(1/900) Bound forms of Ca taken up by the synaptic plasma membrane.
Temperature dependent Ca-binding by the synaptic plasma membrane was increased in the presence of ATP and Mg++. Apparent Km for ATP was about 2.8 X 10(-5) M and optimal concentration of Mg++ was 2 mM in the presence of 2 mM ATP. After preincubation with nonradioactive Ca++, ATP and Mg++ to attain a steady state, addition of 45Ca resulted in remarkable labelling of the membrane, indicating rapid turnover of most of the membrane bound Ca. The presence of oxalate (60 mM) greatly increased Ca up-take on prolonged incubation. The Ca uptake in presence and absence of oxalate had similar substrate specificity and was similarly influenced by various monovalent cations. Furthermore, activities for Ca-uptake in the presence and absence of oxalate could not be separated by sucrose density gradient centrifugation of the synaptic plasma membrane fraction. Accordingly, it was considered that Ca++ in the medium was taken up by surface of the membrane, ATP- and temperature-dependently and then transferred into a cavity where the Ca-oxalate complex is formed. (+info)
(2/900) Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity.
Activity shapes the structure of neurons and their circuits. Two-photon imaging of CA1 neurons expressing enhanced green fluorescent protein in developing hippocampal slices from rat brains was used to characterize dendritic morphogenesis in response to synaptic activity. High-frequency focal synaptic stimulation induced a period (longer than 30 minutes) of enhanced growth of small filopodia-like protrusions (typically less than 5 micrometers long). Synaptically evoked growth was long-lasting and localized to dendritic regions close (less than 50 micrometers) to the stimulating electrode and was prevented by blockade of N-methyl-D-aspartate receptors. Thus, synaptic activation can produce rapid input-specific changes in dendritic structure. Such persistent structural changes could contribute to the development of neural circuitry. (+info)
(3/900) In vivo modulation of rodent glutathione and its role in peroxynitrite-induced neocortical synaptosomal membrane protein damage.
Peroxynitrite, formed by the reaction between nitric oxide and superoxide, leads to the oxidation of proteins, lipids, and DNA, and nitrates thiols such as cysteine and glutathione, and amino acids like tyrosine. Previous in vitro studies have shown glutathione to be an efficient scavenger of peroxynitrite, protecting synaptosomal membranes from protein oxidation, the enzyme glutamine synthetase from inactivation, and preventing the death of hippocampal neurons in culture. The current study was undertaken to see if in vivo modulation of glutathione levels would affect brain cortical synaptosomal membrane proteins and their subsequent reaction with peroxynitrite. Glutathione levels were depleted, in vivo, by injecting animals with 2-cyclohexen-1-one (CHX, 100 mg/kg body weight), and levels of glutathione were enhanced by injecting animals with N-acetylcysteine (NAC, 200 mg/kg body weight), which gets metabolized to cysteine, a precursor of glutathione. Changes in membrane protein conformation and structure in synaptosomes subsequently isolated from these animals were examined using electron paramagnetic resonance, before and after in vitro addition of peroxynitrite. The animals injected with the glutathione depletant CHX showed greater damage to the membrane proteins both before and after peroxynitrite treatment, compared to the non-injected controls. The membrane proteins from animals injected with NAC were comparable to controls before peroxynitrite treatment and were partially protected against peroxynitrite-induced damage. This study showed that modulation of endogenous glutathione levels can affect the degree of peroxynitrite-induced brain membrane damage and may have potential therapeutic significance for oxidative stress-associated neurodegenerative disorders. (+info)
(4/900) Diversity of thyrotropin-releasing hormone receptors in the pituitary and discrete brain regions of rats.
In order to analyze the receptor properties of central nervous system (CNS)-stimulant thyrotropin-releasing hormone (L-pyroglutamyl-L-histidyl-L-prolinamide, TRH), we evaluated the binding of TRH and its analog taltirelin hydrate ((-)-N-[(S)-hexahydro-1-methyl-2,6-dioxo-4-pyrimidinylcarbonyl]-L- histidyl-L-prolinamide tetrahydrate; taltirelin, TA-0910) in rat anterior pituitary and several brain regions. There was a specific binding of [3H]methyl TRH (MeTRH) in the anterior pituitary, hypothalamus, brain stem, cerebral cortex and cerebellum with Kd values of 1.0-1.6 nM. The inhibition of [3H]MeTRH binding by TRH and taltirelin was monophasic in the anterior pituitary, hypothalamus and brain stem with Ki values of 6.3-8.0 nM and 145.5-170.4 nM for TRH and taltirelin, respectively. In contrast, the biphasic inhibition was revealed in the cerebral cortex and cerebellum. The Ki values for TRH and taltirelin were 4.1-4.3 nM and 67.8-73.4 nM for the high affinity binding site and 3.6-4.2 microM and 82.3-197.5 microM for the low affinity binding site, respectively. Addition of 100 microM GTP or its analog 5'-guanylylimidodiphosphate (Gpp[NH]p) affected neither the biphasic inhibition by TRH nor that by taltirelin. Thus the results suggest the presence of distinct high and low affinity TRH receptors in the CNS in contrast to the pituitary. (+info)
(5/900) Studies of excitable membranes. II. A comparison of specializations at neuromuscular junctions and nonjunctional sarcolemmas of mammalian fast and slow twitch muscle fibers.
