Trafficking of synaptic vesicles is changed at the hypothalamus by exposure to an 835 MHz radiofrequency electromagnetic field.

With the rapidly increasing use of mobile phones and their close-contact usage to the brain, there are some concerns about the possible neuronal effects induced by exposure to excessive electromagnetic radiation. Exposure to a radiofrequency electromagnetic field (RF-EMF) of 835 MHz (4.0 W/kg specific absorption rate (SAR) 5 h/day for 12 weeks) may affect hypothalamic presynaptic neurons in C57BL/6 mice. The number and size of the synaptic vesicles (SVs) in the hypothalamic presynaptic terminals were significantly decreased after RF-EMF exposure. Further, the density (SVs numbers/µm) of docking and fusing SVs in the active zones of the presynaptic terminal membrane was significantly decreased in hypothalamic neurons. The expression levels of synapsin I/II and synaptotagmin 1, two regulators of SV trafficking in neurons, were also significantly decreased in the hypothalamus. In parallel, the expression of calcium channel was significantly decreased. These changes in SVs in the active zones may directly decrease the release of neurotransmitters in hypothalamic presynaptic terminals. Therefore, we further studied the possible changes in hypothalamic function by testing the core body temperature and body weight and performed the buried pellet test. The trafficking of SVs was changed by RF-EMF; however, we could not find any significant phenotypical changes in our experimental condition.

Decreased dopamine in striatum and difficult locomotor recovery from MPTP insult after exposure to radiofrequency electromagnetic fields.

Concern is growing about possible neuronal effects of human exposure to radiofrequency electromagnetic fields because of the increasing usage of cell phones and the close proximity of these devices to the brain when in use. We found that exposure to a radiofrequency electromagnetic field (RF-EMF) of 835 MHz (4.0 W/kg specific absorption rate [SAR] for 5 h/day for 12 weeks) affects striatal neurons in C57BL/6 mice. The number of synaptic vesicles (SVs) in striatal presynaptic boutons was significantly decreased after RF-EMF exposure. The expression levels of synapsin I and II were also significantly decreased in the striatum of the RF-EMF-exposed group. RF-EMF exposure led to a reduction in dopamine concentration in the striatum and also to a decrease in the expression of tyrosine hydroxylase in striatal neurons. Furthermore, in behavioral tests, exposure to RF-EMF impeded the recovery of locomotor activities after repeated treatments with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). These results suggest that the observed decrease in dopamine concentration in the striatum was caused by both a reduction in the number of dopaminergic neurons and a decline in the number of SVs. The decreased dopamine neuron numbers and concentration seen after RF-EMF exposure would have caused the difficult recovery after MPTP treatment. In summary, our results strongly suggest that exposing the brain to RF-EMF can decrease the number of SVs and dopaminergic neurons in the striatum. These primary changes impair the recovery of locomotor activities following MPTP damage to the striatum.

Autophagy mediates the degradation of synaptic vesicles: A potential mechanism of synaptic plasticity injury induced by microwave exposure in rats.

To explore how autophagy changes and whether autophagy is involved in the pathophysiological process of synaptic plasticity injury caused by microwave radiation, we established a 30 mW/cm2 microwave-exposure in vivo model, which caused reversible injuries in rat neurons. Microwave radiation induced cognitive impairment in rats and synaptic plasticity injury in rat hippocampal neurons. Autophagy in rat hippocampal neurons was activated following microwave exposure. Additionally, we observed that synaptic vesicles were encapsulated by autophagosomes, a phenomenon more evident in the microwave-exposed group. Colocation of autophagosomes and synaptic vesicles in rat hippocampal neurons increased following microwave exposure. CONCLUSION: microwave exposure led to the activation of autophagy in rat hippocampal neurons, and excessive activation of autophagy might damage synaptic plasticity by mediating synaptic vesicle degradation.

Changes in numbers and size of synaptic vesicles of cortical neurons induced by exposure to 835 MHz radiofrequency-electromagnetic field.

We studied the effects of radiofrequency electromagnetic fields (RF-EMFs) exposure on neuronal functions of mice. Particularly, we focused on RF-EMF effects on synaptic vesicles (SVs), which store neurotransmitters at axon terminals or synaptic boutons. C57 BL/6 mice were exposed to 835 MHz RF-EMF (4.0 W/kg SAR, for 5 h daily) and alterations in SVs at presynaptic terminals in the cerebral cortex were determined. Ultrastructure of randomly selected cortical neurons was observed using typical electron microscopy and bio-high voltage electron microscopy (Bio-HVEM) methods, which enable the estimation of the numbers and size of SVs. The density of the SVs (number /10 µm2 or 40 µm3) was significantly decreased in the presynaptic boutons of cortical neurons after RF-EMF exposure. Furthermore, qPCR and immunoblotting analyses revealed that the expression of synapsins I/II (Syns I/II) genes and proteins were significantly decreased in the cortical neurons of RF-EMF exposed mice. The present study suggested that alteration of SVs and Syn levels may result in alterations of neurotransmitters in the cerebral cortex following RF-EMF exposure.

