AIMTo establish the whole-cell recording techniques of the neuronal alpha4beta2, alpha4beta4, and alpha7-nicotinic acetylcholine receptors heterologously expressed in SH-EP1 cell line and discuss the electrophysiological characteristics of their open states.

METHODSThe cells were cultured with DEME medium(high glucose) and suitable for electrophysiological experiments three days after passage. The receptors were induced from resting states into open states by rapid application of nicotine (alpha4beta2, alpha4beta4) or choline (alpha7).

RESULTSThe SH-EP1 cells cultured by this method were in good conditions and expressed plenty of receptors. Alpha4beta2, alph4beta4 and alpha7 inward currents could be induced by rapid application of agonists but had different dynamic processes against time. All the three types of currents were dose and voltage-dependent and had inward rectification property.

CONCLUSIONThe open states of neuronal alpha4beta2, alpha4beta4, and alpha7-nicotinic acetylcholine receptors and their transitions have distinct characteristics and the inward currents of all this three types of receptors are dose and voltage-dependent and have inward rectification property.

Brain ; cytology ; metabolism ; Cell Line ; Epithelial Cells ; cytology ; Humans ; Membrane Potentials ; physiology ; Neurons ; cytology ; metabolism ; Patch-Clamp Techniques ; Receptors, Nicotinic ; physiology ; Transfection ; alpha7 Nicotinic Acetylcholine Receptor

This study was done to determine the effects of hypothermia on brain cell membrane function and energy metabolism after transient hypoxia-ischemia (HI) in the newborn piglet. Cerebral HI was induced by temporarily complete occlusion of bilateral common carotid arteries with surgical clips and simultaneous breathing with 8% oxygen for 30 min, followed by release of carotid occlusion and normoxic ventilation for 4 hr. Rectal temperature was maintained between 38.0 and 39.0 degrees C in normothermic groups, and between 34.0 and 35.0 degrees C in hypothermic groups for 4 hr after HI. During HI, heart rate, glucose and lactate level in the blood and cerebrospinal fluid increased, and base excess, pH and blood pressure decreased significantly in both normothermic and hypothermic groups. After HI, these abnormalities returned to normal in normothermic group, but lactic acidosis persisted in hypothermic group. Decreased cerebral Na(+),K(+)- ATPase activity and increased lipid peroxidation products, indicative of HI- induced brain injury, were more profound in hypothermic group than in normothermic group. Brain ATP and phosphocreatine levels were not different between normothermic and hypothermic groups. In summary, hypothermia applied immediately after HI for 4 hr did not improve the recovery of brain cell membrane function and energy metabolism in the newborn piglet.
Animal ; Animals, Newborn ; Brain/cytology/*metabolism/physiology ; Cell Membrane/physiology ; Energy Metabolism ; Glucose/metabolism ; *Hypothermia, Induced ; Hypoxia-Ischemia, Brain/*metabolism/physiopathology/therapy ; Lactic Acid/metabolism ; Na(+)-K(+)-Exchanging ATPase/metabolism ; Swine

