial fusion and fission were observed in the brains of cynomolgus macaques that received i.c.v. infusions of AβOs. The mitochondrial fission inhibitor, mdivi-1, alleviated synapse loss and cognitive impairment induced by AβOs in mice. In addition, AβOs failed to cause alterations in expression of mitochondrial dynamics proteins or memory impairment in Il1r1−/− mice.ConclusionThese findings indicate that IL-1β mediates the impact of AβOs on proteins involved in mitochondrial dynamics and that strategies aimed to prevent pathological alterations in those proteins may counteract synapse loss and cognitive impairment in AD.

Roles and Interaction of the MAPK Signaling Cascade in Aβ25-35-Induced Neurotoxicity Using an Isolated Primary Hippocampal Cell Culture System.

Alzheimer's disease (AD) is characterized with increased formation of amyloid-β (Aβ) in the brain. Aβ peptide toxicity is associated with disturbances of several intracellular signaling pathways such as mitogen activated protein kinases (MAPKs). The aim of this study was to investigate the role of MAPKs and their interactions in Aβ-induced neurotoxicity using isolated hippocampal neurons from the rat. Primary hippocampal cells were cultured in neurobasal medium for 4days. Cells were treated with Aβ25-35 and/or MAPKs inhibitors for 24h. Cell viability was determined by an MTT assay and phosphorylated levels of P38, JNK, and ERK were measured by Western blots. Aβ treatment (10-40µM) significantly decreased hippocampal cell viability in a dose-dependent manner. Inhibition of P38 and ERK did not restore cell viability, while JNK inhibition potentiated the Aβ-induced neurotoxicity. Compared to the controls, Aβ treatment increased levels of phosphorylated JNK, ERK, and c-Jun, while it had no effect on levels of phosphorylated P38. In addition, P38 inhibition led to decreased expression levels of phosphorylated ERK; inhibition of JNK resulted in decreased expression of c-Jun; and inhibition of ERK, decreased phosphorylated levels of JNK. These results strongly suggest that P38, ERK, and JNK are not independently involved in Aβ-induced toxicity in the hippocampal cells. In AD, which is a multifactorial disease, inhibiting a single member of the MAPK signaling pathway, does not seem to be sufficient to mitigate Aβ-induced toxicity and thus their interactions with each other or potentially with different signaling pathways should be taken into account.

Lithium for schizophrenia: supporting evidence from a 12-year, nationwide health insurance database and from Akt1-deficient mouse and cellular models

Accumulating evidence suggests AKT1 and DRD2-AKT-GSK3 signaling involvement in schizophrenia. AKT1 activity is also required for lithium, a GSK3 inhibitor, to modulate mood-related behaviors. Notably, GSK3 inhibitor significantly alleviates behavioral deficits in Akt1−/− female mice, whereas typical/atypical antipsychotics have no effect. In agreement with adjunctive therapy with lithium in treating schizophrenia, our data mining indicated that the average utilization rates of lithium in the Taiwan National Health Insurance Research Database from 2002 to 2013 are 10.9% and 6.63% in inpatients and outpatients with schizophrenia, respectively. Given that lithium is commonly used in clinical practice, it is of great interest to evaluate the effect of lithium on alleviating Akt1-related deficits. Taking advantage of Akt1+/− mice to mimic genetic deficiency in patients, behavioral impairments were replicated in female Akt1+/− mice but were alleviated by subchronic lithium treatment for 13 days. Lithium also effectively alleviated the observed reduction in phosphorylated GSK3α/β expression in the brains of Akt1+/− mice. Furthermore, inhibition of Akt expression using an Akt1/2 inhibitor significantly reduced neurite length in P19 cells and primary hippocampal cell cultures, which was also ameliorated by lithium. Collectively, our findings implied the therapeutic potential of lithium and the importance of the AKT1-GSK3 signaling pathway.

Short Term Exposure to Bilirubin Induces Encephalopathy Similar to Alzheimer's Disease in Late Life.