Mammalian fast and slow twitch skeletal muscles are compared by freeze-fracture, thick and thin sectioning, and histochemical techniques using conventional and high voltage electron microscopy. Despite gross morphological differences in endplate structure visualized at relatively low magnifications in this sections, rat extensor digitorum longus (EDL) (fast twitch) and soleus (slow twitch) fibers cannot be distinguished on the basis of size, number, or distribution of molecular specializations of the pre- and postsynaptic junctional membranes exposed by freeze fracturing. Specializations in the cortex of the juxtaneuronal portions of the junctional folds are revealed by high voltage electron stereomicroscopy as a branching, ladder-like filamentous network associated with the putative acetylcholline receptor complexes. These filaments are considered to be involved in restricting the mobility of receptor proteins to the perineuronal aspects of the postynaptic membrane. Although the junctional membranes of both EDL and soleus appear similar, a differential specialization of the secondary synaptic cleft was noted. The extracellular matrix in the bottom of soleus clefts was observed as an ordered system of filamentous "combs," These filamentous arrays have not been detected in EDL junctions. Examination of the extrajunctional sarcolemmas of EDL and soleus reveal additional differences which may be correlated with variations in electrical and contractile properties. For example, particle aggregates termed "square arrays" previously described in the sarcolemmas of some fibers of the rat diaphragm were observed in large numbers in sarcolemmas of EDL fibers but were seldom encountered in soleus fibers. These gross compositional differences in the membranes are discussed in the light of functional differences between fiber types. (+info)
(6/900) Effects of specific modifications of several hydroxyls of tetrodotoxin on its affinity to rat brain membrane.
The widely used sodium channel blocker tetrodotoxin (TTX) is a compound that has six hydroxyl residues at the C-4, C-6, C-8, C-9, C-10, and C-11 positions in addition to a guanidinium group, which is positively charged in biological pH range. Thirteen analogs of this toxin with structural modifications involving one or more of these hydroxyls were examined on their affinity to a rat brain membrane preparation, which is known to contain sodium channels abundantly. The equilibrium dissociation constants associated with the binding of TTX and its analogs to the sodium channels were estimated, from their ability to inhibit the binding of [3H]saxitoxin, as follows (in nM): TTX, 1.8; chiriquitoxin, 1.0; 11-oxoTTX, 1.5; 11-norTTX-6,6-diol, 1.6; 11-norTTX-6(S)-ol, 23; 11-norTTX-6(R)-ol, 31; 11-deoxyTTX, 37; 6-epiTTX, 39; 4-epiTTX, 68; 4,9-anhydroTTX, 180; TTX-8-O-hemisuccinate, >380; TTX-11-carboxylic acid, >2300; tetrodonic acid, >3600; 5,6,11-trideoxyTTX, >5000. The reduction of the affinity observed with the analogs involving reduction or translocation of the hydroxyls at C-6 and C-11 is indicative of the contribution of these residues to the binding to sodium channels as hydrogen bond donors. The especially large value of the dissociation constant for TTX-11-carboxylic acid is consistent with the idea that the C-11-hydroxyl forms a hydrogen bond with a carboxylic acid residue of the channel protein. The markedly low affinity of TTX-8-O-hemisuccinate may possibly be ascribable to intramolecular salt-bridge formation, which neutralizes the positive charge of the guanidinium group. (+info)
(7/900) Empty synaptic vesicles recycle and undergo exocytosis at vesamicol-treated motor nerve terminals.
We investigated whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve-muscle preparations were treated with 5 microM vesamicol to inhibit the vesicular ACh transporter and then were exposed to an elevated potassium solution, 35 mM potassium propionate (35 KP), to release all preformed quanta of ACh. At vesamicol-treated endplates, miniature endplate current (MEPC) frequency increased initially from 0.4 to >300 s-1 in 35 KP but then declined to <1 s-1 by 90 min. The decrease in frequency was not accompanied by a decrease in MEPC average amplitude. Nerve terminals accumulated the activity-dependent dye FM1-43 when exposed to the dye for the final 6 min of a 120-min exposure to 35 KP. Thus synaptic membrane endocytosis continued at a high rate, although MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1-43 and then destained, confirming that exocytosis also still occurred at a high rate. These results demonstrate that recycled cholinergic synaptic vesicles that were not refilled with ACh continued to dock and undergo exocytosis after membrane retrieval. Thus transport of ACh into recycled cholinergic vesicles is not a requirement for repeated cycles of exocytosis and retrieval of synaptic vesicle membrane during prolonged stimulation of motor nerve terminals. (+info)
(8/900) The subcellular localizations of atypical synaptotagmins III and VI. Synaptotagmin III is enriched in synapses and synaptic plasma membranes but not in synaptic vesicles.
Multiple synaptotagmins are expressed in brain, but only synaptotagmins I and II have known functions in fast, synchronous Ca2+-triggered neurotransmitter release. Synaptotagmin III was proposed to regulate other aspects of synaptic vesicle exocytosis, particularly its slow component. Such a function predicts that synaptotagmin III should be an obligatory synaptic vesicle protein, as would also be anticipated from its high homology to synaptotagmins I and II. To test this hypothesis, we studied the distribution, developmental expression, and localization of synaptotagmin III and its closest homolog, synaptotagmin VI. We find that synaptotagmins III and VI are present in all brain regions in heterogeneous distributions and that their levels increase during development in parallel with synaptogenesis. Furthermore, we show by immunocytochemistry that synaptotagmin III is concentrated in synapses, as expected. Surprisingly, however, we observed that synaptotagmin III is highly enriched in synaptic plasma membranes but not in synaptic vesicles. Synaptotagmin VI was also found to be relatively excluded from synaptic vesicles. Our data suggest that synaptotagmins III and VI perform roles in neurons that are not linked to synaptic vesicle exocytosis but to other Ca2+-related nerve terminal events, indicating that the functions of synaptotagmins are more diverse than originally thought. (+info)