In central nervous system only a limited number of vesicles exist in the presynaptic terminals. The size and fusion modes of the vesicles were particularly important because of their potential impact on neuronal communications. Efficient methods were needed to analyze the recycling kinetics of synaptic vesicle and the size of readily releasable pool (RRP). In this study, fluorescent dyes with different affinity for membranes (FM1-43 with high affinity and FM2-10 with low affinity) were used to stain the functional synaptic vesicles of cultured hippocampal neurons and the kinetics of vesicle recycling was measured. The results showed that the destaining proportion was larger for FM2-10 than that for FM1-43 during the first trial, while it was greater for FM1-43 than FM2-10 during the second and third trials (first round, 93.0%+/-5.9% versus 57.9%+/-3.5% for FM2-10 and FM1-43, respectively, P<0.0001; second round, 1.4%+/-3.8% versus 24.0%+/-2.3%, P<0.0001; third round, 2.3%+/-1.6% versus 8.6%+/-1.5%, P=0.005). The results indicated that rapid endocytosis existed not only in the first round but also occurred when the vesicles were reused. Moreover, Both high-frequency stimuli and hypertonic sucrose stimuli were used to estimate the RRP sizes in the mix cultured hippocampal inhibitory neurons at 13-14 days in vitro (DIV). We found that the RRP size estimated by hypertonic sucrose stimuli [(200+/-23.0) pC] was much larger than that estimated by high-frequency stimuli [(51.1+/-10.5) pC]. One possible reason for the discrepancies in RRP estimates is that in mix cultured conditions, one neuron may receive inputs from several neurons and hypertonic sucrose stimuli will cause RRP of all those neurons release, while using dual patch recording, only the connection between two neurons was analyzed. Thus, to exclude out the impacts of inputs numbers on RRP sizes, it is more reasonable to use high-frequency stimuli to estimate the RRP size in mix cultured neurons.
Cells, Cultured ; Endocytosis ; Hippocampus ; cytology ; Neurons ; physiology ; Synaptic Vesicles ; physiology

Cultured primary hippocampal neurons are ideal tool for investigating the subcellular localization and trafficking of neuronal proteins. The aim of the present study was to establish a method to co-culture hippocampal neurons and cortical astrocytes, which would guarantee well conditions of neurons. Newborn Sprague-Dawley (SD) rats were sacrificed by decapitation. The cortex of cerebrum was cut into pieces, and the cortical tissue was digested with trypsin. The liquid suspension of single cells was planted onto a 25 cm² culture flask. On the fourth day of culture, the tissue cells except astrocytes were removed by intensive agitation of culture flask. Purified astrocytes were allowed to grow continuously until they reached most area of flask. At this time point, we replaced the culture media with neuronal cell media containing cytarabine, and planted the primary culture of rat hippocampal neurons onto the feed layer of cortical astrocytes. The microscopic observation results showed that, the astrocytes evenly grew without obvious boundaries between each other, and exhibited good purity. The co-cultured hippocampal neurons were in good condition, developed intertwined network of axons and dendrites, lived for a long time, and could tolerate gene transfection. Above all, this method is relatively simple from a technical point of view, yet provides healthy and reliable neuronal culture.
Animals ; Astrocytes ; cytology ; Cells, Cultured ; Cerebral Cortex ; cytology ; Coculture Techniques ; Culture Media ; Hippocampus ; cytology ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley

BACKGROUNDThe existence of neurogenesis in the hippocampus of adult nonhuman primates has been confirmed in recent years, however, the biological properties of adult neural stem cells or neural progenitor cells (NPCs) from this region remain to be extensively explored. The present work was to investigate on the expansion of NSCs/NPCs from the hippocampus of adult cynomolgus monkeys and the examination of their characteristics in vitro.

METHODSNPCs isolated from the hippocampus of adult cynomolgus monkeys were expanded in vitro in serum-free media containing growth factors, and were then allowed to differentiate by removing mitotic factors. The expansion capacity of NPCs and their differentiation potential were assayed by immunohistochemical and immunocytochemical analysis.

RESULTSDuring primary culture, NPCs underwent cell division, proliferation and aggregation to form neurospheres that were growing in suspension. Without mitotic stimulation, most neurospheres adhered to the culture dish and started to differentiate. Eventually, nearly 12% of the differentiated cells expressed neuron specific marker-beta III-tubulin (Tuj1) and 84% expressed astrocyte specific marker-fibrillary acidic protein (GFAP). In addition, the expression of a neural stem cell marker, nestin, was found both in NPCs and in the subgranular zone of adult monkey hippocampus, where NPCs were originally derived.