Hyperbilirubinemia may increase the risk of Alzheimer's disease (AD) but its mechanistic role in AD pathogenesis remains obscure. Here, we used animal models to investigate the short- and long-term effects of neonatal systemic exposure to bilirubin on brain histology and function as well as the acute effect of lateral ventricle injection of bilirubin in adult rats. We found that three days exposure to bilirubin in newborn rats could induce AD-like pathological changes in late life, including tau protein hyperphosphorylation at multiple sites, increased Aβ production in brain tissues, and spatial learning and memory injury. Bilirubin activated the activities of several protein kinases (GSK-3β, CDK5, and JNK), which were positively correlated with hyperphosphorylated tau; simultaneously increased the expression of AβPP γ-secretase PS2 and decreased the expression of α-secretase ADAM17, which were positively correlated with Aβ production. The above results were well replicated in primary hippocampal cell cultures. These data demonstrate that bilirubin encephalopathy is an AD-like disease, suggesting a potent role of bilirubin in AD.

Growth hormone increases dendritic spine density in primary hippocampal cell cultures

ObjectiveGrowth hormone (GH) is widely known for its peripheral effects during growth and development. However, numerous reports also suggest that GH exert pro-cognitive, restorative, and protective properties in the brain. In in vitro studies, the detection of dendritic spines, small protrusions extending from axons, can act as a marker for cognition-related function as spine formation is considered to be associated with learning and memory. Here we show that an acute 24-hour treatment of GH can increase dendritic spine density in primary hippocampal cell cultures. DesignPrimary hippocampal cells were harvested from embryonic Wistar rats and cultured for 14 days. Cells were treated with supra-physiological doses of GH (10-1000 nM) and subjected to a high-throughput screening protocol. Images were acquired and analyzed using automated image analysis and the number of spines, spines per neurite length, neurite length, and mean area of spines, was reported. ResultsGH treatment (1000 nM) increased the number of dendritic spines by 83% and spines per neurite length by 82% when compared to control. For comparison BDNF, a known inducer of spine densities, produced statistically non-significant increase in this setting. ConclusionThe results was found significant using the highest supra-physiological dose of GH, and the present study further confirms a potential role of the hormone in the treatment of cognitive dysfunction.

Telomerase increasing compound protects hippocampal neurons from amyloid beta toxicity by enhancing the expression of neurotrophins and plasticity related genes

The telomerase reverse transcriptase protein, TERT, is expressed in the adult brain and its exogenic expression protects neurons from oxidative stress and from the cytotoxicity of amyloid beta (Aβ). We previously showed that telomerase increasing compounds (AGS) protected neurons from oxidative stress. Therefore, we suggest that increasing TERT by AGS may protect neurons from the Aβ-induced neurotoxicity by influencing genes and factors that participate in neuronal survival and plasticity. Here we used a primary hippocampal cell culture exposed to aggregated Aβ and hippocampi from adult mice. AGS treatment transiently increased TERT gene expression in hippocampal primary cell cultures in the presence or absence of Aβ and protected neurons from Aβ induced neuronal degradation. An increase in the expression of Growth associated protein 43 (GAP43), and Feminizing locus on X-3 genes (NeuN), in the presence or absence of Aβ, and Synaptophysin (SYP) in the presence of Aβ was observed. GAP43, NeuN, SYP, Neurotrophic factors (NGF, BDNF), beta-catenin and cyclin-D1 expression were increased in the hippocampus of AGS treated mice. This data suggests that increasing TERT by pharmaceutical compounds partially exerts its neuroprotective effect by enhancing the expression of neurotrophic factors and neuronal plasticity genes in a mechanism that involved Wnt/beta-catenin pathway.

Insulin-regulated aminopeptidase inhibitor-mediated increases in dendritic spine density are facilitated by glucose uptake.

