Hippocampal Cell Cultures Research Articles

3-O-sulfated heparan sulfate interactors target synaptic adhesion molecules from neonatal mouse brain and inhibit neural activity and synaptogenesis in vitro

Heparan sulfate (HS) chains, covalently linked to heparan sulfate proteoglycans (HSPG), promote synaptic development and functions by connecting various synaptic adhesion proteins (AP). HS binding to AP could vary according to modifications of HS chains by different sulfotransferases. 3-O-sulfotransferases (Hs3sts) produce rare 3-O-sulfated HSs (3S-HSs), of poorly known functions in the nervous system. Here, we showed that a peptide known to block herpes simplex virus by interfering with 3S-HSs in vitro and in vivo (i.e. G2 peptide), specifically inhibited neural activity, reduced evoked glutamate release, and impaired synaptic assembly in hippocampal cell cultures. A role for 3S-HSs in promoting synaptic assembly and neural activity is consistent with the synaptic interactome of G2 peptide, and with the detection of Hs3sts and their products in synapses of cultured neurons and in synaptosomes prepared from developing brains. Our study suggests that 3S-HSs acting as receptors for herpesviruses might be important regulators of neuronal and synaptic development in vertebrates.

SynaptoPAC, an optogenetic tool for induction of presynaptic plasticity.

Optogenetic manipulations have transformed neuroscience in recent years. While sophisticated tools now exist for controlling the firing patterns of neurons, it remains challenging to optogenetically define the plasticity state of individual synapses. A variety of synapses in the mammalian brain express presynaptic long-term potentiation (LTP) upon elevation of presynaptic cyclic adenosine monophosphate (cAMP), but the molecular expression mechanisms as well as the impact of presynaptic LTP on network activity and behavior are not fully understood. In order to establish optogenetic control of presynaptic cAMP levels and thereby presynaptic potentiation, we developed synaptoPAC, a presynaptically targeted version of the photoactivated adenylyl cyclase bPAC. In cultures of hippocampal granule cells of Wistar rats, activation of synaptoPAC with blue light increased action potential-evoked transmission, an effect not seen in hippocampal cultures of non-granule cells. In acute brain slices of C57BL/6N mice, synaptoPAC activation immediately triggered a strong presynaptic potentiation at mossy fiber synapses in CA3, but not at Schaffer collateral synapses in CA1. Following light-triggered potentiation, mossy fiber transmission decreased within 20min, but remained enhanced still after 30min. The optogenetic potentiation altered the short-term plasticity dynamics of release, reminiscent of presynaptic LTP. Our work establishes synaptoPAC as an optogenetic tool that enables acute light-controlled potentiation of transmitter release at specific synapses in the brain, facilitating studies of the role of presynaptic potentiation in network function and animal behavior in an unprecedented manner. Read the Editorial Highlight for this article on page 270.

Expression of Fbp2, a Newly Discovered Constituent of Memory Formation Mechanisms, Is Regulated by Astrocyte-Neuron Crosstalk.

Fbp2 (muscle isozyme of fructose 1,6-bisphosphatase) is a glyconeogenesis-regulating enzyme and a multifunctional protein indispensable for long-term potentiation (LTP) formation in the hippocampus. Here, we present evidence that expression of Fbp2 in murine hippocampal cell cultures is regulated by crosstalk between neurons and astrocytes. Co-culturing of the two cell types results in a decrease in Fbp2 expression in astrocytes, and its simultaneous increase in neurons, as compared to monocultures. These changes are regulated by paracrine signaling using extracellular vesicle (EV)-packed factors released to the culture medium. It is well accepted that astrocyte–neuron metabolic crosstalk plays a crucial role in shaping neuronal function, and recently we have suggested that Fbp2 is a hub linking neuronal signaling with redox and/or energetic state of brain during the formation of memory traces. Thus, our present results emphasize the importance of astrocyte–neuron crosstalk in the regulation of the cells’ metabolism and synaptic plasticity, and bring us one step closer to a mechanistic understanding of the role of Fbp2 in these processes.

Epileptic seizures of suspected autoimmune origin: a multicentre retrospective study

ObjectiveTo analyse autoantibody status in a well-defined European multicentre cohort of patients with epilepsy of unknown aetiology and to validate the recently proposed Antibody Prevalence in Epilepsy (APE2) and Response...

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.