Various stem cells, including neural stem cells (NSCs), have been extensively studied in stroke models, but how to increase neuronal differentiation rate of NSCs remains unresolved, particularly in a damaged environment. The purpose of this study was to investigate the effects of cerebral microvascular endothelial cells (CMECs) on the neurogenesis of NSCs with or without oxygen-glucose deprivation (OGD). The NSCs acquired from primary culture were immunostained to prove cell purity. Survival and proliferation of NSCs were determined after the co-culture with CMECs for 7 days. After removing the CMECs, NSCs were randomly divided into two groups as follows: OGD and non-OGD groups. Both groups were maintained in differentiation culture for 4 days to evaluate the differentiation rate. Mouse embryo fibroblast (MEF) cells co-cultured with NSCs served as control group. NSCs co-cultured with CMECs had an increase in size (on the 7th day: 89.80±26.12 μm vs. 73.08±15.01 μm, P<0.001) (n=12) and number [on the 7th day: 6.33±5.61/high power objective (HP) vs. 2.23±1.61/HP, P<0.001] (n=12) as compared with those co-cultured with MEF cells. After further differentiation culture for 4 days, NSCs co-cultured with CMECs had an increase in neuronal differentiation rate in OGD and non-OGD groups, but not in the control group (15.16% and 16.07% vs. 8.81%; both P<0.001) (n=6). This study provided evidence that OGD could not alter the effects of CMECs in promoting the neuronal differentiation potential of NSCs. These findings may have important implications for the development of new cell therapies for cerebral vascular diseases.
Animals ; Animals, Newborn ; Brain ; blood supply ; Cell Differentiation ; physiology ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; methods ; Endothelial Cells ; cytology ; metabolism ; Glucose ; metabolism ; Mice ; Mice, Inbred C57BL ; Microvessels ; cytology ; metabolism ; Neural Stem Cells ; cytology ; metabolism ; Oxygen ; metabolism

OBJECTIVE: To identify the subtype of transient receptor potential (TRPs) channel involved in stretch-induced injury of human brain microvascular endothelial cells (HBMEC) and to explore the mechanism responsible for eNOS expression.
 METHODS: TRPs expression was examined by Western blot and immunocytofluoresence in the cultured HBMEC. Mechanical stretch was performed by mini-type multi-functional bio-impact machine. The levels of free calcium ion in cells were examined by the flow cytometry. The eNOS expression was detected by Western blot.
 RESULTS: The mRNA and protein expression of TRPV4 was detected in HBMEC by qRT-PCR, Western blot and immunocytofluoresence. The levels of free calcium ion in the stretch-treated HBMEC was significantly decreased in the presence of TRPV4 specific inhibitor (P<0.001), but there was no difference in calcium levels between the stretch and the control or unspecific inhibitor group (P=0.072 or 0.308). The levels of eNOS protein in the stretch-treated HBMEC were reduced in the presence of TRPV4 specific inhibitor or NOS inhibitor (P<0.05), but it was not changed compared with that in the control group (P>0.05).
 CONCLUSION The eNOS expression is up-regulated under the condition of mechanic stretch, which is related to the activation of TRPV4, resulting in the influx of calcium.
Brain ; cytology ; Calcium ; metabolism ; Cells, Cultured ; Endothelial Cells ; physiology ; Humans ; Nitric Oxide Synthase Type III ; metabolism ; Stress, Mechanical ; TRPV Cation Channels ; physiology

BACKGROUNDNeural stem cells (NSCs) transplantation and gene therapy have been widely investigated for treating the cerebullar and myelonic injuries, however, studies on the ophthalmology are rare. The aim of this study was to investigate the migration and differentiation of brain-derived neurotrophic factor (BDNF) gene transgenic NSCs transplanted into the normal rat retinas.

METHODSNSCs were cultured and purified in vitro and infected with recombinant retrovirus pLXSN-BDNF and pLXSN respectively, to obtain the BDNF overexpressed NSCs (BDNF-NSCs) and control cells (p-NSCs). The expression of BDNF genes in two transgenic NSCs and untreated NSCs were measured by fluorescent quantitative polymerase chain reaction (FQ-PCR) and enzyme-linked immunosorbent assay (ELISA). BDNF-NSCs and NSCs were infected with adeno-associated viruses-enhanced green fluorescent protein (AAV-EGFP) to track them in vivo and served as donor cells for transplantation into the subretinal space of normal rat retinas, phosphated buffer solution (PBS) served as pseudo transplantation for a negative control. Survival, migration, and differentiation of donor cells in host retinas were observed and analyzed with Heidelberg retina angiograph (HRA) and immunohistochemistry, respectively.