Ethyl2-acetylamino-7-hydroxy-4-pyridin-3-yl-4H-chromene-3-carboxylate (HFI-419), the benzopyran-based inhibitor of insulin-regulated aminopeptidase (IRAP), has previously been shown to improve spatial working and recognition memory in rodents. However, the mechanism of its cognitive-enhancing effect remains unknown. There is a close correlation between dendritic spine density and learning in vivo and several studies suggest that increases in neuronal glucose uptake and/or alterations to the activity of matrix metalloproteinases (MMPs) may improve memory and increase dendritic spine density. We aimed to identify the potential mechanism by which HFI-419 enhances memory by utilizing rat primary cultures of hippocampal cells. Alterations to dendritic spine density were assessed in the presence of varying concentrations of HFI-419 at different stages of hippocampal cell development. In addition, glucose uptake and changes to spine density were assessed in the presence of indinavir, an inhibitor of the glucose transporter 4 (GLUT4 ), or the matrix metalloprotease inhibitor CAS 204140-01-2. We confirmed that inhibition of IRAP activity with HFI-419 enhanced spatial working memory in rats, and determined that this enhancement may be driven by GLUT4 -mediated changes to dendritic spine density. We observed that IRAP inhibition increased dendritic spine density prior to peak dendritic growth in hippocampal neurons, and that spine formation was inhibited when GLUT4 -mediated glucose uptake was blocked. In addition, during the peak phase of dendritic spine growth, the effect of IRAP inhibition on enhancement of dendritic spine density resulted specifically in an increase in the proportion of mushroom/stubby-like spines, a morphology associated with memory and learning. Moreover, these spines were deemed to be functional based on their expression of the pre-synaptic markers vesicular glutamate transporter 1 and synapsin. Overall, or findings suggest that IRAP inhibitors may facilitate memory by increasing hippocampal dendritic spine density via a GLUT4 -mediated mechanism. Cover Image for this issue: doi: 10.1111/jnc.14745.

Brain-Derived Neurotrophic Factor in the Nucleus Accumbens Mediates Individual Differences in Behavioral Responses to a Natural, Social Reward.

BDNF-oxytocin interactions in the brain are implicated in mammalian maternal behavior. We found that BDNF gene expression is increased in the hippocampus of rat mothers that show increased pup licking/grooming (high LG mothers) compared to low LG mothers. High LG mothers also showed increased BDNF protein levels in the nucleus accumbens (nAcc). Immunoneutralization of BDNF in the nAcc eliminated the differences in pup LG between high and low LG mothers. Oxytocin antagonist in the ventral hippocampus significantly decreased the frequency of maternal LG behavior. Oxytocin antagonist significantly prevented the oxytocin-induced BDNF gene expression in primary hippocampal cell cultures. We suggest that oxytocin-induced regulation of BDNF in the nAcc provides a neuroendocrine basis for both individual differences in maternal behavior and resilience to the stress of reproduction in female mammals.