Short-term neuronal effects of fumonisin B1 on neuronal activity in rodents

Fumonisin B1 (FB1) is a mycotoxin produced by microscopic fungi (mostly Fusarium species), which may infect our major crops. The toxin inhibits the development of these plants and may also have harmful effects on animals and humans consuming the infected crops.FB1 inhibits sphingolipid biosynthesis which leads to altered membrane characteristics and consequently, altered cellular functions. There are some indications that the toxin has inhibitory effects on neuronal activity in case of repeated consumption, presumably due to sphingolipid depletion. However, according to new literature data, FB1 may have acute excitatory neural effects, too, via different mechanisms of action. Therefore, in the present study, we addressed the neuronal network effects of FB1 following acute treatment, using different electrophysiological techniques in vitro and in vivo.Acute treatments with FB1 (10–100 μM) were carried out on brain slices, tissue cultures and live animals. After direct treatment of samples, electrically evoked or spontaneous field potentials were examined in the hippocampus and the neocortex of rat brain slices and in hippocampal cell cultures. In the hippocampus, a short-term increase in the excitability of neuronal networks and individual cells was observed in response to FB1 treatment. In some cases, the initially enhanced excitation was reversed presumably due to overactivation of neuronal networks. Normal spontaneous activity was found to be stimulated in hippocampal cell cultures. Seizure susceptibility was not affected in the neocortex of brain slices.For the verification of the results caused by direct treatment, effects of systemic administration of FB1 (7.5 mg/kg, i.p.) were also examined. Evoked field potentials recorded in vivo from the somatosensory cortex and cell activation measured by the c-fos technique in hippocampus and somatosensory cortex were analyzed. However, the hippocampal and cortical stimulatory effect detected in vitro could not be demonstrated by these in vivo assays.Altogether, the toxin enhanced the basic excitability of neurons and neuronal networks after direct treatment but there were no effects on the given brain areas after systemic treatment in vivo. Based on the observed in vitro FB1 effects and the lack of data on the penetration of FB1 across the blood-brain barrier, we assume that in vivo consequences of FB1 administration can be more prominent in case of perturbed blood-brain barrier functions.

CEPO (carbamylated erythropoietin)-Fc protects hippocampal cells in culture against beta amyloid-induced apoptosis: considering Akt/GSK-3β and ERK signaling pathways.

The tissue-protective properties of erythropoietin (EPO) have been described in several neurodegenerative diseases models, but erythrocytosis following EPO treatment may lead to deleterious effects. Carbamylated erythropoietin, an EPO derivative lacking hematopoietic side effects, has shown protective properties in some studies. However, it is not known if CEPO protects primary hippocampal cells against Aβ25-35 toxicity. The present study aimed to investigate the effect of CEPO-Fc on biochemical alterations in Akt, GSK-3β, and ERK signaling and cell death induced by Aβ25-35 in isolated hippocampal cell culture. The embryonic hippocampal cells were obtained from 18-19 day rat embryos. The cells were exposed with Aβ25-35 (20 µM) in the absence or presence of CEPO-Fc (1 or 5 IU) and PI3k and ERK inhibitors. CEPO-Fc at the dose of 5 IU significantly prevented the cell loss and caspase-3 cleavage caused by Aβ25-35. Additionally, CEPO-Fc noticeably reversed Aβ mediated decrement of Akt and GSK-3β phosphorylation. With exposure to LY294002, PI3 kinase inhibitor, Akt phosphorylation diminished and CEPO-Fc protective effects disappeared. Furthermore, while CEPO-Fc nullified Aβ-induced increment of phospho-ERK, inhibition of ERK activity by PD98059, had no effect on Aβ25-35-mediated caspase-3 cleavage and cell toxicity. These results imply that protective effects of CEPO-Fc seem to be mainly exerted through the PI3K/Akt pathway rather than ERK signaling. This study suggested that CEPO-Fc prevents Aβ-induced cell toxicity as well as Akt/GSK-3β and ERK alterations in isolated hippocampal cells. These findings might provide a new perspective on CEPO-Fc protective properties as a prospective remedial factor for neurodegenerative diseases like AD.

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.

Visualization, Properties, and Functions of GABAergic Hippocampal Neurons Containing Calcium-Permeable Kainate and AMPA Receptors

