Rapid Ultrastructural Changes in the PSD and Surrounding Membrane after Induction of Structural LTP in Single Dendritic Spines.

The structural plasticity of dendritic spines is considered to be an important basis of synaptic plasticity, learning, and memory. Here, we induced input-specific structural LTP (sLTP) in single dendritic spines in organotypic hippocampal slices from mice of either sex and performed ultrastructural analyses of the spines using efficient correlative light and electron microscopy. We observed reorganization of the PSD nanostructure, such as perforation and segmentation, at 2-3, 20, and 120 min after sLTP induction. In addition, PSD and nonsynaptic axon-spine interface (nsASI) membrane expanded unevenly during sLTP. Specifically, the PSD area showed a transient increase at 2-3 min after sLTP induction. The PSD growth was to a degree less than spine volume growth at 2-3 min and 20 min after sLTP induction but became similar at 120 min. On the other hand, the nsASI area showed a profound and lasting expansion, to a degree similar to spine volume growth throughout the process. These rapid ultrastructural changes in PSD and surrounding membrane may contribute to rapid electrophysiological plasticity during sLTP.SIGNIFICANCE STATEMENT To understand the ultrastructural changes during synaptic plasticity, it is desired to efficiently image single dendritic spines that underwent structural plasticity in electron microscopy. We induced structural long-term potentiation (sLTP) in single dendritic spines by two-photon glutamate uncaging. We then identified the same spines at different phases of sLTP and performed ultrastructural analysis by using an efficient correlative light and electron microscopy method. We found that postsynaptic density undergoes dramatic modification in its structural complexity immediately after sLTP induction. Meanwhile, the nonsynaptic axon-spine interface area shows a rapid and sustained increase throughout sLTP. Our results indicate that the uneven modification of synaptic and nonsynaptic postsynaptic membrane might contribute to rapid electrophysiological plasticity during sLTP.

Ratiometric Detection of γ-Glutamyltransferase in Human Colon Cancer Tissues Using a Two-Photon Probe.

γ-Glutamyltransferase (GGT) plays a role in cleaving the γ-glutamyl bond of glutathione. The GGT is known to be overexpressed in some tumors and has been recognized as a potential biomarker for malignant tumors. Colon cancer is one of the most common cancers worldwide; however, there is no quantitative method for detecting cancer cells in human colon tissues. In this study, we report a ratiometric two-photon probe for GGT that can be applied in human colon tissues. The probe (Probe 2) showed high fluorescence efficiency, marked fluorescence changes, excellent kinetics, and selectivity for the GGT in live colon cells. Additionally, we obtained ratiometric two-photon microscopy images of GGT activity in human colon tissue. We used this method to compare normal and cancer tissues based on their ratio values; the ratio value was higher in cancer tissue than in normal tissue. This study provides a method for quantitative analysis of GGT, particularly in human colon cancer, which will be useful for studying GGT-related diseases and diagnosing colon cancer.

Radiopacity evaluation of calcium aluminate cement containing different radiopacifying agents.

INTRODUCTION: The aim of this study was to evaluate the radiopacity of calcium aluminate cement (EndoBinder) with 3 different radiopacifiers (bismuth oxide, zinc oxide, or zirconium oxide) in comparison with gray mineral trioxide aggregate (GMTA), white MTA, and dental structures (enamel and dentin). METHODS: Eighteen test specimens of each cement with thicknesses of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mm (n = 3) were made by using a stainless steel matrix and were adapted to a standardizing device (8 × 7 cm) with a graduated aluminum stepwedge varying from 2.0-16.0 mm in thickness. To compare the radiopacity of the cements with that of dental structures, slices of first molars with a thickness increasing from 0.5-3.0 mm were obtained and placed on the standardizing device. One occlusal radiograph for each tested cement was taken, with exposure time of 0.1 seconds and focus-film distance of 20 cm. Films were processed in an automatic device, and the mean radiopacity values were obtained by using a photodensitometer. RESULTS: Mean values showed that the thicker the specimen was, the greater was its radiopacity. Only EndoBinder + bismuth oxide (EBBO) and GMTA demonstrated radiopacity values greater than 3.0 mm of the aluminum scale for all thicknesses. When zinc oxide was used as radiopacifier agent, EndoBinder only reached the desired radiopacity with a thickness of 2.0 mm, and with zirconium oxide it was 2.5 mm. CONCLUSIONS: Bismuth oxide was the most efficient radiopacifier for EndoBinder, providing adequate radiopacity in all studied thicknesses, as recommended by ISO 6876, being similar to GMTA.