RESULTSNSCs were purified successfully by limiting dilution assay. The expression of BDNF gene in BDNF-NSCs was the highest among three groups both at mRNA level tested by FQ-PCR (P < 0.05) and at protein level measured by ELISA (P < 0.05), which showed that BDNF was overexpressed in BDNF-NSCs. The results of HRA demonstrated that graft cells could survive well and migrate into the host retinas, while the immunohistochemical analysis revealed that transplanted BDNF-NSCs differentiated into neuron more efficiently compared with the control NSCs 2 months after transplantation.

CONCLUSIONSThe seed cells of NSCs highly secreting BDNF were established. BDNF can promote NSCs to migrate and differentiate into neural cells in the normal host retinas.

Animals ; Brain-Derived Neurotrophic Factor ; genetics ; metabolism ; Cell Differentiation ; physiology ; Cell Movement ; physiology ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Enzyme-Linked Immunosorbent Assay ; Immunohistochemistry ; Neurons ; cytology ; Rats ; Retina ; cytology ; metabolism ; Stem Cell Transplantation

Endothelin (ET)-1 and its receptors (ETA and ETB receptor) are present in the central nervous system. ET exerts biological effects on gliogenesis and glial cell functions. In order to define a possible mechanism of ETA receptor signaling, the distribution of the ETA receptor in developing oligodendrocytes and the effects of ET-1 on the myelination of oligodendrocytes were examined. ETA receptor immunoreactivity was confined to the perivascular elements of the blood vessels during early postnatal development. However later in development, ETA receptor immunoreactivity was no longer observed in the vessels but became localized to the myelinating oligodendrocytes of the primitive corpus callosum of the white matter, apart from the vessels. ET-1 induced myelin basic protein (MBP) in primary oligodendrocyte precursor cell culture though the ETA receptor and was blocked by an ETA receptor antagonist. In addition, ET-1 evoked the release of Ca2+ which is a central regulator of oligodendrocyte differentiation. Our results provide a link between ET-1 and its ETA receptor and myelination during oligodendrocyte differentiation.
Animals ; Brain/pathology ; Calcium/metabolism ; Calcium Signaling ; Cells, Cultured ; Endothelin-1/metabolism/physiology ; Mice ; Mice, Inbred ICR ; Myelin Basic Proteins/genetics/metabolism ; Myelin Sheath/*physiology ; Oligodendroglia/cytology/*metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Endothelin A/metabolism/*physiology

OBJECTIVETo investigate the cell proliferation and differentiation in the developing brain of mouse.

METHODSC57/BL6 mice were divided into 3 groups at random. Bromodeoxyuridine (BrdU) was injected into the brains in different development periods once a day for 7 d. The brains were retrieved 4 weeks after the last BrdU injection. Immunohistochemical and immunofluorescent studies were carried out for detecting cell proliferation (BrdU) and cell differentiation (NeuN, APC, Iba1, and S100beta), respectively.

RESULTSThe number of BrdU labeled cells decreased significantly with the development of the brain. Cell proliferation was prominent in the cortex and striatum. A small portion of BrdU and NeuN double labeled cells could be detected in the cortex at the early stage of development, and in the striatum and CA of the hippocampus in all groups. The majority of BrdU labeled cells were neuroglia, and the number of neuroglia cells decreased dramatically with brain maturation. Neurogenesis is the major cytogenesis in the dentate gyrus.

CONCLUSIONThese results demonstrated that cell proliferation, differentiation and survival were age and brain region related.

Animals ; Animals, Newborn ; Brain ; cytology ; growth & development ; Bromodeoxyuridine ; Cell Count ; Cell Differentiation ; physiology ; Cell Proliferation ; Cerebral Cortex ; cytology ; growth & development ; Corpus Striatum ; cytology ; growth & development ; Fluorescent Antibody Technique ; Hippocampus ; cytology ; growth & development ; Male ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins ; metabolism ; Neuroglia ; cytology ; physiology ; Neurons ; cytology ; physiology ; Nuclear Proteins ; metabolism

OBJECTIVETo investigate the involvement of bone marrow stem cell-derived astrocytes (BMDSCs) in the formation of glia limitans after brain injury.