Calcium-permeable kainate (CP-KARs) and AMPA (CP-AMPARs) receptors of the brain neurons are active participants of synaptic plasticity and neurotransmitter release trigger. In this paper, CP-KARs and CP-AMPARs were identified in hippocampal neuroglial culture on 14–17 day of cultivation by a characteristic Ca2+ response to a selective agonist of CP-KARs and CP-AMPARs, domoic acid (DA), and to a selective agonist of CP-KARs, ATPA. It was shown that DA at a concentration of 300 nM caused a rapid intracellular Ca2+ concentration increase in two minor subpopulations of neurons. Both subpopulations were found to be GABAergic neurons that were positively stained with antibodies against glutamate decarboxylase 65 and 67 (GAD65/67). The antagonist of CP-AMPARs, NASPM, did not suppress Ca2+ response to DA in the neurons of the first subpopulation. The selective agonist of CP-KARs, ATPA, increased [Ca2+]i to the same extent as DA only in the first subpopulation of GABAergic neurons. An inhibitor of GABA(A) receptors, bicuculline, did not increase the amplitude of Ca2+ response to DA in this subpopulation, indicating the absence of CP-KARs in the postsynaptic membrane, where GABA(A) receptors are located. Thus, these GABAergic neurons can be attributed to neurons containing CP-KARs, which are apparently located in the presynaptic membrane of the GABAergic neurons. The [Ca2+]i increase caused by the DA application in the second subpopulation was completely suppressed by NASPM, an inhibitor of CP-AMPARs. NASPM reduced the Ca2+ oscillations amplitude in the same subset, indicating the involvement of CP-AMPARs in the Ca2+ impulse formation during synchronous calcium activity. For this reason, the neurons of this subpopulation can be attributed to the GABAergic neurons containing CP-AMPARs. Most of the neurons in the hippocampal cell culture (70–85%) were not stained with antibodies against GAD65/67 and responded to the DA by increasing the calcium oscillations frequency with a delay. The amplitude of DA-induced oscillations increased in the presence of NASPM in the subpopulation of inhibitory neurons containing CP-KARs, indicating their innervation by inhibitory neurons containing CP-AMRARs. This increase in the Ca2+ oscillation amplitude in the inhibitory neurons containing CP-KARs correlated with a decrease in the amplitude of synchronous calcium activity in a large subpopulation (42 ± 6% of cells) of glutamatergic neurons, suggesting innervation of the latter by inhibitory neurons containing CP-KARs. Thus, GABAergic neurons containing CP-KARs and CP-AMPARs can work in tandem, controlling the activity of individual subpopulations of neurons.

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.

Extracellular vesicles derived from human Wharton\u2019s jelly mesenchymal stem cells protect hippocampal neurons from oxidative stress and synapse damage induced by amyloid-\u03b2 oligomers

BackgroundMesenchymal stem cells (MSCs) have been explored as promising tools for treatment of several neurological and neurodegenerative diseases. MSCs release abundant extracellular vesicles (EVs) containing a variety of biomolecules, including mRNAs, miRNAs, and proteins. We hypothesized that EVs derived from human Wharton’s jelly would act as mediators of the communication between hMSCs and neurons and could protect hippocampal neurons from damage induced by Alzheimer’s disease-linked amyloid beta oligomers (AβOs).MethodsWe isolated and characterized EVs released by human Wharton’s jelly mesenchymal stem cells (hMSC-EVs). The neuroprotective action of hMSC-EVs was investigated in primary hippocampal cultures exposed to AβOs.ResultshMSC-EVs were internalized by hippocampal cells in culture, and this was enhanced in the presence of AβOs in the medium. hMSC-EVs protected hippocampal neurons from oxidative stress and synapse damage induced by AβOs. Neuroprotection by hMSC-EVs was mediated by catalase and was abolished in the presence of the catalase inhibitor, aminotriazole.ConclusionshMSC-EVs protected hippocampal neurons from damage induced by AβOs, and this was related to the transfer of enzymatically active catalase contained in EVs. Results suggest that hMSC-EVs should be further explored as a cell-free therapeutic approach to prevent neuronal damage in Alzheimer’s disease.

Design and synthesis of selective and blood-brain barrier-permeable hydroxamate-based gelatinase inhibitors.

Matrix metalloproteinases (MMPs), a family of zinc-containing endopeptidases involved in the degradation of the extracellular matrix, make a major contribution to the progression of a vast number of diseases, such cancer or epilepsy. Although several MMP inhibitors (MMPi) have been developed to date for the treatment of cancer, they have all failed in clinical trials due to lack of efficacy and, most importantly, the presence of severe side effects. The latter can be explained by their lack of selectivity of these inhibitors. In this regard, MMPs' family members have a high structural homology, which challenge the development of selective inhibitors for a specific MMP. Here, we have used in silico calculations and in vitro data to design MMPi that selectively target gelatinases (MMP-2 and MMP-9) and have the capacity to cross the blood-brain barrier. Following this approach, we obtained compound 40 that shows high proteolytic stability and low cytotoxicity. This compound may be of particular interest for the treatment of central nervous diseases such epilepsy or Alzheimer's disease, where gelatinase activity is increased. Our data show the specificity of compound 40 for recombinant MMP-9 and MMP-2 and endogenous MMP-9 from rat hippocampal cell cultures, and reveals its permeability across the blood-brain barrier in vivo.