Primary Hippocampal Cell Culture and Its Application in Medical Researches

Studies in neuroscience can be performed in vitro and in vivo. In vivo studies will show significant results, but it is difficult to do and time-consuming. Primary hippocampal cell culture widely has used in neurobiological studies such as identifying the cellular mechanism of proteins, neuronal activity, and characteristics. The results of studies conducted on this cell culture will be very useful in discovering pathogenesis of a disease, the effect of a substance on the neuron, and neural basis of memory and learning. However, currently in Indonesia, primary hippocampal cell culture is still rare and difficult to do. The purpose of this study was to demonstrate that primary hippocampal cell culture can be done and developed in Indonesia and to review the application of it in medical researches. The study was an experimental study by obtaining neurons from animal’s hippocampus was conducted in 2015–2018 at Department of Cell Biology, Graduate School of Medicine Osaka University and Faculty of Medicine Universitas Padjadjaran. The experimental animal was mice embryo gathered 17.5-days postcoitus. Enzymatic and mechanical methods collected primary hippocampal cells. The cells counted and cultured, which later were observed to see neuron differentiation. The average number of culture cells from 3 embryonic’s hippocampus were 2.39×106. Neuron differentiation observed on the first day and more visible and numerous on the third day after plating. In conclusion, primary hippocampal cell culture using hippocampus from one hemisphere of embryonic mice brain showed a sufficient number of cells to carry out research and showed neuron differentiation. KULTUR SEL PRIMER HIPOKAMPUS DAN PENGGUNAANNYA DALAM RISET KEDOKTERANPenelitian dalam neurobiologi dapat dilakukan secara in vitro dan in vivo. Penelitian secara in vivo sangat berdampak hasilnya, namun sulit dan memakan waktu yang lama. Kultur sel primer hipokampus banyak digunakan dalam penelitian neurobiologi seperti melihat mekanisme protein seluler, serta aktivitas dan karakteristik neuron. Hasil penelitian yang dilakukan pada kultur sel ini akan sangat bermanfaat dalam menemukan proses suatu penyakit, efek suatu zat terhadap sel saraf, dan kemampuan belajar serta memori. Akan tetapi, saat ini di Indonesia kultur sel primer hipokampus masih jarang dan sulit dilakukan. Tujuan penelitian ini adalah menunjukkan bahwa kultur sel hipokampus primer dapat dilakukan dan dikembangkan di Indonesia, serta meninjau penerapannya dalam riset kedokteran. Penelitian ini merupakan studi eksperimental dengan mengoleksi neuron dari hipokampus hewan coba yang dilakukan pada tahun 2015–2018 di Department of Cell Biology, Graduate School of Medicine Osaka University dan Fakultas Kedokteran Universitas Padjadjaran. Hewan coba berupa embrio mencit hari ke-17,5 pascakoitus. Sel primer hipokampus dikoleksi untuk dihitung dan dikultur menggunakan metode enzimatik dan mekanik. Observasi neuron pada kultur dilanjutkan dengan mengamati diferensiasi neuron. Rerata jumlah sel kultur dari 3 hipokampus adalah 2,39×106. Diferensiasi neuron sudah tampak pada hari pertama dan makin jelas serta tampak pada hari ketiga pascapenanaman. Simpulan, kultur sel primer hipokampus menggunakan hipokampus dari salah satu sisi hemisfer otak menunjukkan jumlah sel yang cukup untuk melakukan suatu penelitian dan menunjukkan diferensiasi dari neuron.

Memantine Is Protective against Cytotoxicity Caused by Lead and Quinolinic Acid in Cultured Rat Embryonic Hippocampal Cells.

Quinolinic acid (QA) is an excitotoxic metabolite of the kynurenine pathway of tryptophan metabolism produced in response to inflammation and oxidative stress. Lead (Pb) causes oxidative stress and thus may produce neurotoxicity by increasing QA production. We investigated the in vitro cytotoxic effects of Pb and QA and the protective effects of the NMDA receptor antagonist memantine. Primary cultures of embryonic hippocampal cells from Wistar rats were treated with different concentrations of Pb, QA, and Pb + QA with and without memantine. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Apoptosis was analyzed by flow cytometry after Annexin-V/propidium iodide staining. The numbers of immunostained neurons (with β3-Tubulin; Tuj1) and astrocytes (with glial fibrillary acidic protein) were counted. Pb at 20 μg/dL (0.97 μM) and QA at 500 nM concentrations showed significant cytotoxic effects, as evidenced by decreased cell viability, increased apoptosis, and a decrease in the number of both astrocytes and neurons. The combination of Pb and QA showed significant synergistic apoptotic effects at lower doses. Memantine (500 nM) was largely protective against the cytotoxic effects of both Pb and QA, suggesting that Pb's and QA's cytotoxicity involves NMDA receptor activation. Whereas the neuroprotection by memantine from QA-induced toxicity has been previously reported, this is the first study reporting the protection by memantine against Pb-induced cytotoxicity in cultured hippocampal cells. Protection by memantine against these neurotoxicants in vivo needs to be investigated.