CryoEM at 100†keV: a demonstration and prospects.

100 kV is investigated as the operating voltage for single-particle electron cryomicroscopy (cryoEM). Reducing the electron energy from the current standard of 300 or 200 keV offers both cost savings and potentially improved imaging. The latter follows from recent measurements of radiation damage to biological specimens by high-energy electrons, which show that at lower energies there is an increased amount of information available per unit damage. For frozen hydrated specimens around 300 Šin thickness, the predicted optimal electron energy for imaging is 100 keV. Currently available electron cryomicroscopes in the 100-120 keV range are not optimized for cryoEM as they lack both the spatially coherent illumination needed for the high defocus used in cryoEM and imaging detectors optimized for 100 keV electrons. To demonstrate the potential of imaging at 100 kV, the voltage of a standard, commercial 200 kV field-emission gun (FEG) microscope was reduced to 100 kV and a side-entry cryoholder was used. As high-efficiency, large-area cameras are not currently available for 100 keV electrons, a commercial hybrid pixel camera designed for X-ray detection was attached to the camera chamber and was used for low-dose data collection. Using this configuration, five single-particle specimens were imaged: hepatitis B virus capsid, bacterial 70S ribosome, catalase, DNA protection during starvation protein and haemoglobin, ranging in size from 4.5 MDa to 64 kDa with corresponding diameters from 320 to 72 Å. These five data sets were used to reconstruct 3D structures with resolutions between 8.4 and 3.4 Å. Based on this work, the practical advantages and current technological limitations to single-particle cryoEM at 100 keV are considered. These results are also discussed in the context of future microscope development towards the goal of rapid, simple and widely available structure determination of any purified biological specimen.

Role of fracture toughness in impact-abrasion wear.

Two new low alloyed steels were developed with different fracture toughness values but at similar level of hardness with same composition and microstructural phase. The steels were subjected to impact-abrasion wear test. This work examines specifically the additional role of toughness during impact-abrasion wear, using a newly developed high toughness steel. Microstructural characterisation of the damaged samples revealed that better toughness helps resist both impact and abrasion damage.

Author Correction: Magnetism and high magnetic-field-induced stability of alloy carbides in Fe-based materials.

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Magnetism and high magnetic-field-induced stability of alloy carbides in Fe-based materials.

Understanding the nature of the magnetic-field-induced precipitation behaviors represents a major step forward towards unravelling the real nature of interesting phenomena in Fe-based alloys and especially towards solving the key materials problem for the development of fusion energy. Experimental results indicate that the applied high magnetic field effectively promotes the precipitation of M23C6 carbides. We build an integrated method, which breaks through the limitations of zero temperature and zero external field, to concentrate on the dependence of the stability induced by the magnetic effect, excluding the thermal effect. We investigate the intimate relationship between the external field and the origins of various magnetics structural characteristics, which are derived from the interactions among the various Wyckoff sites of iron atoms, antiparallel spin of chromium and Fe-C bond distances. The high-magnetic-field-induced exchange coupling increases with the strength of the external field, which then causes an increase in the parallel magnetic moment. The stability of the alloy carbide M23C6 is more dependent on external field effects than thermal effects, whereas that of M2C, M3C and M7C3 is mainly determined by thermal effects.

Bursting response to current-evoked depolarization in rat CA1 pyramidal neurons is correlated with lucifer yellow dye coupling but not with the presence of calbindin-D28k.

Calbindin-D28k (CaBP) immunohistochemistry has been combined with electrophysiological recording and Lucifer Yellow (LY) cell identification in the CA1 region of the rat hippocampal formation. CaBP is shown to be contained within a distinct sub-population of CA1 pyramidal cells which is equivalent to the superficial layer described by Lorente de Nó (1934). The neurogenesis of these CaBP-positive neurons occurs 1-2 days later than the CaBP-negative neurons in the deep pyramidal cell layer, as shown by 3H-thymidine autoradiography. No correlation could be found between the presence or absence of CaBP and the type of electrophysiological response to current-evoked depolarizing pulses. The latter could be separated into bursting or non-bursting types, and the bursting-type response was nearly always found to be associated with the presence of LY dye coupling. Furthermore, when dye coupling involved three neurons, a characteristic pattern was observed which may represent the coupling of phenotypically identical neurons into distinct functional units within the CA1 pyramidal cell layer. In this particular case the three neurons were all likely to be CaBP-positive.

