Characterization of micron-size hydrogen clusters using Mie scattering.

Hydrogen clusters with diameters of a few micrometer range, composed of 108-10 hydrogen molecules, have been produced for the first time in an expansion of supercooled, high-pressure hydrogen gas into a vacuum through a conical nozzle connected to a cryogenic pulsed solenoid valve. The size distribution of the clusters has been evaluated by measuring the angular distribution of laser light scattered from the clusters. The data were analyzed based on the Mie scattering theory combined with the Tikhonov regularization method including the instrumental functions, the validity of which was assessed by performing a calibration study using a reference target consisting of standard micro-particles with two different sizes. The size distribution of the clusters was found discrete peaked at 0.33 ± 0.03, 0.65 ± 0.05, 0.81 ± 0.06, 1.40 ± 0.06 and 2.00 ± 0.13 µm in diameter. The highly reproducible and impurity-free nature of the micron-size hydrogen clusters can be a promising target for laser-driven multi-MeV proton sources with the currently available high power lasers.

Perineuronal nets affect parvalbumin expression in GABAergic neurons of the mouse hippocampus.

Recent studies have suggested that the perineuronal net (PNN), a specialised extracellular matrix structure, and parvalbumin (PV), an EF-hand calcium-binding protein, are involved in the regulation of plasticity of neural circuits. Here, we aimed to quantitatively estimate the relationship between the two plasticity regulators, PV and PNNs, in the hippocampus of young adult mice. Dual fluorescence staining for PV and Wisteria floribunda agglutinin (a broad PNN marker) showed that a substantial population of PV-expressing (PV(+) ) GABAergic neurons lacked PNNs. Optical disector analysis demonstrated that there were fewer PNN(+) neurons than PV(+) neurons. The ratio of PNN expression in PV(+) neurons was generally lower in the dendritic layers than in the principal cell layers, whereas the ratio of PV expression in PNN(+) neurons was effectively 100%. The mean PV fluorescence was significantly higher in PNN(+) /PV(+) neurons than in PNN(-) /PV(+) neurons. Cumulative frequencies for single-cell PV fluorescence indicated that intensely stained PV(+) neurons tend to be enwrapped by PNNs, whereas weakly stained PV(+) neurons are likely to lack PNNs. We digested the PNNs by a unilateral injection of chondroitinase ABC (chABC) into the dorsal CA1 region. Although the densities of PV(+) neurons remained unchanged, the PV fluorescence declined 7 days after chABC injection. Quantitative real-time polymerase chain reaction analysis demonstrated a reduction in PV mRNA expression following chABC injection. These findings indicate that the presence or absence of PNNs affects the relative PV expression in GABAergic neurons in the hippocampus.

Insertable pulse cleaning module with a saturable absorber pair and a compensating amplifier for high-intensity ultrashort-pulse lasers.

We demonstrate the performance of an efficient insertable pulse cleaning module (IPCM) that uses a saturable absorber (SA) pair with a compensating multi-pass amplifier. IPCM consists of a first SA, a grating compressor, a second SA, a stretcher and a compensating Ti:sapphire amplifier. It is implemented with a conventional chirped pulse amplification (CPA) Ti:sapphire laser system, resulting in a double CPA system architecture, and suppresses the amplified spontaneous emission (ASE) level of the pulse pedestal by about three orders of magnitude while preserving the output pulse energy and repetition-rate of the overall laser system. The duration of recompressed cleaned pulses is comparable to that obtained without the cleaning module. The effectiveness of the cleaning module is confirmed in laser-driven proton acceleration experiments. At the 10(9) W/cm2 pedestal level, the surface structure and electrical resistivity of an insulator target (100 nm silicon nitride) are preserved prior to the arrival of the intense ultrashort pulse.

Mie scattering from submicron-sized CO2 clusters formed in a supersonic expansion of a gas mixture.

