John E. Chad

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.

Neuroprotection by both NMDA and non-NMDA receptor antagonists in in vitro ischemia

We have investigated the relative contributions of oxygen and glucose deprivation to ischaemic neurodegeneration in organotypic hippocampal slice cultures. Cultures prepared from 10-day-old rats were maintained in vitro for 14 days and then deprived of either oxygen (hypoxia), glucose (hypoglycaemia), or both oxygen and glucose (ischaemia). Hypoxia alone induced degeneration selectively in CA1 pyramidal cells and this was greatly potentiated if glucose was removed from the medium. We have also characterised the effects of both pre- and post-treatment using glutamate receptor antagonists and the sodium channel blocker tetrodotoxin (TTX). Neuronal death following either hypoxia or ischaemia was prevented by pre-incubation with CNQX, MK-801 or tetrodotoxin. MK-801 or CNQX also prevented death induced by either hypoxia or ischaemia if added immediately post-insult, however, post-insult addition of TTX prevented hypoxic but not ischaemic damage. Organotypic hippocampal slice cultures are sensitive to both NMDA and non-NMDA glutamate receptor blockade and thus represent a useful in vitro system for the study of ischaemic neurodegeneration paralleling results reported using in vivo models of ischaemia.

Inactivation of calcium channels

Rapid progress in our understanding of the properties and functions of voltage-gated calcium channels had produced the need for an update to our previous review of calcium inactivation. The major elements of change included in this review are: 1. The existence of multiple forms of voltage-sensitive Ca+ channels, with distinctive single channel properties, thus necessitating a reappraisal of properties deduced from macroscopic current recordings, particularly of the processes of activation and inactivation. 2. The differences in biochemical properties between channel types are reflected in their differences in divalent selectivity, their requirement for metabolic maintenance and their mechanism of inactivation. These properties appear to divide the channels into two categories which may relate to their molecular structures. Further subgroupings, based upon the voltage thresholds, have also been observed. 3. Molecular properties of one class of channels have been elucidated, which correlate with the observed biochemistry of channel modulation and inactivation. 4. An enzymatic process underlying the mechanism of Ca2+-dependent inactivation has been elucidated and may serve as a model for other modulatory systems. The interweaving of the properties of these Ca2+ channels, with their spatial distributions and their influence upon other channel types, acts to transduce and integrate information within cells.

Kinetics of calcium‐dependent inactivation of calcium current in voltage‐clamped neurones of Aplysia californica.

Ca currents flowing during voltage‐clamp depolarizations were examined in axotomized Aplysia neurones under conditions that virtually eliminated other currents. Moderate to large currents exhibited a two‐component time course of relaxation that can be approximated reasonably well by the sum of two exponentials. The rapid phase (tau 1 approximately equal to 70 ms at 0 mV) plus the slower phase (tau 2 approximately equal to 300 ms at 0 mV) ride upon a steady, non‐inactivating current, I infinity. Conditions that diminish the peak current amplitude, such as reduced stimulus depolarization, inactivation remaining from a prior depolarization, or partial blockade of the Ca conductance by Cd, slowed both phases of inactivation, and all selectively eliminated the tau 1 phase, such that weak currents exhibited only the slower phase of decline. Injection of EGTA slowed both phases of inactivation, decreased the extent of the tau 1 phase, and increased the intensity of I infinity and of the current during the tau 2 phase. For a given voltage, the rate of inactivation increased as the peak current strength was increased, and decreased as the peak current strength was decreased. For a given peak current the rate of inactivation decreased as depolarization was increased. The relation of inactivation to prior Ca2+ entry was essentially linear for small currents, but decreased in slope with time during strong currents. The relation also became shallower with increasing depolarization, suggesting an apparent decrease in the efficacy of Ca in causing inactivation at more positive potentials. The basic kinetics of Ca current inactivation along with experimentally induced changes in those kinetics were simulated with a binding‐site model in which inactivation develops during current flow as a function of the entry and accumulation of free Ca2+. This demonstrated that a single Ca‐mediated process can account for the two‐component time course of inactivation, and that the nearly bi‐exponential shape need not arise from two separate processes. The two‐component time course emerges as a consequence of a postulated hyperbolic reaction between diminishing probability of channels remaining open and the accumulation of intracellular free Ca2+. The occurrence of a single‐ or a two‐component time course of inactivation thus appears to depend on the levels of internal free Ca2+ traversed during current flow.