The V-ATPase membrane domain is a sensor of granular pH that controls the exocytotic machinery.

Several studies have suggested that the V0 domain of the vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) is directly implicated in secretory vesicle exocytosis through a role in membrane fusion. We report in this paper that there was a rapid decrease in neurotransmitter release after acute photoinactivation of the V0 a1-I subunit in neuronal pairs. Likewise, inactivation of the V0 a1-I subunit in chromaffin cells resulted in a decreased frequency and prolonged kinetics of amperometric spikes induced by depolarization, with shortening of the fusion pore open time. Dissipation of the granular pH gradient was associated with an inhibition of exocytosis and correlated with the V1-V0 association status in secretory granules. We thus conclude that V0 serves as a sensor of intragranular pH that controls exocytosis and synaptic transmission via the reversible dissociation of V1 at acidic pH. Hence, the V-ATPase membrane domain would allow the exocytotic machinery to discriminate fully loaded and acidified vesicles from vesicles undergoing neurotransmitter reloading.

Low (60 cGy) doses of (56)Fe HZE-particle radiation lead to a persistent reduction in the glutamatergic readily releasable pool in rat hippocampal synaptosomes.

Exposure to galactic cosmic radiation (GCR) is considered to be a potential health risk in long-term space travel, and it represents a significant risk to the central nervous system (CNS). The most harmful component of GCR is the HZE [high-mass, highly charged (Z), high-energy] particles, e.g. (56)Fe. In ground-based experiments, exposure to HZE-particle radiation induces pronounced deficits in hippocampus-dependent learning and memory in rodents. The mechanisms underlying these impairments are mostly unknown, but some studies suggest that HZE-particle exposure perturbs the regulation of long-term potentiation (LTP) at the CA1 synapse in the hippocampus. In this study, we irradiated rats with 60 cGy of 1 GeV (56)Fe-particle radiation and established its impact on hippocampal glutamatergic neurotransmissions at 3 and 6 months after exposure. Exposure to 60 cGy (56)Fe-particle radiation significantly (P < 0.05) reduced hyperosmotic sucrose evoked [(3)H]-glutamate release from hippocampal synaptosomes, a measure of the readily releasable vesicular pool (RRP). This HZE-particle-induced reduction in the glutamatergic RRP persisted for at least 6 months after exposure. At 90 days postirradiation, there was a significant reduction in the expression of the NR1, NR2A and NR2B subunits of the glutamatergic NMDA receptor. The level of the NR2A protein remained suppressed at 180 days postirradiation, but the level of NR2B and NR1 proteins returned to or exceeded normal levels, respectively. Overall, this study shows that hippocampal glutamatergic transmission is sensitive to relative low doses of (56)Fe particles. Whether the observed HZE-particle-induced change in glutamate transmission, which plays a critical role in learning and memory, is the cause of HZE-particle-induced neurocognitive impairment requires further investigation.

Abnormality of synaptic vesicular associated proteins in cerebral cortex and hippocampus after microwave exposure.

Studies were performed to determine the effects of microwave on synaptic vesicles and the expression of synaptic vesicular associated proteins including synapsin I, VAMP-2, syntaxin, and synaptophysin. 25 Wistar rats were exposed to microwave which the average power density was 30 mW/cm(2), and whole body average specific absorption rate was 14.1 W/kg for 5 min. Synaptosome preparations in the cerebral cortex and hippocampus were obtained by isotonic Percoll/sucrose discontinuous gradients at 6 h, 1, 3, and 7 days after radiation. The expression of synaptic vesicular associated proteins was measured using Western blots and image analysis. The interaction between VAMP-2 and syntaxin was examined by coimmunoprecipitation analysis. Synapsin I in the cerebral cortex were decreased at 3 days (P < 0.01) after radiation and in the hippocampus increased at 1 day (P < 0.01), decreased at 3 days (P < 0.01), increased again at 7 days (P < 0.01) after exposure, compared with the sham-treated controls. Synaptophysin were increased in 1-7 days (P < 0.01) after exposure in the cerebral cortex and hippocampus. VAMP-2 were decreased at 1 and 3 days (P < 0.01) and syntaxin were decreased in 6 h to 3 days (P < 0.01) after radiation in the cerebral cortex and hippocampus. The interactions between VAMP-2 and syntaxin were decreased at 3-7 days (P < 0.01) after radiation in the cerebral cortex and hippocampus, compared with the sham-treated controls. These results suggest that 30 mW/cm(2) (SAR 14.1 W/kg) microwave radiation can result in the perturbation of the synaptic vesicles associated proteins: synapsin I, synaptophysin, VAMP-2, and syntaxin. The perturbation could induce the deposit of synaptic vesicle, which might be relative to the dysfunction of the synaptic transmission, even the cognition deficit.

