Quantitative and correlative extreme ultraviolet coherent imaging of mouse hippocampal neurons at high resolution.

Microscopy with extreme ultraviolet (EUV) light can provide many advantages over optical, hard x-ray or electron-based techniques. However, traditional EUV sources and optics have large disadvantages of scale and cost. Here, we demonstrate the use of a laboratory-scale, coherent EUV source to image biological samples-mouse hippocampal neurons-providing quantitative phase and amplitude transmission information with a lateral resolution of 80 nm and an axial sensitivity of ~1 nm. A comparison with fluorescence imaging of the same samples demonstrated EUV imaging was able to identify, without the need for staining or superresolution techniques, <100-nm-wide and <10-nm-thick structures not observable from the fluorescence images. Unlike hard x-ray microscopy, no damage is observed of the delicate neuron structure. The combination of previously demonstrated tomographic imaging techniques with the latest advances in laser technologies and coherent EUV sources has the potential for high-resolution element-specific imaging within biological structures in 3D.

Bispyridinium Compounds Inhibit Both Muscle and Neuronal Nicotinic Acetylcholine Receptors in Human Cell Lines.

Standard treatment of poisoning by organophosphorus anticholinesterases uses atropine to reduce the muscarinic effects of acetylcholine accumulation and oximes to reactivate acetylcholinesterase (the effectiveness of which depends on the specific anticholinesterase), but does not directly address the nicotinic effects of poisoning. Bispyridinium molecules which act as noncompetitive antagonists at nicotinic acetylcholine receptors have been identified as promising compounds and one has been shown to improve survival following organophosphorus poisoning in guinea-pigs. Here, we have investigated the structural requirements for antagonism and compared inhibitory potency of these compounds at muscle and neuronal nicotinic receptors and acetylcholinesterase. A series of compounds was synthesised, in which the length of the polymethylene linker between the two pyridinium moieties was increased sequentially from one to ten carbon atoms. Their effects on nicotinic receptor-mediated calcium responses were tested in muscle-derived (CN21) and neuronal (SH-SY5Y) cells. Their ability to inhibit acetylcholinesterase activity was tested using human erythrocyte ghosts. In both cell lines, the nicotinic response was inhibited in a dose-dependent manner and the inhibitory potency of the compounds increased with greater linker length between the two pyridinium moieties, as did their inhibitory potency for human acetylcholinesterase activity in vitro. These results demonstrate that bispyridinium compounds inhibit both neuronal and muscle nicotinic receptors and that their potency depends on the length of the hydrocarbon chain linking the two pyridinium moieties. Knowledge of structure-activity relationships will aid the optimisation of molecular structures for therapeutic use against the nicotinic effects of organophosphorus poisoning.

A super-oscillatory lens optical microscope for subwavelength imaging.

The past decade has seen an intensive effort to achieve optical imaging resolution beyond the diffraction limit. Apart from the Pendry-Veselago negative index superlens, implementation of which in optics faces challenges of losses and as yet unattainable fabrication finesse, other super-resolution approaches necessitate the lens either to be in the near proximity of the object or manufactured on it, or work only for a narrow class of samples, such as intensely luminescent or sparse objects. Here we report a new super-resolution microscope for optical imaging that beats the diffraction limit of conventional instruments and the recently demonstrated near-field optical superlens and hyperlens. This non-invasive subwavelength imaging paradigm uses a binary amplitude mask for direct focusing of laser light into a subwavelength spot in the post-evanescent field by precisely tailoring the interference of a large number of beams diffracted from a nanostructured mask. The new technology, which--in principle--has no physical limits on resolution, could be universally used for imaging at any wavelength and does not depend on the luminescence of the object, which can be tens of micrometres away from the mask. It has been implemented as a straightforward modification of a conventional microscope showing resolution better than λ/6.

In vitro CNS tissue analogues formed by self-organisation of reaggregated post-natal brain tissue.