A detailed mathematical model is presented for a submicron-sized cluster formation in a binary gas mixture flowing through a three-staged conical nozzle. By measuring the angular distribution of light scattered from the clusters, the size of CO(2) clusters, produced in a supersonic expansion of the mixture gas of CO(2)(30%)/H(2)(70%) or CO(2)(10%)/He(90%), has been evaluated using the Mie scattering method. The mean sizes of CO(2) clusters are estimated to be 0.28 ± 0.03 µm for CO(2)/H(2) and 0.26 ± 0.04 µm for CO(2)/He, respectively. In addition, total gas density profiles in radial direction of the gas jet, measuring the phase shift of the light passing through the target by utilizing an interferometer, are found to be agreed with the numerical modeling within a factor of two. The dryness (= monomer/(monomer + cluster) ratio) in the targets is found to support the numerical modeling. The apparatus developed to evaluate the cluster-gas targets proved that our mathematical model of cluster formation is reliable enough for the binary gas mixture.

Reduction of noise induced by power supply lines using phase-locked loop.

An experimental implementation for the reduction of power-line noise in delicate signal detection is presented. This implementation improves the signal-to-noise ratio without limiting the bandwidth of the measurement. A sinusoidal wave and its harmonics, both synchronized with the frequency of the power line, are used to cancel out the power supply noise induced in the measurement signal. The wave and the harmonics are generated via a phase-locked loop implementation. Their amplitude and phase are adjusted, and then they are added to the measurement signal using a series of operational amplifiers to compensate for the noise. Although we applied this method to the particular case of scanning tunneling microscopy measurements, considerably improving the image quality, our implementation can be applied to other measurement systems for which noise from the power lines can compromise the signal detection.

Isolation of a mammalian homologue of a fission yeast differentiation regulator.

In the fission yeast Schizosaccharomyces pombe the nrd1(+) gene encoding an RNA binding protein negatively regulates the onset of differentiation. Its biological role is to block differentiation by repressing a subset of the Ste11-regulated genes essential for conjugation and meiosis until the cells reach a critical level of nutrient starvation. By using the phenotypic suppression of the S. pombe temperature-sensitive pat1 mutant that commits lethal haploid meiosis at the restrictive temperature, we have cloned ROD1, a functional homologue of nrd1(+), from rat and human cDNA libraries. Like nrd1(+), ROD1 encodes a protein with four repeats of typical RNA binding domains, though its amino acid homology to Nrd1 is limited. When expressed in the fission yeast, ROD1 behaves in a way that is functionally similar to nrd1(+), being able to repress Ste11-regulated genes and to inhibit conjugation upon overexpression. ROD1 is predominantly expressed in hematopoietic cells or organs of adult and embryonic rat. Like nrd1(+) for fission yeast differentiation, overexpressed ROD1 effectively blocks both 12-O-tetradecanoyl phorbol-13-acetate-induced megakaryocytic and sodium butyrate-induced erythroid differentiation of the K562 human leukemia cells without affecting their proliferative ability. These results suggest a role for ROD1 in differentiation control in mammalian cells. We discuss the possibility that a differentiation control system found in the fission yeast might well be conserved in more complex organisms, including mammals.

The biological properties of a novel ethyl methacrylate resin.

A novel ethyl methacrylate (EMA) resin was developed to overcome the tissue, organ and systemic damage associated with the residual monomer of conventional methyl methacrylate (MMA) resin bone cement. EMA resin is a chemical/ photopolymerizable material and is easy to handle during clinical procedures. The biocompatibility of EMA was evaluated in accordance with ISO10993-6. No inflammatory response was observed 1 and 9 weeks after implantation in the dorsal subcutaneous tissue of ddY mice. EMA resin also demonstrated better biocompatibility when compared with conventional bone cements. Poly-L-lactic acid (PLLA) was used as a carrier for bone morphogenetic protein (BMP) and added to the EMA slurry. The EMA-PLLA composite membrane was sticky and BMP readily adhered to its surface. The EMA-PLLA-BMP composite membrane induced new bone formation, the new bone growing in the shape of the EMA in the thigh muscle pouch of ddY mice. This novel EMA resin has many potential clinical applications.