The dual phosphatase activity of synaptojanin1 is required for both efficient synaptic vesicle endocytosis and reavailability at nerve terminals.

Phosphoinositides have been implicated in synaptic vesicle recycling largely based on studies of enzymes that regulate phosphoinositide synthesis and hydrolysis. One such enzyme is synaptojanin1, a multifunctional protein conserved from yeast to humans, which contains two phosphoinositol phosphatase domains and a proline-rich domain. Genetic ablation of synaptojanin1 leads to pleiotropic defects in presynaptic function, including accumulation of free clathrin-coated vesicles and delayed vesicle reavailability, implicating this enzyme in postendocytic uncoating of vesicles. To further elucidate the role of synaptojanin1 at nerve terminals, we performed quantitative synaptic vesicle recycling assays in synj1(-/-) neurons. Our studies show that synaptojanin1 is also required for normal vesicle endocytosis. Defects in both endocytosis and postendocytic vesicle reavailability can be fully restored upon reintroduction of synaptojanin1. However, expression of synaptojanin1 with mutations abolishing catalytic activity of each phosphatase domain reveals that the dual action of both domains is required for normal synaptic vesicle internalization and reavailability.

Influence of synapsin I on synaptic vesicles: an analysis by force-volume mode of the atomic force microscope and dynamic light scattering.

Synaptic vesicles (SVs) are small neuronal organelles that store neurotransmitters and release them by exocytosis into the synaptic cleft for signal transmission between nerve cells. They consist of a highly curved membrane composed of different lipids containing several proteins with specific functions. A family of abundant extrinsic SV proteins, the synapsins, interact with SV proteins and phospholipids and play an important role in the regulation of SV trafficking and stability. We investigated the interactions of one these proteins with the SV membrane using atomic force microscope and dynamic light scattering. We examined SVs isolated from rat forebrain both under native conditions and after depletion of endogenous synapsin I. We used the atomic force microscope in two modes: imaging mode for characterizing the shape and size of SVs, and force-volume mode for characterizing their stiffness. Synapsin-depleted SVs were larger in size and showed a higher tendency to aggregate than native vesicles, although their stiffness was not significantly different. Because synapsins are believed to cross-link SV to each other and to the actin cytoskeleton, we also measured the SV aggregation kinetics induced by synapsin I by dynamic light scattering and atomic force microscopy and found that the addition of synapsin I promotes a rapid aggregation of SVs. The data indicate that synapsin directly affects SV stability and aggregation state and support the physiological role of synapsins in the assembly and regulation of SV pools within nerve terminals.

Release and recycling of the readily releasable vesicle population in a synapse possessing no reserve population.

We previously demonstrated that the tergotrochanteral muscle (TTM) of Drosophila is innervated by unique synapses that possess a small readily releasable/recycling vesicle population (active zone population), but not the larger reserve vesicle population observed at other neuromuscular junctions in this animal. Using light and electron microscopic techniques and intracellular recording from the G1 muscle fiber of the TTM, the release and recycling characteristics of the readily releasable/recycling population were observed without any possible contribution from a reserve population. Our results indicate 1) the total number of vesicles in synapses presynaptic to the G1 fiber correlates with the total number of quanta that can be released onto this fiber; 2) the number of quanta released by a single action potential onto the G1 fiber is about one half the number of morphologically "docked" vesicles in active zones onto the G1, and this ratio decreases in a partially depleted state; 3) the recycling rate at 1-Hz stimulation, a frequency that does not cause any depression, is 0.24 recycled vesicle/active zone/s; and 4) normal-appearing spontaneous release occurs from the active zone vesicle population and, unlike synapses that possess a reserve population, the frequency of this release is reduced after high-frequency evoked activity.

Relationship of the reserve vesicle population to synaptic depression in the tergotrochanteral and dorsal longitudinal muscles of Drosophila.