CONCLUSIONSNPCs from the hippocampus of adult cynomolgus monkeys can be expanded to some extent in vitro and are capable of differentiating into neurons and astrocytes. Further experiments to promote the in vitro proliferation capacity of NPCs will be required before adult NPCs can be used as a useful cell model for studying adult neurogenesis and cell replacement therapy using adult stem cells.

Animals ; Cell Differentiation ; Cells, Cultured ; Hippocampus ; cytology ; Immunohistochemistry ; Macaca fascicularis ; Male ; Stem Cells ; cytology

AIMTo investigate the effects of oxygen-glucose deprivation on cultured rat hippocampal neurons.

METHODSThe hippocampal neurons cultured for 12 d were exposed to combined oxygen-glucose deprivation for 0.5 - 4 h and then cultured with original medium in normoxia for 28 h. Necrotic neurons were identified by 0.4% trypan blue staining and apoptotic neurons were detected by a TUNEL technique. Meanwhile, the area, perimeter and circle diameter of cell bodies were measured respectively by a photography analysis system.

RESULTSThe percentage of necrotic cells in cultured hippocampal neurons increased significantly during oxygen-glucose deprivation, but the percentage of apoptotic cells increased significantly after 28 h oxygen-glucose recovery. Photography analysis showed that area, perimeter and circle diameter of the necrotic cell bodies were larger than those of the apoptotic ones.

CONCLUSIONOxygen-glucose deprivation can lead to severe damage of cultured hippocampal neurons. The necrosis is major during acute oxygen-glucose deprivation, while the apoptosis is major 28 h after oxygen-glucose recovery.

Animals ; Cell Hypoxia ; Cells, Cultured ; Glucose ; deficiency ; Hippocampus ; cytology ; Neurons ; cytology ; Oxygen ; metabolism ; Rats ; Rats, Wistar

AIMIn order to explore the neurobiological mechanism of morphine addiction and treatment methods, the acute and chronic effects of morphine on the intracellular free calcium concentration ([Ca2+]i) in cultured hippocampal neurons were investigated.

METHODSChanges of [Ca2+]i induced by morphine in primarily cultured hippocampal neurons were measured by confocal laser scanning microscopy using Ca(2+) -sensitive dye fluo-4 as the calcium fluorescent probe.

RESULTSMorphine actually induced the increase in [Ca2+]i of hippocampal neurons. This process could be blocked by naltrindole (delta opioid receptor antagonist) pretreatment, but not by CTOP (micro opioid receptor antagonist) pretreatment. Pretreatment of the cells with thapsigargin almost completely blocked morphine-evoked response; while pretreatment of the cells with verapamil partially inhibited this response. After exposure to 100 micromol/L morphine for 24 h, intracellular [Ca2+]i increased and the increase could be intensified after adding 10 micromol/L naloxone to the medium.

CONCLUSIONMorphine induced the release of Ca2+ is mainly from inositol 1, 4, 5-trisphosphate (IP3) sensitive stores in hippocampal neuron of rats through activation of delta2 subtype opioid receptor.

Animals ; Calcium ; metabolism ; Cells, Cultured ; Hippocampus ; cytology ; Male ; Microscopy, Confocal ; Morphine ; pharmacology ; Neurons ; cytology ; drug effects ; Rats ; Rats, Wistar

AIMTo establish the model of oxygen-glucose deprivation in vitro rat hippocampal neurons.

METHODSThe hippocampal neurons cultured for 12 d were exposed to combined oxygen-glucose deprivation for 0.5-4 h and then cultured with original medium in normoxia for 24 h. Auto-biochemical analyzer determined LDH activity. The change of neuronal morphology and neuron survival were observed by converted contrast microscope and assessed by photography analysis system. Neuron apoptosis was detected by using the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nickel end labeling (TUNEL) method.

RESULTSThe neurons swelled, LDH release increased and neuron survival decreased after gradually oxygen-glucose deprivation. The percentage of apoptosis increased obviously 24 h after recovering the supply of oxygen and glucose.

CONCLUSIONThe model of oxygen-glucose deprivation in vitro rat hippocampal neurons is established successfully by using the modified ACSF (artificial cerebral spinal fluid) with serum and glucose free.

