The existence of NMDA receptor and a new organizer protein, Shank, in the postsynaptic density was studied with the cultured hippocampal neurons using by double immunofluorescence method. The hippocampi from embryonic 18 days were dissected and hippocampal neurons were obtained from dissociated hippocampi with 0.25% trypsin and 0.1% DNase in PBS. The hippocampal neurons were plated with density 3,600/cm2 on the poly-L-lysine coated coverglass and cultured 37degrees C, 5% CO2 incubator for 5 weeks. The N2 supplemented MEM was used as a culture medium. Following results are obtained from experiments: 1. The 3~5 minor processes from the cell body of hippocampal neurons were observed at 20 hr in vitro. One of the minor processes was elongated and looked like an axon, and another minor processes showed dendritic branching pattern with slender in thickness. 2. The excitatory NMDA receptor colocalized with PSD-95 which is the postsynaptic density protein. The presynaptic protein, synapsin 1, was closely apposed with PSD-95. 3. Shank which is an organizer protein colocalize with NMDA receptor/PSD-95 complex in the postsynaptic density. Shank proteins may be concerned with the cluster formation of NMDA receptor/PSD-95 in the postsynaptic membrane.
Axons ; Deoxyribonucleases ; Fluorescent Antibody Technique ; Immunohistochemistry ; Incubators ; Membranes ; N-Methylaspartate* ; Neurons* ; Post-Synaptic Density ; Trypsin

Alpha-synuclein is a small neuronal protein that is closely associated with the etiology of Parkinson's disease. Mutations in and alterations in expression levels of alpha-synuclein cause autosomal dominant early onset heredity forms of Parkinson's disease, and sporadic Parkinson's disease is defined in part by the presence of Lewy bodies and Lewy neurites that are composed primarily of alpha-synuclein deposited in an aggregated amyloid fibril state. The normal function of alpha-synuclein is poorly understood, and the precise mechanisms by which it leads to toxicity and cell death are also unclear. Although alpha-synuclein is a highly soluble, cytoplasmic protein, it binds to a variety of cellular membranes of different properties and compositions. These interactions are considered critical for at least some normal functions of alpha-synuclein, and may well play critical roles in both the aggregation of the protein and its mechanisms of toxicity. Here we review the known features of alpha-synuclein membrane interactions in the context of both the putative functions of the protein and of its pathological roles in disease.
alpha-Synuclein* ; Amyloid ; Cell Death ; Cytoplasm ; Heredity ; Lewy Bodies ; Membranes* ; Neurites ; Neurons ; Parkinson Disease ; Synaptic Transmission

Epilepsy is one of the most common episodic neurological diseases, and patients with epilepsy may experience a range of neurological, psychological, and behavioral problems. Recurring seizures potentially contribute to the progressive severity of epilepsy, cognitive and behavioral consequences. The clinical and experimental evidences involving radiological, pathological, and biochemical studies suggest that seizures can potentially injure the brain via a number of diverse molecular, cellular, and network mechanisms. The damage includes neuronal death, axodendritic changes, molecular changes of synaptic membrane, and gliosis and increased neurogenesis. Those changes induce rewiring of the network and reorganization of synapses, causing alteration of the functional and morphological properties as the mechanism of epilepsy. As the most overt form of alterations, the neuronal death may result from the execution of cellular programs that are similar to the molecular machinery of programmed cell death including the caspases and bcl-2 family proteins. In epileptic seizure, the neurons are overexcited and run out of energy. The low energy state is closely related with the necrotic pathway. The features suggest that the neuronal death in epilepsy may follow characteristic mechanism, suggesting necrotic programmed cell death pathway. Therapeutic modification of seizure-induced death could open new strategy in epilepsy treatment.
Brain ; Caspases ; Cell Death ; Epilepsy* ; Gliosis ; Humans ; Neurogenesis ; Neurons* ; Seizures ; Synapses ; Synaptic Membranes

Previous studies have suggested that brain stem noradrenergic inputs differentially modulate neurons in the paraventricular nucleus (PVN). Here, we compared the effects of norepinephrine (NE) on spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) in identified PVN neurons using slice patch technique. In 17 of 18 type I neurons, NE (30-100microM) reversibly decreased sIPSC frequency to 41+/-7% of the baseline value (4.4+/-0.8 Hz, p<0.001). This effect was blocked by yohimbine (2-20microM), an alpha2-adrenoceptor antagonist and mimicked by clonidine (50 microM), an alpha2-adrenoceptor agonist. In contrast, NE increased sIPSC frequency to 248+/-32% of the control (3.06+/-0.37 Hz, p<0.001) in 31 of 54 type II neurons, but decreased the frequency to 41+/-7% of the control (5.5+/-1.3 Hz) in the rest of type II neurons (p<0.001). In both types of PVN neurons, NE did not affect the mean amplitude and decay time constant of sIPSCs. In addition, membrane input resistance and amplitude of sIPSC of type I neurons were larger than those of type II neurons tested (1209 vs. 736 M omega, p<0.001; 110 vs. 81 pS, p<0.001). The results suggest that noradrenergic modulation of inhibitory synaptic transmission in the PVN decreases the neuronal excitability in most type I neurons via alpha2-adrenoceptor, however, either increases in about 60% or decreases in 40% of type II neurons.
Brain Stem ; Clonidine ; Inhibitory Postsynaptic Potentials* ; Membranes ; Neurons ; Norepinephrine ; Paraventricular Hypothalamic Nucleus* ; Synaptic Transmission ; Yohimbine