We have previously demonstrated that Drosophila synapses possess two vesicle populations-a small active zone population replenished by "fast" recycling and a much larger reserve population replenished by a slower recycling mechanism that includes endosomal intermediates. In this paper, we demonstrate that the synapses onto the tergotrochanteral muscle (TTM) are very unusual in that they possess only the active zone vesicle population but not the reserve population. The depression characteristics to repetitive stimulation of the TTM were compared with those of the dorsal longitudinal muscle (DLM), the synapses of which possess both an active zone and a reserve population. It was observed that the TTM response depressed more quickly than that of the DLM. To further explore the possible contribution of the reserve population to release, using the shibire mutant, DLM synapses were experimentally constructed that possess only the active zone population, and their depression characteristics were compared with those of the same synapses possessing both populations. It was observed that responses from DLM synapses possessing only the active zone population depressed more quickly than the same synapses possessing both populations. These experiments were conducted under conditions of blocked recycling so that the difference in stimulation tolerance represents the contribution of the reserve population to release. Furthermore, the depression curve of the DLM synapses lacking a reserve population now closely approximated that of the TTM synapses. These data suggest that the reserve vesicle population of DLM synapses may contribute to transmitter release during repetitive firing at physiological frequencies (5-10 Hz).

Activity-dependent liberation of synaptic neuropeptide vesicles.

Despite the importance of neuropeptide release, which is evoked by long bouts of action potential activity and which regulates behavior, peptidergic vesicle movement has not been examined in living nerve terminals. Previous in vitro studies have found that secretory vesicle motion at many sites of release is constitutive: Ca(2+) does not affect the movement of small synaptic vesicles in nerve terminals or the movement of large dense core vesicles in growth cones and endocrine cells. However, in vivo imaging of a neuropeptide, atrial natriuretic factor, tagged with green fluorescent protein in larval Drosophila melanogaster neuromuscular junctions shows that peptidergic vesicle behavior in nerve terminals is sensitive to activity-induced Ca(2+) influx. Specifically, peptidergic vesicles are immobile in resting synaptic boutons but become mobile after seconds of stimulation. Vesicle movement is undirected, occurs without the use of axonal transport motors or F-actin, and aids in the depletion of undocked neuropeptide vesicles. Peptidergic vesicle mobilization and post-tetanic potentiation of neuropeptide release are sustained for minutes.

Multiple roles for the active zone protein RIM1alpha in late stages of neurotransmitter release.

The active zone protein RIM1alpha interacts with multiple active zone and synaptic vesicle proteins and is implicated in short- and long-term synaptic plasticity, but it is unclear how RIM1alpha's biochemical interactions translate into physiological functions. To address this question, we analyzed synaptic transmission in autaptic neurons cultured from RIM1alpha-/- mice. Deletion of RIM1alpha causes a large reduction in the readily releasable pool of vesicles, alters short-term plasticity, and changes the properties of evoked asynchronous release. Lack of RIM1alpha, however, had no effect on synapse formation, spontaneous release, overall Ca2+ sensitivity of release, or synaptic vesicle recycling. These results suggest that RIM1alpha modulates sequential steps in synaptic vesicle exocytosis through serial protein-protein interactions and that this modulation is the basis for RIM1alpha's role in synaptic plasticity.

Frequency dependence of synaptic vesicle exocytosis in aortic baroreceptor neurons and the role of group III mGluRs.

Synaptic transmission between baroreceptor afferents and the nucleus tractus solitarius (NTS) is essential for reflex regulation of blood pressure. High frequency stimulation of the afferents in vivo leads to a decrease in synaptic strength and is generally attributed to reduction in presynaptic neurotransmitter release. It has been hypothesized that during high frequency stimulation glutamate a major neurotransmitter at the baroreceptor afferent terminals inhibits its own release via presynaptic group III metabotropic glutamate receptors (mGluRs). A key player in modulation of presynaptic release is vesicle exocytosis. The present study utilized cultured aortic baroreceptor neurons and the styryl dye FM2-10 to characterize (1) the dependence of exocytosis at these afferent nerve terminals on the frequency of neuronal activation, (2) the effect of duration of stimulation on the rate of exocytosis and (3) the role of mGluRs in the frequency-dependent modulation of exocytosis. Destaining in the FM2-10 loaded boutons during 3 min of stimulation, a measure of exocytosis, progressively decreased with increasing frequency (0.5, 1.0 and 10 Hz). Blockade of group III mGluRs with 300 microM (RS)-cyclopropyl-4-phosphonophenylglycine (CPPG) facilitated exocytosis evoked by 10 Hz stimulation but not at 0.5 Hz. The data suggest that aortic baroreceptor terminals exhibit frequency-dependent depression of exocytosis and support a role for group III mGluRs in the frequency-dependent modulation of exocytosis.