Delirium during intravenous sedation with midazolam alone and with propofol in dental treatment.

A 62-year-old man visited our clinic for dental implantation under intravenous sedation. He demonstrated increased psychomotor activity and incomprehensible verbal contact during intravenous sedation. Although delirium caused by midazolam or propofol in different patients has been reported, the present case represents a delirium that developed from both drugs in the same patient, possibly because of the patient's smaller tolerance to midazolam and propofol.

Cdk6-cyclin D3 complex evades inhibition by inhibitor proteins and uniquely controls cell's proliferation competence.

Mammalian cells require a cyclin D-dependent kinase for the cell cycle start, yet many mesenchymal cells express three seemingly redundant D cyclins and similarly, seemingly redundant Cdk4 and Cdk6 as their kinase partners. We have found that the Cdk6-cyclin D3 complex is unique among the D cyclin and kinase combinations in the ability to promote the cell cycle start. In an anchorage-minus G(1)-arrested rat fibroblast, only Cdk6-D3 retains kinase activity due mainly to its ability to evade inhibition by p27(KIP1) and p21(CIP1) with a resemblance to viral cyclin-bound Cdk6. Rodent fibroblasts engineered to overexpress both Cdk6 and cyclin D3 highly resist serum starvation- or cell-cell contact-imposed G(1)-arrest. In BALB/c 3T3 cells, D3 is constitutively expressed, but Cdk6 is markedly induced with concomitant activation upon stimulation with a growth-promoting factor. These results suggest a role for the Cdk6-D3 complex in regulating cell's proliferation ability in response to external stimuli.

Cell cycle start from quiescence controlled by tyrosine phosphorylation of Cdk4.

In mammals Cdk4 (or Cdk6 in some cell types) is required for starting the cell cycle. Recently we showed that Cdk4 is regulated by tyrosine phosphorylation and dephosphorylation, and that this regulation is required for a DNA damage-induced G1 arrest. We report here that a generic anti-phosphotyrosine antibody can detect tyrosine-phosphorylated Cdk4 and that as revealed by immunoblot detection and kinase assay, this regulation is employed for DNA damage-responsive checkpoint control during cell cycle start from quiescence. In rat fibroblasts traversing G1 or arrested in G1 by deprivation of anchorage, Cdk4 does not undergo tyrosine phosphorylation. Tyrosine phosphorylation occurs only during cell's arrest in quiescence and dephosphorylation during their cell cycle start. Ultraviolet irradiation blocks dephosphorylation and concomitant activation of Cdk4, thereby preventing the start of cell cycling. Thus, unlike tyrosine phosphorylation of Cdc2, which controls phase transition in the regular cell cycle, tyrosine phosphorylation of Cdk4 is employed for controlling cell cycle start from quiescence in a rat fibroblast.

Cyclin G: a new mammalian cyclin with homology to fission yeast Cig1.

A new gene encoding a cyclin-like protein has been isolated from a rat fibroblast cDNA library by cross-hybridization with a mixture of c-src family proto-oncogene kinase domains as a probe. This putative cyclin, called cyclin G, contains a typical cyclin box at the N-terminus but no apparent 'destruction box' or 'PEST' sequence. Interestingly, in its C-terminus region, it has a sequence homologous with a tyrosine phosphorylation site of the epidermal growth factor receptor. Although this cyclin is phylogenetically related to HCS26 of Saccharomyces cerevisiae, it most resembles Cig1, a B-type cyclin, of Schizosaccharomyces pombe, which has been suggested to act at the G1/S phase of the cell cycle. Cyclin G mRNA is induced within 3 h after growth stimulation and remains elevated with no apparent cell cycle dependency, indicating its close association with growth stimuli but not with the cell cycle.

Postsynaptic and extrasynaptic localization of Kv4.2 channels in the mouse hippocampal region, with special reference to targeted clustering at gabaergic synapses.