Laser photolysis of caged compounds at 405 nm: photochemical advantages, localisation, phototoxicity and methods for calibration.

Rapid, localised photolytic release of neurotransmitters from caged precursors at synaptic regions in the extracellular space is greatly hampered at irradiation wavelengths in the near-UV, close to the wavelength of maximum absorption of the caged precursor, because of inner-filtering by strong absorption of light in the cage solution between the objective and cell. For this reason two-photon excitation is commonly used for photolysis, particularly at multiple points distributed over large fields; or, with near-UV, if combined with local perfusion of the cage. These methods each have problems: the small cross-sections of common cages with two-photon excitation require high cage concentrations and light intensities near the phototoxic limit, while local perfusion gives non-uniform cage concentrations over the field of view. Single-photon photolysis at 405 nm, although less efficient than at 330-350 nm, with present cages is more efficient than two-photon photolysis. The reduced light absorption in the bulk cage solution permits efficient wide-field uncaging at non-toxic intensities with uniform cage concentration. Full photolysis of MNI-glutamate with 100 micros pulses required intensities of 2 mW microm(-2) at the preparation, shown to be non-toxic with repeated exposures. Light scattering at 405 nm was estimated as 50% at 18 microm depth in 21-day rat cerebellum. Methods are described for: (1) varying the laser spot size; (2) photolysis calibration in the microscope with the caged fluorophore NPE-HPTS over the wavelength range 347-405 nm; and (3) determining the point-spread function of excitation. Furthermore, DM-Nitrophen photolysis at 405 nm was efficient for intracellular investigations of Ca2+-dependent processes.

A novel control software that improves the experimental workflow of scanning photostimulation experiments.

Optical uncaging of caged compounds is a well-established method to study the functional anatomy of a brain region on the circuit level. We present an alternative approach to existing experimental setups. Using a low-magnification objective we acquire images for planning the spatial patterns of stimulation. Then high-magnification objectives are used during laser stimulation providing a laser spot between 2 microm and 20 microm size. The core of this system is a video-based control software that monitors and controls the connected devices, allows for planning of the experiment, coordinates the stimulation process and manages automatic data storage. This combines a high-resolution analysis of neuronal circuits with flexible and efficient online planning and execution of a grid of spatial stimulation patterns on a larger scale. The software offers special optical features that enable the system to achieve a maximum degree of spatial reliability. The hardware is mainly built upon standard laboratory devices and thus ideally suited to cost-effectively complement existing electrophysiological setups with a minimal amount of additional equipment. Finally, we demonstrate the performance of the system by mapping the excitatory and inhibitory connections of entorhinal cortex layer II stellate neurons and present an approach for the analysis of photo-induced synaptic responses in high spontaneous activity.

Physical and chemical stability of pemetrexed in infusion solutions.