Hippocampal neurons transplanted into ischemically lesioned hippocampus: electroresponsiveness and reestablishment of circuitries.

Severe forebrain ischemia was used to damage selectively the CA1 region of the rat hippocampal formation. One week later the CA1 region was repopulated with suspensions of 18 day old fetal hippocampal tissue. Intracellular recordings were made from single units within the transplants by using the "in vitro" slice preparation, two to nine months following transplantation. Based on firing characteristics during depolarizing current injection, pyramidal-like and interneuron-like cells were identified within the transplants. Synaptic potentials could be evoked in the pyramidal-like neurons by stratum radiatum and stratum oriens stimulation demonstrating that normal afferent contacts had been made. Local inhibitory circuits were not obvious within the transplanted regions as demonstrated by prolonged EPSP's and the absence of early or late after-hyperpolarization. This was supported by the lack of conductance fluctuation in the active membrane when compared with the resting cell. Antidromic spikes could be evoked by applying shocks to the stratum oriens, towards the fimbria and subiculum, suggesting that the transplanted neurons were projecting basal neurites, quite long distances, along the normal efferent pathways. Thus, the transplanted neurons have the capacity to reconstruct damaged circuitries and develop intrinsic properties similar to their normal counterparts.

Synthesis, resolution, and absolute configuration of the isomers of the neuronal excitant 1-amino-1,3-cyclopentanedicarboxylic acid.

The endogenous amino acids glutamate and aspartate depolarize mammalian neurons to produce excitation, and the rigid glutamate analogue 1-amino-1,3-cyclopentanedicarboxylic acid also has this effect. This compound exists as two pairs of geometric isomers, and in the present study the absolute configuration of the four isomers is assigned. The known (+)-S and (-)-R isomers of 3-oxocyclopentanecarboxylic acid were used as the basis for the synthesis. The cis and trans amino acids were obtained by fractional crystallization. Spectral data, including optical rotation, circular dichroism, and 13C nuclear magnetic resonance, are presented. The compounds were evaluated as excitants by microiontophoretic ejection into the dendritic region of impaled CA1 pyramidal neurons of rat hippocampal slices. One isomer, cis-1R,3R, mimicked completely the actions elicited by N-methyl-D-aspartic acid; the other three isomers were alpha-kainic acid like.

Structural requirements for activation of excitatory amino acid receptors in the rat spinal cord in vitro.

The conformational requirements for activation of N-methyl-D-aspartate (NMDA) and quisqualate (QUIS) excitatory amino acid receptors on rat spinal neurones in vitro have been examined using a number of conformationally restricted compounds related to L-glutamate (L-GLU). The excitants were assigned to a receptor type on the basis of their susceptibility to blockade by D (-)-2-amino-5-phosphonvalerate (DAPV) and kynurenate (KYNA). When iontophoretically applied to unidentified neurones in the dorsal horn of spinal cord slices maintained in vitro, three of the isomers of 1-amino-1,3-cyclopentane dicarboxylate (ACPD) evoked excitations which were DAPV-sensitive and therefore were probably elicited via NMDA receptors. The fourth isomer (D-trans-(1R,3S)-ACPD) resembled quinolinate (QUIN) in its actions, and differed from both NMDA and QUIS. Several pyridine derivatives in addition to QUIN were tested, and both the 2,5- and 2,6-pyridine dicarboxylates evoked excitations which, like those produced by QUIS and L-GLU, were largely unaffected by both DAPV and KYNA and thus appeared due to activation of the QUIS receptor. 2,4-Pyridine dicarboxylate acted as a weak and unselective antagonist of amino acid-induced excitations. The results support an earlier conclusion that compounds reacting with the NMDA receptor do so in an extended configuration whereas the QUIS receptor has a more folded template. The possibility that QUIN reacts with a receptor different from those activated by other amino acids is considered.

The action of quinolinate in the rat spinal cord in vitro.