Voltage-dependent potassium (Kv) channels in the CNS are involved in regulation of subthreshold membrane potentials, and thus reception and integration of synaptic signals. Although such features are particularly important for induction of hippocampal synaptic plasticity, relatively little is known about their subcellular localization. Here we analyzed the detailed distribution of Kv4.2 potassium channels in the mouse hippocampal region using confocal and electron microscopy. At the light microscopic level, the Kv4.2 immunoreactivity occurred in a punctate fashion in the whole area of the hippocampal region. In the hippocampus proper, most of the Kv4.2-positive puncta were small, and they were abundant at the dendritic compartments of pyramidal neurons. High-resolution confocal microscopy revealed that there was no apparent association between Kv4.2-positive puncta with major synaptic markers, such as vesicular glutamate transporters and glutamic acid decarboxylase. In the subicular complex and dentate gyrus, we encountered large distinct Kv4.2-positive puncta at the perimeter of somata and proximal dendrites of principal cells. These puncta were often in contact with glutamic acid decarboxylase-positive boutons, but showed no apparent association with vesicular glutamate transporters. The glutamic acid decarboxylase-positive boutons apposing to Kv4.2-positive puncta were parvalbumin-positive. Quantitative image analysis showed that approximately half of Kv4.2-positive puncta were closely apposed to glutamic acid decarboxylase-positive boutons in the parasubiculum and dentate gyrus. Electron microscopic examination substantiated the presence of large Kv4.2-positive patches at postsynaptic sites of symmetric synapses and small patches at extrasynaptic sites. No presynaptic terminals were labeled. The present findings indicate targeted clustering of Kv4.2 potassium channels at postsynaptic sites of GABAergic synapses and extrasynaptic sites, and provide some key to understand their role in the hippocampal region.

Stability of the synaptic structure in the hippocampus of BALB/c mice with allergic rhinitis.

OBJECTIVE: The aim of this study was to determine whether allergic rhinitis can induce structural changes in the synapse formation in the hippocampus of BALB/c mice immunocytochemically. METHODS: Allergic rhinitis was induced in mice by two intra-peritoneal injections of ovalbumin administered with a one-week interval. After two weeks, the sensitised mice were challenged with an intra-nasal injection of ovalbumin for two weeks. To analyse the hippocampal synaptic structures, sections were immunostained with antibodies against glutamic acid decarboxylase 65 and glutamic acid decarboxylase 67 (for γ-aminobutyric acid-ergic terminals), synaptophysin (for glutamatergic and γ-aminobutyric acid-ergic terminals) and spinophilin (for dendritic spines). The number of nasal rubbing movements was significantly greater in the allergic rhinitis mice than in the control mice. However, the expression patterns of the four above-mentioned synaptic markers in the hippocampus showed no detectable difference between the allergic rhinitis and control mice. RESULTS AND CONCLUSION: These data indicate that the synaptic structure in the hippocampus might remain unaltered in allergic rhinitis patients.

Colocalization of parvalbumin and somatostatin-like immunoreactivity in the mouse hippocampus: quantitative analysis with optical dissector.