BACKGROUND: Pemetrexed is a multitargeted, antifolate, antineoplastic agent that is indicated for single-agent use in locally advanced or metastatic non-small-cell lung cancer after prior chemotherapy and in combination with cisplatin for the treatment of malignant pleural mesothelioma not treatable by surgery. Currently, there is no information on the long-term stability of pemetrexed beyond 24 hours. OBJECTIVE: To evaluate the longer-term physical and chemical stability of pemetrexed 2, 10, and 20 mg/mL in polyvinyl chloride (PVC) bags of dextrose 5% injection and NaCl 0.9% injection. METHODS: Triplicate samples of pemetrexed were prepared in the concentrations and infusion solutions required. Evaluations for physical and chemical stability were performed initially and over 2 days at 23 degrees C protected from light and exposed to fluorescent light, and over 31 days of storage at 4 degrees C protected from light. Physical stability was assessed using turbidimetric and particulate measurement as well as visual observation. Chemical stability was evaluated by HPLC. RESULTS: All pemetrexed solutions remained chemically stable, with little or no loss of pemetrexed over 2 days at 23 degrees C, protected from light and exposed to fluorescent light, and over 31 days of storage at 4 degrees C, protected from light. The room temperature samples were physically stable throughout the 48 hour test period. However, pemetrexed admixtures developed large numbers of microparticulates during refrigerated storage exceeding 24 hours. CONCLUSIONS: Pemetrexed is chemically stable for 2 days at room temperature and 31 days refrigerated in dextrose 5% injection and NaCl 0.9% injection. However, substantial numbers of microparticulates may form in pemetrexed diluted in the infusion solutions in PVC bags, especially during longer periods of refrigerated storage. Limiting the refrigerated storage period to the manufacturer-recommended 24 hours will limit particulate formation.

Distribution and properties of functional postsynaptic kainate receptors on neocortical layer V pyramidal neurons.

The distribution of glutamate receptor subtypes on the surface of neurons is highly relevant for synaptic transmission and signal processing. In the present study we investigated the location and properties of functional kainate receptors (KARs) on the somatodendritic membrane of rat neocortical layer V pyramidal neurons. Infrared-guided laser stimulation was used to apply glutamate photolytically to the soma and various sites along the apical dendrite. Electrical currents, resulting from the activation of pharmacologically isolated KARs, were measured by whole-cell patch-clamp recording. In addition, KARs on somatic and dendritic outside-out patches were activated while still within the brain tissue. We found that functional KARs are located on the entire somatodendritic membrane that was examined. Fast kinetics, a linear I-V relationship, and a relatively high single-channel conductance are characteristic features of these receptors. We provide evidence that the unitary properties of somatic and dendritic KARs are identical. Regarding the subcellular distribution of KARs, our results indicate that the density of these receptors increases toward the distal dendrite. They are located mainly at extrasynaptic sites but also mediate fast synaptic signaling triggered by afferent stimulation. The differential distribution speaks in favor of a selective targeting of KARs on central neurons and may reflect a mechanism for a location-dependent regulation of synaptic efficacy. Furthermore, it is feasible to assume that extrasynaptic KARs could be activated by a "spillover" of synaptically released glutamate, ambient glutamate in the CSF, or glutamate released from adjacent astrocytes.

Circuit analysis of experience-dependent plasticity in the developing rat barrel cortex.

Sensory deprivation during a critical period reduces spine motility and disrupts receptive field structure of layer 2/3 neurons in rat barrel cortex. To determine the locus of plasticity, we used laser scanning photostimulation, allowing us to rapidly map intracortical synaptic connectivity in brain slices. Layer 2/3 neurons differed in their spatial distributions of presynaptic partners: neurons directly above barrels received, on average, significantly more layer 4 input than those above the septa separating barrels. Complementary connectivity was found in deprived cortex: neurons above septa were now strongly coupled to septal regions, while connectivity between barrel regions and layer 2/3 was reduced. These results reveal competitive interactions between barrel and septal circuits in the establishment of precise intracortical circuits.

Neocortical long-term potentiation and long-term depression: site of expression investigated by infrared-guided laser stimulation.

The synaptic site of expression of long-term potentiation (LTP) and long-term depression (LTD) is still a matter of debate. To address the question of presynaptic versus postsynaptic expression of neocortical LTP and LTD in a direct approach, we measured the glutamate sensitivity of apical dendrites of layer 5 pyramidal neurons during LTP and LTD. We used infrared-guided laser stimulation to release glutamate from its "caged" form with high spatial and temporal resolution. Responses to photolytically released glutamate and synaptically evoked EPSPs were recorded with patch-clamp pipettes from the neuronal somata. LTP and LTD could be induced by electrical stimulation at the same synapses in succession. The NMDA receptor-dependent LTD was accompanied by a decrease in the dendritic glutamate sensitivity, suggesting a postsynaptic expression of neocortical LTD. In contrast, LTP was never accompanied by a change in the dendritic glutamate sensitivity. A possible explanation for this finding is a presynaptic expression of neocortical LTP. Another set of experiments corroborated these results: Photolytic application of glutamate with a frequency of 5 Hz caused a long-lasting Ca2+ and NMDA receptor-dependent decrease in the dendritic glutamate sensitivity. In contrast, LTP of dendritic glutamate sensitivity was never induced by photostimulation, despite several experimental modifications to prevent washout of the induction mechanism and to induce a stronger postsynaptic Ca2+ influx. In conclusion, our findings provide strong evidence for a postsynaptic expression of neocortical LTD and favor a primarily presynaptic locus of neocortical LTP.