METHODSIn a female SD rat model of brain injury, green fluorescence protein (GFP)-labeled BMDSCs from male SD rats were transplanted via the caudal vein 24 h after the injury. The rats were sacrificed at 2, 4 and 8 weeks after the transplantation, and immunohistochemistry for glial fibrillary acidic protein (GFAP) was performed to observe the astrocytes. The fluorescence emitted by GFP was observed to identify the presence of the bone marrow-derived stem cells, and the GFAP(+)/GFP(+) cells in the glia limitnas were detected under fluorescence microscopy. RESULTS The GFAP(+)/GFP(+) cells were found in the glia limitans between the brain lesion and normal brain tissue.

CONCLUSIONBone marrow stem cell-derived astrocytes is involved in glia limitans formation after brain injury, which can be of significance in brain injury recovery and implantation of engineered materials.

Animals ; Astrocytes ; cytology ; physiology ; Bone Marrow Cells ; cytology ; metabolism ; Brain Injuries ; pathology ; Female ; Glial Fibrillary Acidic Protein ; metabolism ; Green Fluorescent Proteins ; Male ; Mesenchymal Stromal Cells ; cytology ; Neuroglia ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley

Animals choose among sleep, courtship, and feeding behaviors based on the integration of both external sensory cues and internal states; such choices are essential for survival and reproduction. These competing behaviors are closely related and controlled by distinct neural circuits, but whether they are also regulated by shared neural nodes is unclear. Here, we investigated how a set of male-specific P1 neurons controls sleep, courtship, and feeding behaviors in Drosophila males. We found that mild activation of P1 neurons was sufficient to affect sleep, but not courtship or feeding, while stronger activation of P1 neurons labeled by four out of five independent drivers induced courtship, but only the driver that targeted the largest number of P1 neurons affected feeding. These results reveal a common neural node that affects sleep, courtship, and feeding in a threshold-dependent manner, and provide insights into how competing behaviors can be regulated by a shared neural node.
Animals ; Animals, Genetically Modified ; Brain ; cytology ; Courtship ; Drosophila ; Drosophila Proteins ; genetics ; metabolism ; Feeding Behavior ; physiology ; Locomotion ; Male ; Neural Inhibition ; physiology ; Neural Pathways ; physiology ; Neurons ; physiology ; Sex Factors ; Sleep ; physiology

OBJECTIVETo investigate the morphology alterations and proteomics changes in primary astrocytes following fluid percussion injury.

METHODSPrimary cultures of astrocytes were prepared from cerebral hemispheres of 1-3 d-old SD rats, then, astrocytes were randomly divided into control group and injury group which were subjected to (0.2 +/- 0.01) MPa fluid percussion injury. The changes of protein expression pattern in astrocytes between injury and control groups were monitored with two dimensional gel electrophoresis.

RESULTSAstrocytes' s abnonmalities of morphology after injury were apparent. The fluid percussion injury caused astrocytes edema, shrinkage, cell junction disconnection and necrosis at 2 h after injury. 24 h and 48 h after injury, most part of astrocytes's dendrites and soma became hypertrophy and showed a higher rate of cell proliferation. The dynamic proteomics changes were identified and total different 13 spots were detected in this study from the 2DE gels. The different displayed 5 spots were identified via MALDI-TOF: cofilin 1, destrin, phosphoglycerate mutase 1, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10, annexin 1.

CONCLUSIONThe obvious alteration of morphology and protein expression pattern in primary cultured astrocytes could be induced after fluid percussion injury. The differential proteins detected were probably related to stress responses.

Animals ; Animals, Newborn ; Astrocytes ; cytology ; metabolism ; Brain Injuries ; metabolism ; physiopathology ; Cerebral Cortex ; cytology ; Female ; Male ; Primary Cell Culture ; methods ; Proteins ; metabolism ; Proteome ; analysis ; Proteomics ; methods ; Rats ; Rats, Sprague-Dawley ; Stress, Physiological ; physiology