The N-methyl-D-aspartate (NMDA) receptor-mediated glutamatergic neurotransmission is involved in synaptic plasticity, developmental processes, learning and memory and many neuropathological disorders including age-related diseases. In the present study, regulation of the NMDA receptor properties by various ligands was investigated using (3H)MK-801 binding studies in the synaptic membranes of young and aged rat forebrains. The binding in the presence of glutamate and glycine increased dramatically with growth between 1 and 6 weeks old, and thereafter declined gradually with aging. Glutamate, glycine or spermine respectively increased the binding with growth. Glutamate maintained the binding during aging, while glycine or spermine significantly decreased the binding in the aged brain. The maximum stimulation by glycine varied depending on the ages of brains. Greater sensitivity to glycine was observed at 1 week and 3 months and the sensitivity was significantly reduced in the aged brain. In contrast, spermine showed similar stimulation patterns in young and aged rats. These results indicated that the functional properties of the NMDA receptor-ion channel complex in young and aged rat forebrains are differentially regulated by agonists, and the reduction of the receptor function with normal aging may be, in some degree, due to the reduction of the receptor sensitivity to glycine.
Aging ; Animals ; Brain ; Dizocilpine Maleate ; Glutamic Acid* ; Glycine* ; Learning ; Ligands ; Memory ; N-Methylaspartate ; Plastics ; Prosencephalon* ; Rats* ; Spermine* ; Synaptic Membranes* ; Synaptic Transmission

Introduction of patch-clamp techniques allowed an increase in resolution of membrane current recordings. However, the technique was limited by apparent need for direct contact of pipette with cell membrane. Thus, this technique was restricted to isolated or cultured cell preparation. Although much has been achieved with such preparations, the studies of synapsis between cultured cells are undefined. Many of these problems were overcome by application of patch-clamp techniques to brain-slices. The use of high-resolution optics allowed visualization of cells to be recorded. It was possible to remove tissue covering cells and record currents in synaptically connected neurons. The brain-slice technique has greatly facilitated the investigation of electrical properties of neurons and the analysis of synaptic transmission between neurons. "Blow and seal"technique, when combined with infrared differential interference contrast video microscopy, permits recording of membrane potential and currents, not only from large cell body of neurons, but also from small processes. The technique offers many advantages, such as the case with which patch-pipette recordings can be made, the possibility of identifying cell type prior to recording and finally, the ability to visualize and record electrical activity from different compartments or from more than one site in the same neuron.
Anesthetics, General ; Cell Membrane ; Cells, Cultured ; Chromosome Pairing ; Membrane Potentials ; Membranes ; Microscopy, Video ; Neurons ; Patch-Clamp Techniques ; Synaptic Transmission

Ginseng root, as a folk medicine, has been used in for eastern countries for thousands of years. Ginseng extract has been shown to have a variety of effects on the activity of the central nervous system, promoting simulation as well as inhibition of the cortical activity. A survey of the relevant literatures has indicated that the putative anxiolytic activity of red ginseng has not been scientifically investigated. Therefore, the present study was designed to assess anxiolytic effect of ginseng total saponinis(GTS). The putative anxiolytic effects of several fractions of GTS were investigated in mice using an elevated plus maze paradigm. Single dose administration of TS Fr.- I showed anxiolytic action in mice. Anxiolytic effect induced by TS Fr.-I was similar to that induced by diazepam. TS Fr.-II, TS Fr.-III and TS Fr.-IV did not show the anxiolytic action compared with that of TS Fr.-I. It was suggested that regulation of GABAergic neurotransmission may be important in the action of GTS. The Interaction of GTS fractions with benzodiazepine receptor was performed using rat cortical membranes. GTS inhibited the binding of [3H] Rp 15-1788 on the benzodiazepine receptor. Among from TS fractions, the binding activity of GTS in the TS Fr.-IV was highest, which did not show the anxiolytic activity. From these results, we conclude that GTS has anxiolytic action, and the is not related to benzodiazepine receptor binding activity.
Animals ; Anti-Anxiety Agents* ; Central Nervous System ; Diazepam ; Medicine, Traditional ; Membranes ; Mice ; Panax* ; Rats ; Receptors, GABA-A ; Saponins* ; Synaptic Transmission