Temporal integration can readily switch between sublinear and supralinear summation.

Temporal summation at dendrites of cultured rat hippocampal neurons was examined as a function of the interval separating two dendritic inputs. A novel method that relies on single-mode optical fibers to achieve rapid photorelease of glutamate was developed. Dendritic excitation achieved with this approach resembles that associated with miniature excitatory postsynaptic currents (mEPSCs), but the strengths, sites, and timing of the inputs can be precisely controlled. Dendritic summation deviated markedly from behavior predicted by passive cable theory. Subthreshold temporal summation varied as a triphasic function of the interpulse interval. As the interpulse interval decreased, local dendritic Na+ conductances were recruited to generate a marked transition from sublinear to supralinear summation. These results suggest that active dendritic conductances acting in concert with passive cable properties may serve to boost coincident synaptic inputs and attenuate noncoincident inputs.

Epidermal N-acetylation of p-aminobenzoyl glutamic acid: difference in response to ultraviolet B irradiation.

Arylamine N-acetyltransferases (EC 2.3.1.5) are conjugating drug-metabolizing enzymes and consist of two major isozymes, NAT1 and NAT2. As they have different substrate specificity and expression of polymorphism, distribution of the isozymes may be detected by investigating acetylation. p-Aminobenzoyl glutamic acid (pABG), one of the specific substrates for NAT1 in human pro-monocytic cell-line, was metabolized through acetylation by 9000 x g supernatant fraction from human epidermal keratinocytes and the effect of ultraviolet B (UVB) irradiation on the acetylation was also studied. Forty-eight hours after irradiation of UVB (200 J/m2), the activity was not increased (114 +/- 8.3%, n = 3), while increase in N-acetylating capacity for 2-aminofluorene (substrate for NAT1 and NAT2) amounted to 201 +/- 16% (n = 3). These results suggest that there are at least two isozymes for N-acetylation in the human epidermic and NAT2 may be affected by UVB irradiation.

Focal photolysis of caged glutamate produces long-term depression of hippocampal glutamate receptors.

Separating contributions of pre- and postsynaptic factors to the maintenance of long-term potentiation (LTP) and long-term depression (LTD) has been confounded by their experimental interdependence. To isolate the postsynaptic contribution, glutamate-receptor-mediated currents were elicited by localized photolysis of caged glutamate in small spots along the dendrites of CA1 hippocampal pyramidal cells. With synaptic transmission blocked, pairing depolarization of pyramidal cells with repeated photolysis of caged glutamate at one site markedly and persistently depressed subsequent responses to glutamate; responses at a second, unpaired site were unchanged. Like synaptically induced LTD at the CA3-CA1 synapse, this depression was site specific, NMDA-receptor dependent and blocked by protein-phosphatase inhibitors. Thus, robust, persistent alterations of postsynaptic glutamate receptor efficacy can occur without presynaptic neurotransmitter release.

[The question of amino acid isomerization in the microwave field. Results of a model study with standard solutions]. / Zur Frage der Aminosäureisomerisierung im Mikrowellenfeld. Ergebnisse eines Modellversuches mit Standardlösungen.

Aqueous standard-solutions of L-alanine, L-glutamic acid, and L-proline do not reveal any increase of D-enantiomers after 30 min heating--neither by the conventional method on a hotplate, nor in a standard microwave oven. A specific "microwave effect" and, hence, a special consumer risk is, in contrast to recent assumptions, not detectable. Effects on the amino acids which were observed in conventionally heated samples are explained by higher heat-exposure during the treatment of these samples.