The responses of dorsal horn neurones to the excitatory amino acids quisqualate, kainate, N-methyl-D-aspartate (NMDA), and quinolinate have been examined in an in vitro preparation of the rat spinal cord. The antagonism of these responses by iontophoretically applied D-(-)-2-amino-5-phosphonovalerate (DAPV), kynurenate, and acridinate was tested, and the results were compared with data obtained from the spinal cord in vivo. The pattern of antagonism was similar in both preparations, although the potencies of agonists and antagonists were found to be significantly greater in vitro. The antagonism of amino acid induced firing of neurones was also recorded during the application of DAPV and kynurenate in the bathing medium. Dose-response curves and IC50 values were determined for these antagonists against all four agonists. The responses to quinolinate were antagonized differently from those to NMDA, quisqualate, or kainate, suggesting that quinolinate does not act specifically through the NMDA receptor as it does in other regions, nor does it appear to act via two or more of the three archetypal amino acid receptors. These findings suggest that a fourth amino acid receptor responsible for quinolinate's action in the spinal cord may exist.

Excitation of rat hippocampal neurones by the stereoisomers of cis- and trans-1-amino-1,3-cyclopentane dicarboxylate.

Intracellular recordings were obtained from rat hippocampal neurons during the microiontophoretic ejection of the stereoisomers of cis- and trans-1-amino-1,3-cyclopentane dicarboxylate into the dendritic region (stratum radiatum) of the impaled cells. L-(+)-cis-1-Amino-1,3-cyclopentane dicarboxylate, D(+)-trans-1-amino-1,3-cyclopentane dicarboxylate, and L-(-)-trans-1-amino-1,3-cyclopentane dicarboxylate all evoked patterns of excitation resembling that elicited by kainate. All of these responses were unaffected by D-(-)-2-amino-5-phosphonovalerate but were antagonized at comparable currents by kynurenate. The excitation produced by D-(-)-cis-1-amino-1,3-cyclopentane dicarboxylate was similar to that evoked by N-methyl-D-aspartate. At low ejection currents a slow depolarization triggered rhythmic burst firing, each burst consisting of a depolarizing shift in membrane potential upon which were superimposed four to five action potentials. These responses were antagonized both by D-(-)-2-amino-5-phosphonovalerate and by kynurenate. The results are discussed with respect to the conformational requirements considered to be necessary for interaction at the kainate and N-methyl-D-aspartate receptors on CA1 pyramidal neurones. It is important to note that the isopropylene side chain of kainate is absent from the 1-amino-1-3-cyclopentane dicarboxylate molecule.

The N-methyl-D-aspartate receptor and burst firing of CA1 hippocampal pyramidal neurons.

Previous intracellular investigations in the rat hippocampus have demonstrated that N-methyl-D-aspartate, ibotenate and 2,3-pyridine dicarboxylate (quinolinate) all evoke burst firing of CA1 pyramidal neurons, whereas kainate and quisqualate, which are thought to react with different receptors, do not. The purpose of the present study has been to investigate the ability of a series of compounds either to trigger burst firing or to antagonize this pattern of excitation. We report here that N-methyl-L-aspartate, 1,2-benzene dicarboxylate (phthalate) and methylene succinate (itaconate) are also capable of evoking burst firing. The results of this investigation suggest that since both quinolinate and phthalate are rigid planar molecules and only the 2 and 3 positioning of the carboxylates of pyridine was active, a cis configuration of the carboxyls with respect to the 2,3 carbon bond appears to be necessary for excitation. While a nitrogen atom is not necessary for activity (this is absent in phthalate and itaconate) a third functional group, bearing at least a partial positive charge, and in a position alpha to one of the carboxyl groups is required. The requirements for pyridine derivatives to trigger burst firing is similar to that reported as necessary for evoking convulsions and neurotoxicity after intrahippocampal infusion and a correlation between N-methyl-D-aspartate-like burst firing and depolarization and this neuropathology is considered. An important observation has been that the addition of a benzene ring to either quinolinate or phthalate to yield 2,3-quinoline dicarboxylate and 2,3-napthalene dicarboxylate, respectively, converted these excitants into antagonists of burst firing.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel spinal cord slice preparation from the rat.

A spinal cord slice preparation is described. The lumbar enlargement of weanling rats is exposed by laminectomy, the dorsal and ventral roots cut and a portion of cord consisting of about 4 segments removed and chilled to 3 degrees C. A modified tissue chopper is used to produce 400 micron dorsoventral longitudinal slices. The slices are maintained in a low-volume, continuously perfused tissue chamber at the interface between warm moist 95% O2/5% CO2 and oxygenated artificial cerebrospinal fluid, and remain viable for over 8 h. An extracellular recording of excitatory amino acid-induced activity of a dorsal horn lamina IV cell is shown. Excitatory amino acid antagonists applied iontophoretically and in the perfusate have actions similar to those in the adult spinal cord in vivo. This in vitro preparation of the cord has permitted stable extracellular recordings from single cells of 2 h or more, and has the potential for intracellular investigation of spinal cord neurones.