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.

Synthesis and In Vitro Neuroprotective Activity of Analogs with Various Spacer Lengths of a Dimeric Dipeptide Mimetic (GK-2) of Nerve Growth Factor

Dimeric dipeptide mimetic GK-2 or hexamethylenediamide bis-(N-monosuccinyl-L-glutamyl-L-lysine), which activated TrkA receptors and exhibited neuroprotective activity in vitro (10–5 – 10–9 M) and in vivo (0.05 – 5 mg/kg i.p.), was designed by us earlier based on the beta-turn of the nerve growth factor (NGF) fourth loop. The effect of dimerizing spacer length on the manifestation of neuroprotective activity was studied by synthesizing GK-2 (11 σ-bonds) analogs tetramethylenediamide bis-(N-monosuccinyl-L-glutamyl-Llysine) (GK-2c); pentamethylenediamide bis-(N-monosuccinyl-L-glutamyl-L-lysine) (GK-2b); and trimethylenediamide bis-(N-monosuccinyl-L-glutamyl-L-lysyl-6-aminohexanoic acid) (GK-2a) containing 9, 10, and 22 σ-bonds, respectively, between the lysine Cα-atoms of the dipeptide fragments. Neuroprotective activity was investigated in vitro using HT-22 mouse hippocampal neuronal cell culture under H2O2-induced oxidative stress and showed that the neuroprotective activity as measured by the minimum effective concentration was 104 times less if the distance between the dipeptide fragments increased from 11 to 22 σ-bonds. The neuroprotective activity decreased insignificantly if this distance was decreased from 11 to 10 σ bonds (GK-2b) and disappeared with 9 σ -bonds (GK-2c). Therefore, the hexamethylene spacer was optimal although the distance between the corresponding amino-acid moieties of natural NGF from an x-ray structure analysis was considerably greater at 33 σ-bonds. This contradiction could have arisen because two ionically bound mimetic molecules interacted with TrkA receptors. Another possible explanation was that TrkA molecules could approach closer in the presence of bis-dipeptide GK-2 than NGF.

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.

Влияние соединения ФС-ЛХТ-317 на активность NMDA-рецепторов в смешанной нейроглиальной культуре гиппокампа крысы

В опытах на нейроглиальной культуре клеток гиппокампа крыс Spraque Dawley методом флуоресцентной визуализации оценили влияние соединения ФС-ЛХТ-317 (магния бис-ацетаминоэтансульфоната), обладающего церебропротекторным действием, на естественную инактивацию кальциевых каналов NMDA-рецепторов. Установлено, что это вещество в течение 0,2 – 0,4 с после аппликации оказывает ингибиторный эффект, направленный на подавление активности NMDA-рецепторов. Данный эффект формируется в диапазоне концентраций от 1 до 10 мМ и является обратимым.

The positive allosteric modulation of GABAA receptors mRNA in immature hippocampal rat neurons by midazolam affects receptor expression and induces apoptosis

Background: Numerous experimental studies show that anesthetics are potentially toxic to the immature brain. Even though benzodiazepines are widely used in pediatric anesthesia and intensive care medicine, only a few studies examine the effects of these drugs on immature neurons.Methods: Hippocampal neuronal cell cultures of embryonic Wistar rats (15 days in culture) were incubated with midazolam 100 or 300 nM for either 30 min or 4 h. The time course of the mRNA expression of the glutamate receptors subunits NR1, NR2A and NR2B of the NMDA receptor, the GluA-1 and A-2 subunits of the AMPA receptor as well as the alpha 1 subunit of the GABAA receptor were examined by PCR. Apoptosis was detected using Western blot analysis for BAX, Bcl-2 and Caspase-3.Results: Midazolam at 100 and 300 nM applied for 30 min and 100 nM for 4 h affected glutamate receptor and GABAA receptor subunit expression. However, these effects were reversible within 72 h following washout. When 300 nM midazolam was applied for 4 h a significant increase in the NR 1 and NR 2A mRNA subunit expression could be detected. The increase in NR 2B receptor subunit expression as well as the GluA1 subunit expression was not reversible within 72 h following washout. This increase in mRNA glutamate receptor subunit expression was associated with a significant increase in neuronal apoptosis.Conclusion: In immature neurons midazolam altered GABA and glutamate mRNA receptor subunit expression. Prolonged increase in midazolam-induced glutamate receptor expression was associated with apoptosis.

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.