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A quantitative measure of the flash-evoked after-discharge (FEAD) in the rat was derived using power spectral analysis. The utility of this method of measurement was demonstrated by quantifying the suppression of FEAD by the drug yohimbine. This method of quantification would facilitate the use of FEAD for the purpose of screening antiepileptic drugs.
Intertrial EP variance measured in response to a brief binocularly presented flash of light decreases with a time-course that overlaps the average EP. This decrease in variance is not exclusive to the alpha frequency band (8-12 c/sec) but it is selective for it. Subjects with large initial variance tend to show a large decrease in variance. There is no relationship between the magnitude of the decrease in variance and the consistency of the EP. The phenomenon reflected by the decrease in variance is probably identical with 'event-related desynchronization.' The results support the hypothesis that an EP may consist of two processes that overlap in time: a reduction in background EEG activity and an almost deterministic transient potential change.
An accurate method for measuring flash evoked alpha attenuation has been derived, based on a model of the alpha generator. The method has been applied to 10 healthy volunteers in order to determine their alpha attenuation latencies (AALs). For the 9 subjects which showed at least some alpha rhythm, the AAL group mean is 95 msec. The AAL inaccuracy (S.D. 5 msec) is smaller than interindividual differences (S.D. 13 msec). There exists no relationship between the AAL and the frequency of the alpha rhythm.
Flash VECPs have been reported in 3 albino babies ranging in age from 1 to 7 months. The responses were dominated by a large positive component preceded by an earlier negative. The amplitude of this positive component measured from the peak of the preceding negative wave had a greater amplitude over the contralateral hemisphere on monocular stimulation indicative of the misrouting present in the albino visual system.
Flash visual evoked potentials (VEP) and concurrent multiple unit activity (MUA) were recorded from closely spaced intracortical sites in unanesthetized monkeys before and after intracortical injection of the GABAA antagonist bicuculline. Laminar VEP profiles were subjected to current source density (CSD) analysis to localize the transmembrane current flows contributing to the generation of the field potentials. Before bicuculline, the first large VEP component, N40, was generated principally within the parvocellular thalamorecipient sublamina 4Cb. After bicuculline injection, the current sinks associated with N40 spread throughout lamina 4, consistent with a release of intracortical inhibition mediated by GABA. A subsequent component, P65, believed to represent recurrent inhibitory activity within 4Cb, was greatly diminished in size after bicuculline injection. The laminar pattern of current sources and sinks coincident with this component was more complicated after bicuculline, reflecting the summation of current flows associated with disinhibited lamina 4 activity. Bicuculline also altered the responses of neuronal elements in laminae 3 and 5, evidenced by large increases in MUA in these laminae that began approximately 50 msec after stimulation. Finally, bicuculline diminished the degree of intracortical ocular dominance, implicating GABAergic mechanisms in the maintenance and refinement of ocular input segregation within cortical columns.
The oscillatory potentials (OPs) of the flash-elicited electroretinogram b-wave have been identified as sensitive indices of abnormalities within the retinal circulation. A recent study by Lovasik and Kothe has identified the rod system as being more vulnerable than the cone system to transient alterations of the retinal vascular perfusion pressure (RVPP). In view of these previous findings, we investigated the susceptibility of the scotopic OPs to altered retinal perfusion, in 10 normotensive paid volunteers between 21 and 31 years of age. A transient increase in RVPP was effected by body inversion, while a transient decrease in RVPP was obtained by suction ophthalmodynamometry. Group averaged data showed that OP-1 to OP-4 decreased in amplitude with either an increase or a decrease in RVPP. In contrast to all other OPs, OP-5 was reduced to a greater degree when RVPP was decreased and showed a pronounced gain in amplitude as RVPP was increased. Our study has isolated a component-specific vulnerability to altered retinal perfusion. This finding may be interpreted as indicating a different retinal site of origin for the generators of OP-5. The heightened sensitivity of OP-5 to alteration of the RVPP may offer diagnostic advantage for investigating chronic diseases causing retinal ischemia.
We have developed the technique of flash visual evoked potentials (VEPs) triggered by periodic synchronous discharges (PSDs) to investigate the interaction between PSDs and VEPs in 3 patients with Creutzfeldt-Jakob disease (CJD). This technique enables us to explore the cerebral pathophysiology underlying CJD. Unexpectedly, we found that PSDs and VEPs did not interact even when VEPs were evoked in close temporal proximity to PSDs. Thus, it appears that PSDs are generated by a different neuronal mechanism from that involved in VEP generation.