Pro-epileptic changes in synaptic function can be accompanied by pro-epileptic changes in neuronal excitability

Repetitive sensory input, stroboscopic lights or repeated sounds can induce epileptic seizures in susceptible individuals. In order to understand the process we have to consider multiple factors. The output of a set of neurones is determined by the amount of excitatory synaptic input, the degree of positive feedback and their inherent electrical excitability, which can be modified by synaptic inhibition. Recent research has shown that it is possible to separate these phenomena, and that they do not always behave in unison.

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°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.

The actions of muscle relaxants at nicotinic acetylcholine receptor isoforms

The pharmacological diversity of the different isoforms of the nicotinic acetylcholine receptor arises from the diversity of the subunits that assemble to form the native receptors. The aim of this study was to investigate the actions of the muscle relaxants d-tubocurarine, pancuronium and vecuronium on different isoforms of nicotinic acetylcholine receptors (mouse foetal muscle, mouse adult muscle and a rat neuronal), using the Xenopus oocyte expression system. Oocytes were injected with cRNAs for alpha, beta, gamma, delta subunits (the native foetal muscle subunit combination), or with cRNAs for alpha, beta, epsilon, delta subunits (the native adult muscle subunit combination), or with cRNAs for alpha4beta2 subunits (a putative native neuronal subunit combination). Acetylcholine had a similar potency at all three subunit combinations (EC50 11.6, 17.4 and 19.1 microM, respectively). At all three receptor types, d-tubocurarine and pancuronium blocked the responses elicited by acetylcholine in a reversible manner. Furthermore, the inhibition of the acetylcholine currents for the foetal and adult nicotinic acetylcholine receptor by pancuronium and d-tubocurarine was independent of the holding voltage over the range -100 to -40 mV. In oocytes expressing the foetal muscle nicotinic acetylcholine receptors the inhibition of the current in response to 100 microM acetylcholine by 10 nM d-tubocurarine was 29 +/- 5% (mean +/- S.E.M.; n = 7), and the inhibition by 10 nM pancuronium was 39 +/- 6% (mean +/- S.E.M.; n = 8; P > 0.05 vs. d-tubocurarine). However, in the adult form of the muscle nicotinic acetylcholine receptor, 10 nM d-tubocurarine and 10 nM pancuronium were both more effective at blocking the response to 100 microM acetylcholine compared to the foetal muscle nicotinic acetylcholine receptor, with values of 55 +/- 5% (P < 0.01; n = 12) and 60 +/- 4% (P < 0.001; n = 10), respectively. Thus the developmental switch from the gamma to the epsilon subunit alters the antagonism of the nicotinic acetylcholine receptor for both pancuronium and d-tubocurarine. Vecuronium was more potent than pancuronium. One nM vecuronium reduced the response to 100 microM acetylcholine by 71 +- 6% (n = 10) for foetal and 63 +/- 5% (n = 4) for adult nicotinic acetylcholine receptors. In the alpha4beta2 neuronal nicotinic acetylcholine receptor combination, 10 nM pancuronium was a more effective antagonist of the response to 100 microM acetylcholine (69 +/- 6%, n = 6) than 10 nM d-tubocurarine (30 +/- 5%; n = 6; P < 0.05 compared to pancuronium). This is in contrast to the adult muscle nicotinic acetylcholine receptor, where pancuronium and d-tubocurarine were equieffective. The expression of the beta2 subunit with muscle alpha, epsilon and delta subunits formed a functional receptor which was blocked by pancuronium and d-tubocurarine in a similar manner to the alphabeta1epsilondelta subunit consistent with the hypothesis that the beta subunit is not a major determinant in the action of this drug at the adult muscle nicotinic acetylcholine receptor.