Microwave fixation and localization of calcium in synaptic vesicles.

The distribution of Ca2+ ions is demonstrated in the synaptic terminals by means of a 2-step chemical precipitation of Ca2+ ions in nervous tissue. K-oxalate/K-antimonate chemical replacement with simultaneous computerized microwave irradiation is used. This precipitate in cell structures was investigated by computerized electron probe X-ray micro-analysis. The calculated values (from the theoretical, standards and sections), elemental binding ratios and elemental molecular weight ratios were compared. Each calculated value coincided with the theoretical value. This method can reliably detect Ca2+ ions at the micromolar level. Ca2+ ions were distributed in the synaptic vesicles and surrounding membranes. Further progress is expected in freeze-substitution and in the application and propagation of the EELS-Imaging system in calcium determinations.

[Quantitative changes in the ultrastructural elements of presynapses exposed to low-intensity laser radiation within the framework of a factorial model]. / Kolichestvennye izmeneniia élementov ul'trastruktury obluchennykh nizkointensivnym lazerom presinapsov v ramkakh faktornoi modeli.

The ultrastructure elements of presynaptic terminals (PT) in a dorsal horn of cat spinal cord were studied morphometrically in norm and after helium-neon laser irradiation. The disperse computer analysis showed changes in a median terminal radius, the number and localization of synaptic vesicles, and no changes in the shape and length of the plasmalemma profile of the irradiated PT.

A quantitative study of synaptic ribbons in pinealocytes of adult Chinese hamsters (Cricetulus griseus) under different photoperiodic conditions.

Synaptic ribbons (SR) in pinealocytes of adult (120-130 day-old) male Chinese hamsters (Cricetulus griseus) were classified into types 1, 2 and 3; these have a central dense structure showing rod-like, various and ring-like profiles, respectively. The central structure of the type-2 SR usually appeared as round, oval or comma-like bodies, and occasionally as plates showing various profiles or club-shaped bodies. The quantity of each type of SR, expressed as the SR index, was determined over a 24-h period under a light/dark regime (LD) 12:12 or LD 14:10. On comparing the results obtained from adults with previously published data from young (60-70-days-old) animals under LD 12:12, it was found that, in both young and adult animals, the type-1 and type-3 SR indices exhibited different 24-h variations, whereas the type-2 SR index remained constant over a 24-h period. In addition, the indices of the type-2 SR, but not those of the other SR types, were found to be significantly larger in adult than in young animals. In adult animals, the effects of the photoperiod were different between the three types of SR. A nocturnal increase in the type-1 SR index was observed under both LD 12:12 and LD 14:10, its time course being different for each of these photoperiods. Under LD 14:10, the type-2 SR index showed a significant 24-h rhythm with larger values during the dark period; this was not observed under LD 12:12. The type-3 SR index was almost the same under LD 12:12 and LD 14:10.(ABSTRACT TRUNCATED AT 250 WORDS)

Free amino acids in synaptic vesicles isolated from the cerebellum and cerebral hemispheres of control and neonatally X-irradiated rats.

X-irradiation of the rat brain (1000R, at two days of age), suppresses the normal age-related increase in the weight of the cerebellum and cerebral hemispheres and influences amino acid levels. The decrease in glutamic acid concentration, particularly in the cerebellum, supports the previously advanced proposition that this amino acid may be associated with or may be the transmitter of, the rat cerebellar granule cells. Subfractionation of the cerebellar tissue reveals that the decrease in the glutamic acid level consequent to the loss of granule cells, is reflected in the cytoplasmic fraction but not in the synaptic vesicle subfraction, where glutamic acid was increased. The reduced weight gain in the cerebral hemispheres after irradiation, is accompanied by a significant decrease of aspartate in the cytoplasmic fraction, changes which suggest that a specific cell type, with aspartic acid as its neurotransmitter (possibly in the hippocampus), may also be radiosensitive in the early postnatal period. In contrast, in the synaptic vesicle fraction from cerebral hemispheres, all free amino acids, with the exception of glutamine, increased significantly. Overall, the changes in free amino acid concentration induced by X-irradiation in the cytoplasmic fraction in both brain regions studied are opposite to those found in the synaptic vesicle fraction and although they may indicate changes in specific cell populations, as proposed above, they could also reflect changes in cellular compartmentalization and metabolism or changes in the relative axonal arborization of the affected regions.