In this paper, we report the characterization of 'Hi-Spot' cultures formed by the re-aggregation of dissociated postnatal CNS tissue grown at an air-liquid interface. This produces a self-organised, dense, organotypic cellular network. Western blot, immunohistochemical, viral transfection and electron microscopy analyses reveal neuronal and glial populations, and the development of a synaptic network. Multi-electrode array recordings show synaptically driven network activity that develops through time from single unit spiking activity to global network bursting events. This activity is blocked by tetanus toxin and modified by antagonists of glutamatergic and GABAergic receptors suggesting tonic activity of excitatory and inhibitory synaptic signaling. The tissue-like properties of these cultures has been further demonstrated by their relative insensitivity to glutamate toxicity. Exposure to millimolar concentrations of glutamate for hours is necessary to produce significant excitotoxic neuronal death, as in vivo. We conclude that 'Hi-Spots' are biological analogues of CNS tissue at a level of complexity that allows for detailed functional analyses of emergent neuronal network properties.

Kainic acid induces rapid cell death followed by transiently reduced cell proliferation in the immature granule cell layer of rat organotypic hippocampal slice cultures.

Brain injury due to seizures results in transiently increased cell proliferation and neurogenesis in the subgranular zone of the adult dentate gyrus. In contrast, the immature postnatal brain appears to be more resistant to cell death after seizure-induced brain injury and paradoxically reacts to seizures by reducing SGZ proliferation. Organotypic hippocampal slice cultures are a useful paradigm for modelling the early postnatal hippocampus. We have investigated the temporal relationship between cell death and cell proliferation after kainate in the granule cell layer of rat organotypic hippocampal slice cultures equivalent to post natal day 11 animals. We found stable numbers and densities of mature thionine stained cells in the granule cell layer over 72 h in control cultures grown in defined medium. We also found a slowly declining cell proliferation rate over the same time period under control conditions. We report evidence of early cell death in the granule cell layer after just 2 h exposure to 5 microM kainate, followed by a significant decrease in cell proliferation in the granule cell layer at 24 h. In contrast to control conditions, cell proliferation rose significantly in the kainate exposed cultures by 72 h back to levels seen at 2 h. There were no significant changes in cell labelling with antibody to activated caspase-3 between kainate treated and control cultures at any time point examined. Our results suggest that kainate-induced injury in the early postnatal hippocampus damages precursor cells contributing to a reduction in granule layer cell proliferation.

Intraischaemic hypothermia reduces free radical production and protects against ischaemic insults in cultured hippocampal slices.

Hypothermia has been demonstrated to be an effective neuroprotective strategy in a number of models of ischaemic and excitotoxic neurodegeneration in vitro and in vivo. Reduced glutamate release and free radical production have been postulated as potential mechanisms underlying this effect but no definitive mechanism has yet been reported. In the current study, we have used oxygen-glucose deprivation in organotypic hippocampal slice cultures as an in vitro model of cerebral ischaemia. When assessed by propidium iodide fluorescence, reducing the temperature during oxygen-glucose deprivation to 31-33 degrees C was significantly neuroprotective but this effect was lost if the initiation of hypothermia was delayed until the post-insult recovery period. The neuroprotective effects of hypothermia were associated with a significant decrease in both nitric oxide production, as assessed by 3-amino-4-aminomethyl-2',7'-difluorofluorescein fluorescence, and superoxide formation. Further, hypothermia significantly attenuated NMDA-induced nitric oxide formation in the absence of hypoxia/hypoglycaemia. We conclude that the neuroprotective effects of hypothermia are mediated through a reduction in nitric oxide and superoxide formation and that this effect is likely to be downstream of NMDA receptor activation.

4,5-diaminofluoroscein imaging of nitric oxide synthesis in crayfish terminal ganglia.

We have analyzed the synthesis of nitric oxide in the terminal abdominal ganglion of the crayfish using the fluorescent probe 4,5-Diaminofluoroscein diacetate, DAF-2 DA. Following DAF-2 loading, ganglia showed cell-specific patterns of fluorescence in which the occurrence of strongly fluorescent cell bodies was highest in specific anterior, central, and posterior regions. We found that preincubation with the nitric oxide synthase (NOS) inhibitor L-NAME prevented much of the initial development of DAF-2 fluorescence, whereas the inactive isomer D-NAME had no effect. Washout of preincubated L-NAME caused increased cell-specific fluorescence due to endogenous NOS activity. Application of the NOS substrate L-arginine also resulted in an increase of DAF-2 fluorescence in a cell-specific manner. We bath applied the NO donor SNAP to increase exogenous NO levels which resulted in DAF-2 fluorescence increases in most cells. We therefore presume that the cell-specific pattern of DAF-2 fluorescence indicates the distribution of neurones actively synthesizing NO. The similarity between the DAF-2 staining pattern and previously published studies of NOS activity are discussed.