The colocalization of parvalbumin (PV) and somatostatin (SS)-like immunoreactivity was studied quantitatively in the mouse hippocampus, with particular reference to their areal and dorsoventral differences. The optical disector method was applied by using a confocal laser scanning microscope with immunofluorescent double-labeling. In the present study, we found a particular subpopulation of hippocampal nonprincipal neurons that contained both PV and SS-like immunoreactivity, i.e., PV-immunoreactive (IR)/SS-like immunoreactive (LIR) neurons. In the CA1 region, PV-IR/SS-LIR neurons were restricted to the stratum oriens (SO). In the CA3 region, they were scattered in the SO, stratum pyramidale (SP), and stratum radiatum (SR). However, they were rarely seen in the dentate gyrus (DG). The proportion of PV-IR/SS-LIR neurons in the PV-IR neurons or SS-LIR neurons was about 10% in the CA1 region, 15-30% in the CA3 region, 0-5% in the DG, and 10-20% in total. Laminar analysis revealed that the proportions of PV-IR/SS-LIR neurons in the PV-IR neurons were high in the SO (about 25%) of the CA1 region, and in the SO (about 50%) and SR (30-45%) of the CA3 region. The proportion of PV-IR/SS-LIR neurons in the SS-LIR neurons was low in the SO of the CA1 region (about 10%), but high in the SO (35-65%) and SR (35-45%) of the CA3 region. Morphologically, medium-sized horizontal fusiform and multipolar PV-IR/SS-LIR neurons were frequently observed, and they showed weak immunoreactivity for PV. Large-sized vertical bitufted and triangular PV-IR neurons lacked SS-like immunoreactivity, and most of them showed moderate to intense immunoreactivity for PV. In addition, we provide direct evidence that some PV-IR/SS-LIR neurons projected to the medial septum by using retrograde labeling with Fluoro-Gold injection. These observations indicate that PV-IR/SS-LIR neurons constitute a particular subpopulation of hippocampal nonprincipal neurons.

Quantitative analysis of neuronal nitric oxide synthase-immunoreactive neurons in the mouse hippocampus with optical disector.

A detailed quantitative analysis of immunocytochemically identified nonprincipal neurons containing neuronal nitric oxide synthase (nNOS) was performed on the mouse hippocampus, with particular reference to the dorsoventral gradient. The present study applied two variations of a stereologic technique, the optical disector--one that used confocal laser-scanning microscope optical sections to examine colocalization of nNOS and glutamic acid decarboxylase 67 (GAD67), and the other that used conventional thick sections to examine numerical densities (NDs) and cell sizes of nNOS-immunoreactive (IR) neurons. Colocalization analysis indicated that practically all nNOS-IR neurons (97.6%) were GAD67-IR, whereas a part of the GAD67-IR neurons (about 30%) were nNOS-IR in the whole hippocampus at both dorsal and ventral levels. The percentages of GAD67-IR neurons containing nNOS were higher in the dentate gyrus (DG, about 50%), and lower in the Ammon's horn (about 20%). Laminar analysis revealed that the majority of GAD67-IR neurons contained nNOS in the stratum lacunosum-moleculare of the CA3 region (about 60%) and in the molecular layer of the DG (about 80%). The NDs of nNOS-IR neurons in the whole hippocampus showed a dorsoventral gradient, which increased from dorsal (1.6 x 10(3)/mm3) to ventral (2.2 x 10(3)/mm3) levels. The NDs were relatively higher in the principal cell layers, where about 40% of nNOS-IR neurons were situated both in the Ammon's horn and DG. The mean cell sizes of nNOS-IR neurons showed no remarkable laminar differences or dorsoventral gradient in the Ammon's horn, but they were extensively larger in the hilus of the DG than in other layers. These results indicate that nNOS-IR neurons in the mouse hippocampus represent a subpopulation of gamma-aminobutyric acid (GABA)ergic neurons and suggest that the laminar distributions of nNOS-IR neurons related to possible functional heterogeneity of GABAergic neurons in each hippocampal layer.

Induction of DNA polymerase beta and gamma in the lungs of age-related oxygen tolerant rats.