Acridinic acid: a new antagonist of amino acid-induced excitations of central neurones.

The actions of acridinic acid (2,3-quinoline dicarboxylic acid), a new derivative of quinolinic acid, as an antagonist of amino acid-induced excitations are described. Acridinate, like kynurenate, in the cerebral cortex reduced the effects of all amino acids equally, but in the spinal cord was significantly less active against quisqualate.

2-Amino-5-phosphonovalerate and Co2+ selectively block depolarization and burst firing of rat hippocampal CA1 pyramidal neurones by N-methyl-D-aspartate.

Intracellular recordings from pyramidal neurones during microiontophoretic ejection of N-methyl-D-aspartate and quisqualate into the pyramidal cell layer of the CA1 region of the rat hippocampal slice showed that both amino acids caused depolarization and evoked spike activity. Whereas quisqualate evoked tetrodotoxin-sensitive spikes, those produced by N-methyl-D-aspartate consisted of bursts of tetrodotoxin-sensitive action potentials superimposed on an underlying depolarizing shift of membrane potential. Both membrane depolarization and the superimposed depolarizing shifts associated with N-methyl-D-aspartate excitation were selectively and reversibly antagonized by the D(-) isomer of 2-amino-5-phosphonovalerate and Co2+. Both amino acids caused an increase in membrane conductance when small ejection currents were used, and the depolarizing response to these compounds was prevented by current injection. However, only the increase by N-methyl-D-aspartate was blocked by 2-amino-5-phosphonovalerate and Co2+. These results provide evidence to support the suggestion that different mechanisms underlie the excitatory response to N-methyl-D-aspartate and quisqualate in CA1 pyramidal neurones.

Ca2+-dependent depolarization and burst firing of rat CA1 pyramidal neurones induced by N-methyl-D-aspartic acid and quinolinic acid: antagonism by 2-amino-5-phosphonovaleric and kynurenic acids.

The excitatory effects of microiontophoretically applied quisqualic (QUIS), N-methyl-D-aspartic (NMDA), and quinolinic (QUIN) acids were investigated using intracellular recording from CAl pyramidal neurones in slices of rat hippocampus. QUIS evoked only simple action potentials superimposed upon a depolarization which attained a clear plateau. When this level had been reached, increased ejecting currents did not produce further depolarization. By contrast, with low currents NMDA and QUIN elicited small membrane depolarizations which triggered bursts of action potentials superimposed upon rhythmically occurring depolarizing shifts. Larger currents caused depolarization which if sufficiently large completely blocked spike activity. Tetrodotoxin (TTX) prevented the spikes evoked by QUIS and the bursts of action potentials seen with NMDA and QUIN, and the rhythmic depolarizing shifts then appeared as broad spikes of up to 50 mV in amplitude. These and the underlying membrane depolarization were blocked by Co2+, by the NMDA antagonist D(-)-2-amino-5-phosphonovaleric acid (DAPV), and by kynurenic acid (KYNU). It thus appears that the depolarization and burst firing of rat CAl pyramidal neurones elicited by NMDA and QUIN are Ca2+ dependent while the actions of QUIS are not.

Pre-and postsynaptic actions of baclofen: blockade of the late synaptically-evoked hyperpolarization of CA1 hippocampal neurones.

Using intracellular recording techniques, the effects of beta-p-chlorophenyl-GABA (baclofen) on passive membrane properties and postsynaptic potentials of CA1 pyramidal neurones were investigated. In experiments where only the hyperpolarizing action of baclofen was precluded by conventional current clamp techniques, 20 microM ( +/- ) baclofen blocked the early GABA-mediated IPSP and also a late hyperpolarization which, since it could be evoked by orthodromic stimulation subthreshold for spike firing, would not be expected to be produced by a Ca2+-activated increase in potassium conductance (AHP), but to be a transmitter-mediated event. In addition the conductance increase associated with this late IPSP evoked by subthreshold stimulation and also that associated with the AHP produced by spike activation were abolished. Baclofen also appeared to increase the duration of EPSPs, an event possibly related to loss of IPSPs. The hyperpolarization produced by baclofen was associated with an increased conductance of the resting membrane, an event possibly associated with an elevated potassium flux. To preclude this postsynaptic effect as a cause of reduced synaptic responses, tetraethylammonium chloride (TEA), a compound which decreases conductance and depolarizes the membrane of CA1 pyramidal neurones by a reduction of a 'leak' or resting potassium conductance (gK), was added to the bathing medium.(ABSTRACT TRUNCATED AT 250 WORDS)

4,5,6,7-Tetrahydroisothiazolo[5,4-c]pyridin-3-ol and related analogues of THIP. Synthesis and biological activity.