Animals ; Animals, Newborn ; Cell Hypoxia ; Cells, Cultured ; Glucose ; deficiency ; Hippocampus ; cytology ; Neurons ; cytology ; Oxygen ; physiology ; Rats ; Rats, Wistar

It was reported that α7 nicotinic acetylcholine receptor (α7-nAChR) knockout (α7 KO) mice showed few functional phenotypes. The purpose of this study was to investigate the effect of α7 KO on the electrophysiological characteristics of hippocampus in mice. The effect of α7 KO on hippocampal CA3-CA1 synaptic transmission in mice was evaluated by standard extracellular field potential recordings. The electrophysiological phenotype of γ-aminobutyrate A receptors (GABA-Rs) of single hippocampal neuron was detected by perforated patch-clamp recordings. The results showed that, the slope of field excitatory postsynaptic potential (fEPSP) and carbachol-induced theta oscillation were significantly decreased in the hippocampal CA1 neurons of α7 KO mice, compared with those of wild type mice. Under the treatment of GABA-R agonist muscimol, the I-V curves of both the hippocampal CA1 and CA3 neurons of α7 KO mice shifted towards depolarizing direction obviously, compared with those of wild type mice. These results suggest that the hippocampal CA3-CA1 synaptic transmission in α7 KO mice was significantly impaired and GABA-R maturation was significantly delayed, indicating that the deletion of α7-nAChR gene could significantly change the electrophysiological function of the hippocampus. The results may provide a new understanding of the role of α7-nAChR in hippocampal function and associated diseases.
Animals ; Hippocampus ; cytology ; Mice ; Mice, Knockout ; Neurons ; physiology ; Phenotype ; Synaptic Transmission ; alpha7 Nicotinic Acetylcholine Receptor ; physiology

OBJECTIVETo explore the effect of low doses X-ray on proliferation of hippocampal pyramidal cell in the area of CA1 in prenatal rat and its relevant mechanism.

METHODSA total of 25 pregnant rats were randomly divided into four experimental groups and one control group. The experimental groups, in a duration of consistent 18 days, respectively received different doses as follows: 0.015 mGy/d, 0.03 mGy/d, 0.06 mGy/d and 0.09 mGy/d. The control group received sham radiation. To observe the density and width of hippocampal pyramidal cell in the area of CA1 by HE stained and observe the expression of the ERK1/2 by IHM.

RESULTS(1) Except C group, all other groups presented increment in width of the level of hippocampal pyramidal cell, compared with C group; H group, M group, L1 group and L2 group were higher than that (F value respectively were 8.475, 33.42, 14.395, 44.955; P value respectively were 0.002, 0.048, 0.030, 0.012). But the phenomenon of inhomogeneity in width in H group was observed, at the same time, the density of cell in H group became looser (F = 4.466, P = 0.017). (2) The expression of ERK1/2 in the hippocampus CA1 was seen in cytoplasm of every group, the average optical density of positive ERK1/2 protein significantly increased in L1 group and L2 group, compared with control group respectively (F value respectively were 4.561, 4.103, P value respectively were 0.044, 0.035).

CONCLUSIONLow doses X-ray could promote proliferation of hippocampus CA1 cell in prenatal. The reason could be the increment of the ERK1/2 protein induced by X-ray. When the doses reached 0.09 mGy/d, the excesses proliferation phenomenon was observed.

Animals ; Cell Proliferation ; radiation effects ; Female ; Hippocampus ; cytology ; radiation effects ; Male ; Maternal Exposure ; Neurons ; cytology ; radiation effects ; Pregnancy ; Pyramidal Cells ; cytology ; radiation effects ; Radiation, Ionizing ; Rats ; X-Rays

Angelica ; chemistry ; Animals ; Animals, Newborn ; Cell Count ; Female ; Fetal Hypoxia ; pathology ; Hippocampus ; cytology ; drug effects ; Male ; Neuroglia ; cytology ; Neurons ; cytology ; Pregnancy ; Rats ; Rats, Sprague-Dawley

In the adult brain, neural stem cells have been found in two major niches: the dentate gyrus and the subventricular zone [corrected]. Neurons derived from these stem cells contribute to learning, memory, and the autonomous repair of the brain under pathological conditions. Hence, the physiology of adult neural stem cells has become a significant component of research on synaptic plasticity and neuronal disorders. In addition, the recently developed induced pluripotent stem cell technique provides a powerful tool for researchers engaged in the pathological and pharmacological study of neuronal disorders. In this review, we briefly summarize the research progress in neural stem cells in the adult brain and in the neuropathological application of the induced pluripotent stem cell technique.
Hippocampus ; cytology ; metabolism ; Humans ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; Models, Biological ; Neural Stem Cells ; cytology ; metabolism ; transplantation ; Neurodegenerative Diseases ; metabolism ; pathology ; prevention & control ; Neurogenesis ; Signal Transduction