To clarify a mechanism for oxygen tolerance in young rats, 3 and 8 week-old rats were exposed to 100% oxygen. All 8 week-old (8W) rats died between 48 and 72h, whereas most 3 week-old (3W) rats survived for more than 72 h under hyperoxia. It was assumed that this difference is attributable to oxygen tolerance in 3W rats compared with 8W rats. To clarify this difference, we measured the change in the activity of DNA polymerase, which is related to the final step of DNA repair. DNA polymerase activity in crude lung extracts from 3W rats increased up to 72 h after oxygen exposure. On the other hand, the activity in 8W rats was decreased at 24 h and 48 h. The activity of DNA polymerase beta, which is related to nuclear DNA (nDNA) repair, was approximately seven times higher in 3W rats than in 8W rats. DNA polymerase beta activities in 3W rats decreased up to 48 h with oxygen exposure, but recovered to pre-exposure levels by 72 h. Moreover, an induction of DNA polymerase gamma, which is related to mitochondrial DNA (mtDNA) replication and/or repair, was observed only in 3W rat lungs after 24 h of oxygen exposure. From these results, we conclude that the induction of DNA polymerase beta and DNA polymerase gamma in lung tissue plays a key role in oxygen tolerance in very young rats.

Multiple mechanisms for the effects of capsaicin, bradykinin and nicotine on CGRP release from tracheal afferent nerves: role of prostaglandins, sympathetic nerves and mast cells.

Application of capsaicin (CAP), bradykinin (BK) or nicotine (NIC) to intraluminally perfused rat tracheas induced an increase in calcitonin gene-related peptide (CGRP) levels in the perfusates. Depletion of sensory afferent CGRP with systemic CAP pretreatment resulted in a significant reduction of CGRP release evoked by CAP, BK or NIC. Chemical destruction of sympathetic nerve fibres by systemic pretreatment with 6-hydroxydopamine reduced CGRP release evoked by NIC, but did not alter the release produced by CAP or BK. Elimination of the tracheal mast cell population by pretreatment with compound 48/80 did not alter the effects of CAP, BK or NIC. CGRP release evoked by BK and NIC, but not CAP, was diminished by indomethacin, suggesting that cyclooxygenase products mediate the actions of BK and NIC. Prostaglandins, PGE1, PGE2, PGF2 alpha and PGI2, displayed stimulatory effects on CGRP release in the trachea. There are evidently multiple mechanisms mediating CGRP release from sensory terminals in rat trachea. It appears that CAP exerts a direct action on sensory nerves, while the effects of BK and NIC are mediated by PG synthesis. Sympathetic activation may be involved in NIC, but not BK, induced PG-mediated CGRP release.

Heterogeneous expression of the cholecystokinin-like immunoreactivity in the mouse hippocampus, with special reference to the dorsoventral difference.

The neuropeptide cholecystokinin (CCK) is widely distributed in the CNS. We herein investigated the immunocytochemical localization of CCK in the glutamatergic excitatory pathways in the mouse hippocampus, with particular reference to the dorsoventral difference. The intense CCK-like immunoreactivity (CCK-LI) was found in the mossy fiber pathway (stratum lucidum and dentate hilus) and in the inner molecular layer of the dentate gyrus. In the mossy fiber pathway, the CCK-LI was more intense at the ventral level than at the dorsal level. On the other hand, the CCK-LI in the stratum lucidum was more intense in the distal portion than in the proximal portion, both at the dorsal and ventral levels. High-resolution three-dimensional image analysis revealed the coexpression of CCK and synaptoporin (SPO) in the single mossy terminal, where they were spatially segregated but adjacent to each other. Quantitative image analysis indicated the difference in the amount of CCK within the mossy terminals along the dorsoventral and transverse axes of the hippocampus. On the other hand, in the inner molecular layer, CCK- and SPO-positive elements appeared to have little relation to each other. We also examined the postnatal development of the CCK-LI in the mouse hippocampus. The CCK-LI was detected in the inner molecular layer of the ventral dentate gyrus at postnatal day (P) 7. In the mossy fiber pathway, the CCK-LI was first evident at P 14, but it was restricted to the distal portion of the stratum lucidum in the ventral hippocampus. Interestingly, the distributions of the SPO immunoreactivity at P 7 were already similar to those of adult mice. The patterns of expression of CCK-LI at P 28 were almost similar to those of adult mice. The present data demonstrate the heterogeneous expression of CCK-LI in the mouse hippocampus, and provide a baseline to understand the role of CCK in the mouse brain.