Nitric oxide (NO) is a small molecule (mol. wt. 30 Da) and oxidative free radical. It is uncharged and can therefore diffuse freely within and between cells across membrane. Such characteristics make it a biologically important messenger in physiologic processes such as neurotransmission and the control of vascular tone. NO is also highly toxic and is known to acts as a mediator of cytotoxicity during host defense. NO is synthesized by nitric oxide synthase (NOS) through L-arginine/nitric oxide pathway which is a dioxygenation process. NO synthesis involves several participants, three co-substrates, five electrons, five co-factors and two prosthetic groups. Under normal condition, low levels of NO are synthesized by type I and III NOS for a short period of time and mediates many physiologic processes. Under condition of oxidant stress, high levels of NO are synthesized by type II NOS and inhibits a variety of metabolic processes and can also cause direct damage to DNA. Such interaction result in cytostasis, energy depletion and ultimately cell death. NO has the potential to interact with a variety of intercellular targets producing diverse array of metabolic effects. It is known that NO is involved in hemodynamic regulation, neurogenic inflammation, re-innervation, management of dentin hypersensitivity on teeth. Under basal condition of pulpal blood flow, NO provides constant vasodilator tone acting against sympathetic vasoconstriction. Substance P, a well known vasodilator, was reported to be mediated partly by NO, while calcitonin-gene related peptide has provided no evidence of its relation with NO. This review describes the roles of NO in dental pulp in addition to the known general roles of it.
Cell Death ; Dental Pulp ; Dentin Sensitivity ; DNA ; Electrons ; Hemodynamics ; Membranes ; Neurogenic Inflammation ; Nitric Oxide ; Nitric Oxide Synthase ; Oxygen ; Substance P ; Synaptic Transmission ; Tooth ; Vasoconstriction

Reactive oxygen species (ROS) and nitrogen species (RNS) are implicated in cellular signaling processes and as a cause of oxidative stress. Recent studies indicate that ROS and RNS are important signaling molecules involved in nociceptive transmission. Xanthine oxidase (XO) system is a well-known system for superoxide anions (O2(.-)) generation, and sodium nitroprusside (SNP) is a representative nitric oxide (NO) donor. Patch clamp recording in spinal slices was used to investigate the role of O2(.-) and NO on substantia gelatinosa (SG) neuronal excitability. Application of xanthine and xanthine oxidase (X/XO) compound induced membrane depolarization. Low concentration SNP (10 microM) induced depolarization of the membrane, whereas high concentration SNP (1 mM) evoked membrane hyperpolarization. These responses were significantly decreased by pretreatment with phenyl N-tert-butylnitrone (PBN; nonspecific ROS and RNS scavenger). Addition of thapsigargin to an external calcium free solution for blocking synaptic transmission, led to significantly decreased X/XO-induced responses. Additionally, X/XO and SNP-induced responses were unchanged in the presence of intracellular applied PBN, indicative of the involvement of presynaptic action. Inclusion of GDP-beta-S or suramin (G protein inhibitors) in the patch pipette decreased SNP-induced responses, whereas it failed to decrease X/XO-induced responses. Pretreatment with n-ethylmaleimide (NEM; thiol-alkylating agent) decreased the effects of SNP, suggesting that these responses were mediated by direct oxidation of channel protein, whereas X/XO-induced responses were unchanged. These data suggested that ROS and RNS play distinct roles in the regulation of the membrane excitability of SG neurons related to the pain transmission.
Animals ; Calcium ; Ethylmaleimide ; Humans ; Membranes ; Neurons* ; Nitric Oxide ; Nitrogen* ; Nitroprusside ; Oxidative Stress ; Rats* ; Reactive Oxygen Species* ; Substantia Gelatinosa* ; Superoxides ; Suramin ; Synaptic Transmission ; Thapsigargin ; Tissue Donors ; Xanthine ; Xanthine Oxidase

PURPOSE: Recombinant human growth hormone (rhGH) has been widely used to treat short stature. However, there are some concerns that growth hormone treatment may induce skeletal maturation and early onset of puberty. In this study, we investigated whether rhGH can directly affect the neuronal activities of of gonadotropin-releasing hormone (GnRH). METHODS: We performed brain slice gramicidin-perforated current clamp recording to examine the direct membrane effects of rhGH on GnRH neurons, and a whole-cell voltage-clamp recording to examine the effects of rhGH on spontaneous postsynaptic events and holding currents in immature (postnatal days 13-21) and adult (postnatal days 42-73) mice. RESULTS: In immature mice, all 5 GnRH neurons recorded in gramicidin-perforated current clamp mode showed no membrane potential changes on application of rhGH (0.4, 1 microgram/mL). In adult GnRH neurons, 7 (78%) of 9 neurons tested showed no response to rhGH (0.2-1 microgram/mL) and 2 neurons showed slight depolarization. In 9 (90%) of 10 immature neurons tested, rhGH did not induce any membrane holding current changes or spontaneous postsynaptic currents (sPSCs). There was no change in sPSCs and holding current in 4 of 5 adult GnRH neurons. CONCLUSION: These findings demonstrate that rhGH does not directly affect the GnRH neuronal activities in our experimental model.
Adult ; Animals ; Brain ; Gonadotropin-Releasing Hormone ; Growth Hormone ; Human Growth Hormone ; Humans ; Membrane Potentials ; Membranes ; Mice ; Models, Theoretical ; Neurons ; Puberty ; Synaptic Potentials