The thio analogues of the GABA (gamma-aminobutyric acid) agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), the GABA uptake inhibitor THPO (4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol), and the glycine antagonist THAZ (5,6,7,8-tetrahydro-4H-isoxazolo[4,5-d]azepin-3-ol) have been synthesized and tested biologically on single neurons in the cat spinal cord and in vitro by using synaptic membrane preparations obtained from rat brains. In contrast to THIP, thio-THIP (4,5,6,7-tetrahydroisothiazolo[5,4-c]pyridin-3-ol, 5) was only a weak GABA agonist. Thio-THPO (4,5,6,7-tetrahydroisothiazolo[4,5-c]pyridin-3-ol, 10) was slightly weaker than THPO as an inhibitor of GABA uptake in vitro, and these two compounds were approximately equipotent in enhancing the inhibition of the firing of cat spinal neurons by GABA. Like THAZ and structurally related bicyclic isoxazole zwitterions, thio-THAZ (5,6,7,8-tetrahydro-4H-isothiazolo[4,5-d]azepin-3-ol, 15) was an antagonist at glycine receptors on cat spinal neurons. The I/U ratios, which reflect the ability of neutral amino acids to penetrate the blood-brain barrier (BBB), were calculated for 5 (I/U = 16), 10 (63), and 15 (200). These low I/U ratios, compared with the findings that THIP (I/U = 500 or 1500) and THPO (I/U = 2500) enter the brain after systemic administration, suggest that the thio analogues may penetrate the BBB very easily.

Spinal interneurone depression by DS103-282.

The depressant effect of 5-chloro-4-(2-imidazolin-2-yl-amino)-2,1,3-benzothiodiazole (DS103-282) on the polysynaptic excitation of interneurones in the cat spinal cord appears to be related to a postsynaptic reduction in the effectiveness of excitatory transmitters than to interference with their presynaptic release.

Antagonists of synaptic and amino acid excitation of neurones in the cat spinal cord.

In the spinal cord of the anaesthetized cat microelectrophoretically administered (+/-)-cis-2,3-piperidine dicarboxylate (2,3-PDA), (+/-)-cis-2,5-piperidine dicarboxylate (2,5-PDA), gamma-D-glutamylglycine (gamma DGG), beta-D-aspartyl-beta-alanine (beta DAA), (+/-)-2-amino-4-phosphonobutyrate (2-APB), (+/-)-2-amino-5-phosphonovalerate (2-APV) and (+/-)-2-amino-7-phosphonoheptanoate (2-APH) were assessed as antagonists of chemical excitation of dorsal horn interneurones and Renshaw cells by N-methyl-D-aspartate (NMDA), L-aspartate, quisqualate (QUIS), kainate and L-glutamate, and of monosynaptic and polysynaptic excitation by impulses in primary afferent fibres of muscle and cutaneous origin. Whereas polysynaptic excitation of interneurones was readily and reversibly depressed by 2-APV, 2-APH, beta DAA, gamma DGG and 2,3-PDA, all of which also reduced excitation by NMDA (and L-aspartate) more than that by QUIS (and L-glutamate), no selective antagonism of monosynaptic excitation could be demonstrated. In particular, 2,3-PDA, which depressed excitation by kainate to a greater extent than that by either QUIS or NMDA, appeared to have no effect on monosynaptic excitation. The results support the involvement of L-aspartate as the transmitter of some spinal excitatory interneurones, but none of the antagonists tested were considered suitable for assessing the role of L-glutamate as the transmitter of some spinal primary afferent fibres.

Post-excitatory depression of neuronal firing by acidic amino acids and acetylcholine in the cat spinal cord.

In the spinal cord of cats anaesthetized with pentobarbitone, the excitation of interneurones and Renshaw cells by acidic amino acids or acetylcholine (Renshaw cells) is followed by a period of depressed excitability. This depression appears to be the consequence of prolonged repetitive firing rather than of the enzymic or chemical conversion of the excitants to neuronal depressants.