Enhancement by 5-hydroxytryptamine and analogues of desensitization of neuronal and muscle nicotinic receptors expressed in Xenopus oocytes

1 The action of 5‐hydroxytryptamine (5‐HT) on neuronal and muscle nicotinic acetylcholine receptors (nicotinic AChR) expressed in Xenopus oocytes was studied. 2 5‐HT enhanced the rate of desensitization of the acetylcholine (ACh) current response in all receptor subtypes investigated (muscle, αβ2γδ and α4β2), acting in a dose‐dependent manner. 3 5‐HT also reduced the peak current elicited by ACh in a dose‐dependent manner. The IC50 value for the muscle type receptor was 227 ± 0.44 μm, and 166 ± 0.47 μm and 283 ± 0.28 μm for the combinations αβ2γ and α4β2 respectively. 4 The effect of 5‐HT on the responses to ACh (10 μm) was found to be independent of membrane voltage over the range tested (−80 to −10 mV), and to be readily reversed by washout. 5 The action of 5‐HT could be mimicked by structurally similar molecules. The homologue tryptamine was less potent than 5‐HT in blocking the ACh current, with an IC50 of 1.0 ± 0.02 mM. Ketanserin, a 5‐HT2 receptor antagonist, was more potent than 5‐HT, the IC50 being 49.0 ± 1.4 μm. 6 We postulate that a highly conserved portion of the tertiary structure of nicotinic AChRs, which includes some part of the ACh binding site, has affinity for 5‐HT and structural analogues.

Synaptic release rather than failure in the conditioning pulse results in paired-pulse facilitation during minimal synaptic stimulation in the rat hippocampal CA1 neurones

In this paper we report our observations of the relationship of paired-pulse facilitation (PPF) with synaptic release or release failure at small numbers of synaptic sites. Minimal stimulation protocols were employed to enable the activation of only one or a few axons which synapse onto CA1 pyramidal cells in the hippocampus. Excitatory postsynaptic currents (EPSCs) in response to paired stimulation were measured. On the analysis of the data, we examined the effects of failure and synaptic release on PPF, and found that PPF was observed as a decrease in failures of synaptic release in response to test stimuli which were preceded by conditioning stimuli that evoked synaptic release. The test-pulse failure rate following conditioning pulse failures was indistinguishable from the overall conditioning pulse failure rate. It is postulated that the mechanism of paired-pulse facilitation is presynaptic and associated with successful synaptic release in response to the conditioning stimulus.

Bicuculline enhances the late GABAB receptor-mediated paired-pulse inhibition observed in rat hippocampal slices

The inhibition of CA1 pyramidal neurones in rat hippocampal slices was studied using extracellular recordings of population spike potential responses to paired orthodromic stimulation. Variation of the interpulse interval allowed the separation of an early phase of inhibition (interpulse interval 5-20 ms), blocked by the GABAA receptor antagonist bicuculline (1 microM; n = 11), and a late phase (interpulse interval 200-400 ms) blocked by the GABAB receptor antagonist phaclofen (1 mM; n = 5) but enhanced by bicuculline (n = 11). Similar enhancement was not observed when conditioning response amplitudes were increased by increasing the stimulus strength, rather than bicuculline. Orthodromic stimulation leads to synaptic excitation of both pyramidal neurones and inhibitory interneurones, and may also lead to activation of inhibitory inputs onto interneurones. Bicuculline could prevent inhibition of the interneurones, and hence enhance the late, GABAB receptor-mediated inhibition. Conversely, the therapeutic administration of benzodiazepines would be postulated to enhance the inhibition of inhibitory interneurones, leading to an iatrogenic decrease in GABAB receptor-mediated inhibition.