Patterns of colocalization of neuronal nitric oxide synthase and somatostatin-like immunoreactivity in the mouse hippocampus: quantitative analysis with optical disector.

In some brain regions, previous studies reported the frequent coexistence between neuronal nitric oxide synthase (nNOS) and somatostatin (SOM). In the hippocampus, nNOS and SOM were mainly expressed in GABAergic nonprincipal neurons. Here we estimated the immunocytochemical colocalization of nNOS and SOM in the mouse hippocampus using the optical disector. Both in the Ammon's horn and dentate gyrus, we encountered only a few nNOS-immunoreactive (IR)/SOM-like immunoreactive (LIR) neurons. They were mainly located in the stratum oriens of the Ammon's horn and in the dentate hilus. The nNOS-IR/SOM-LIR neurons usually showed characteristic large somata with thick dendrites, whereas the majority of nNOS-IR/SOM-negative neurons showed small somata with thin dendrites. Quantitative data revealed that the double-labeled cells represented only 4% and 7% of nNOS-IR neurons and SOM-LIR neurons, respectively, in the whole area of the hippocampus. We also found the laminar and dorsoventral differences in the degree of colocalization between nNOS and SOM. The percentages of nNOS-IR neurons containing SOM-like immunoreactivity were relatively high in the stratum oriens of the ventral CA1 region (24%), stratum lucidum of the dorsal CA3 region (29%) and dorsal dentate hilus (32%), but they were quite low in the other layers. On the other hand, the percentages of SOM-LIR neurons containing nNOS immunoreactivity were somewhat high in the stratum lucidum of the dorsal CA3 region (19%) and dorsal dentate hilus (28%), whereas they were very low in the other layers. Immunofluorescent triple labeling of axon terminals for nNOS, SOM and glutamic acid decarboxylase indicated that some nNOS-IR/SOM-LIR neurons might be dendritic inhibitory cells. The present results show the infrequent colocalization of nNOS and SOM in the mouse hippocampus, and also suggest that the double-labeled cells may be a particular subpopulation of hippocampal GABAergic nonprincipal neurons.

Immunocytochemical localization of neuronal calcium sensor-1 in the hippocampus and cerebellum of the mouse, with special reference to presynaptic terminals.

Neuronal calcium sensor-1 (NCS-1) is a member of the EF-hand calcium-binding protein superfamily, which is considered to modulate synaptic transmission and plasticity. In this work, we first examined the distribution patterns of NCS-1 in the hippocampus and cerebellum. The intense NCS-1-immunoreactive (IR) elements in the hippocampus were restricted to dendritic layers, while those in the cerebellum occurred in both dendritic and cellular layers. Then, we examined the exact localization of NCS-1 using immunofluorescent double labeling for NCS-1 and synaptophysin, a marker of presynaptic terminals. In the hippocampus, the mossy fiber systems (terminals and bundles) exhibited intense NCS-1 immunoreactivity. On the other hand, the presumed principal cell dendrites were also NCS-1-IR in the stratum lacunosum-moleculare of Ammon's horn and molecular layer of the dentate gyrus, where NCS-1-IR elements and synaptophysin-IR presynaptic terminals showed characteristic complementary distribution patterns. In the cerebellum, some of the basket cell axon terminals surrounding the somata of Purkinje cells exhibited NCS-1 immunoreactivity, while the pinceau showed consistent labeling for NCS-1. Higher magnification observations revealed that the NCS-1-IR presumed granule cell dendrites and synaptophysin-IR mossy fiber terminals in the glomeruli of the cerebellum showed characteristic complementary distribution patterns. Furthermore, we estimated quantitatively the relative amount of NCS-1 in the presynaptic terminals in individual layers, and confirmed that the mossy fiber terminals in the hippocampus contained comparatively high amounts of NCS-1. These results showed the diverse localization of NCS-1 in pre- and/or postsynaptic elements of the hippocampus and cerebellum, and suggest potential roles in specific synaptic transmission.