[Effect of pyridoxal phosphate on gamma-aminobutyric acid metabolism in different sections of the brain in irradiated animals]. / Vliianie piridoksal'fosfata na obmen gamma-aminomaslianoi kisloty v razlichnykh otdelakh golovnogo mozga obluchennykh zhivotnykh.

A study was made of the effect of X-rays (4,5 Gy) and pyridoxal phosphate (3 mg/kg, v/v) on the activity of pyridoxal enzymes of GABA metabolism (e.g. glutamate decarboxylase, E.C. 4.1.1.15) and aminobutyrate aminotransferase (GABA-T, E.C. 2.6.1.19), as well as on GABA and glutamate content of the hemisphere cortex, brain stem and cerebellum of rabbits 6 and 10 days following irradiation and injection of a coenzyme. The height of the radiation sickness in rabbits was characterized by the manifest changes in glutamate decarboxylase and GABA-T activity, as well as in GABA and glutamate content of various brain parts differing in the structural and functional functions. The administration of pyridoxal phosphate produced pronounced activation of glutamate decarboxylase, particularly 6 days after irradiation and administration of the co-enzyme, and, to a lesser extent, influenced GABA-T function. Pyridoxal phosphate favored maintaining the GABA level above the control level in the hemisphere cortex and brain stem 6 and 10 days after exposure. The injection of pyridoxal phosphate did not normalize the glutamate content of the brain parts 6 days after exposure, but favored the normalization of GABA-T activity on day 10.

Chemical evolution of the citric acid cycle: sunlight photolysis of the amino acids glutamate and aspartate.

Sunlight photolysis of the amino acids glutamate and aspartate were carried out on 0.1 M aqueous solutions at pH = 7.0. The non-volatile products were identified by GC-MS analysis of derived methyl esters. The major product from glutamic acid was succinic acid, and, analogously, aspartic acid photolyzed to malonic acid. The photochemical oxidative decarboxylation of glutamate parallels its metabolism in modern cells and may provide an evolutionary link between simple amino acids and reactions of the citric acid cycle.

[Early changes in GABA and glutamate levels and aminotransferase activity in parts of the rat brain following whole-body gamma irradiation at an absolutely lethal dose]. / Rannie izmeneniia urovnia GAMK, glutamata i aminotransferaznoi aktivnosti v otdelakh golovnogo mozga krys posle obshchego gamma-oblucheniia v absoliutno letal'noi doze.

The contents of gamma-aminobutyric acid (GABA) and glutamate (GL) as well as GABA-aspartate- and alanine aminotransferase activities were measured in rat cerebellum, cerebral cortex and truncus cerebri 1, 3, 6, 24 and 48 hr following total-body gamma-irradiation (60Co) with a dose of 30 Gy. All the indices under study changed in a similar way in the cortex and truncus cerebri while in the cerebellum, GABA level increased and GABA-alpha-ketoglutarate aminotransfearse activity decreased 60 min after irradiation. The levels of GABA and GL in the cortex and truncus cerebri decreased immediately and increased 24 hr after irradiation. Activity of aminotransferases changed in a phase manner: changes in aspartate- and alanine aminotransferase activity were more pronounced than those of GABA-alpha-ketoglutarate aminotransferase activity and correlated with the glutamate level changes.

[Enzyme activity of glutamic acid metabolism and the Krebs cycle in the brain of rats laser-irradiated against a background of altered adrenoreceptor function]. / Aktivnost' nekotorykh fermentov obmena glutaminovoi kisloty i tsikla Krebsa v golovnom mozge krys pri lazernom obluchenii na fone izmenennogo funktsional'nogo sostoianiia adrenoretseptorov.

In the in vivo experiments it was demonstrated that the effect of a helium-neon laser (lambda = 632.8 nm), at the background of altered functional status of adrenoreceptors, changes the activity of some enzymes of the glutamic acid metabolism and the Krebs cycle. This may be attributed to both the direct effect of laser radiation and the indirect effect via the adrenergic system.