To characterize the dependencies of event-related potentials (ERPs) in lower vertebrates and brain levels upon recent history and sequences of stimuli, trains of flashes were delivered at various frequencies to unanesthetized rays while recording in optic tectum and telencephalon. ERPs to repetitive stimuli cannot be understood in terms of simple refractoriness and recovery. Processes must be invoked such as simultaneous excitation and suppression, facilitation and its opposite, rebound and induced rhythms, each with development and decay times and non-linearities. Some of these processes are uncovered by omitting a stimulus from a train. Omitted stimulus potentials (OSPs) act as though the brain expects a stimulus within 5-7 msec of the interstimulus interval (ISI) of the train. Very few ISIs suffice. The effect upon visual evoked potential (VEP) form and duration of the number of stimuli in short trains, before the steady state response (SSR) is established, is complex. Alternation of the amplitude of successive VEPs (1 large every 2 VEPs, 1 in 3, 1 in 4) is one indication of complexities in the SSR. OSPs also alternate. A single extra stimulus interpolated into a regular train causes distinct effects according to its position. Sharp discontinuities in these effects appear with < 5 msec shifts. Total power of the SSR decreases with stimulation frequency but there is a large peak of increased power at 7 Hz and another at 12 Hz. Induced rhythms are a labile, late phase of OSPs as well as of rested VEPs and of the off response to a long light pulse. Jittered ISI experiments show that the apparent expectation of the OSP is little affected and that the intervals in the last few hundred milliseconds are most influential. The OSP studied here (ISI < 0.5 s) is quite different from that so far studied in human subjects (ISI > 1 sec). We predict further similarities when each taxon is tested in the other ISI range. A major category of response characteristics, besides sensitivity, receptive fields and recovery times, is dependence upon recent history of iterative events, including intervals, delays omissions and perhaps multiple facilitating and forgetting time constants. The variables examined parametrically in this study are only some of those available. Such dynamical characteristics are important neglected properties of afferent systems at each level.
Diazo-2 is a calcium chelator based on BAPTA [(1989) J. Biol. Chem., in press], whose electron withdrawing diazoacetyl group may be rapidly (2000 s-1) converted photochemically to an electron donating carboxymethyl group by exposure to near ultraviolet light, producing an increase in its calcium affinity (Kd changes from 2.2 microM to 0.073 microM) without steric modification of the metal binding site. Photolysis of a 2 mM solution of this compound with a brief flash of light from a frequency-doubled ruby laser (347 nm) caused single skinned muscle fibres from the semitendinosus muscle of the frog Rana temporaria to relax with a mean half-time of 60.4 +/- 5 ms (range 30-100 ms, n = 15) at 12 degrees C, which is faster than the relaxation observed in intact muscles (half-time 133 ms at 14 degrees C [(1986) J. Mol. Biol. 188, 325-342]) and similar to the rate of the fast phase of tension decay in intact single fibres (20 s-1 at 10 degrees C [(1982) J. Physiol. 329, 1-20]).
Laser flash photolysis of alpha-tocopherol in methanol and in aqueous micellar solutions has been shown to produce the alpha-tocopheroxyl radical. The reaction between the alpha-tocopheroxyl radical and ascorbate in positively charged hexadecyltrimethylammonium chloride (HTAC) micelles occurred with a second order rate constant of 7.2 x 10(7) M-1.s-1, whereas in negatively charged sodium dodecyl sulphate (SDS) micelles the rats constant was only 3.8 x 10(4) M-1.s-1. The alpha-tocopheroxyl radical was found to be relatively long-lived in HTAC micelles (t1/2 greater than or equal to 5 min), allowing the slow disappearance of the alpha-tocopheroxyl radical by reaction with glutathione to be observed.
Ligand binding kinetics to heme-proteins between 40 and 300 K point to a regulatory role of protein dynamics. A protein-specific susceptibility of the heme-iron reactivity to dynamic fluctuations emerges from the distribution of reaction enthalpies derived from flash-photolysis measurements below ca. 180 K; we quantify it in terms of 'intramolecular viscosity', postulating that narrow low-temperature enthalpy distributions correspond to low internal viscosity and vice versa. The thermal evolution of ligand binding kinetics suggests, with other results, an interplay between high-frequency transitions of the amino acid side chains and low-frequency collective motions as a possible regulatory mechanism of protein dynamics.
The binding and interaction of carbamoylcholine with the nicotinic acetylcholine receptor was investigated using photolytically released carbamoylcholine ('caged' carbamoylcholine). Upon UV flash activation of this photolabile substrate analog, characteristic changes in the IR absorbance spectrum were detected. Apart from difference bands arising from the changes of molecular structure upon photolytical release, spectral features can be attributed to the agonist upon binding to the receptor as well as to conformational changes of the receptor itself. The two photo-labile agonist analogs N-[1-(2-nitrophenyl)ethyl] carbamoylcholine iodide (cage I) and N-(alpha-carboxy-2-nitrobenzyl) carbamoylcholine trifluoroacetate (cage II), with different structures for comparison of the 1680-1540 cm-1 region sensitive for protein conformation, yielded consistent results. A preliminary interpretation in terms of substrate binding and local conformational changes of the receptor upon carbamoylcholine binding is provided, in analogy to the binding of acetylcholine, activation, and subsequent deactivation taking place during signal transduction.
Electron transfer from P700 in photosystem I (PSI) particles from spinach to Anabaena sp. PCC 7119 flavodoxin has been studied using laser flash absorption spectroscopy. A non-linear protein concentration dependence of the rate constants was obtained, suggesting a two-step mechanism involving complex formation (k = 3.6 x 10(7) M-1.s-1) followed by intracomplex electron transfer (k = 270 s-1). The observed rate constants had a biphasic dependence on the concentrations of NaCl or MgCl2, with maximum values in the 40-80 mM range for NaCl and 4-12 mM for MgCl2. To our knowledge, this is the first time that the kinetics of PSI-dependent flavodoxin photoreduction have been determined.
Flash-induced single-electron reduction of cytochrome c oxidase. Compound F (oxoferryl state) by RuII(2,2'-bipyridyl)3(2+) [Nilsson (1992) Proc. Natl. Acad. Sci. USA 89, 6497-6501] gives rise to three phases of membrane potential generation in proteoliposomes with tau values and contributions of ca. 45 microsecond (20%), 1 ms (20%) and 5 ms (60%). The rapid phase is not sensitive to the binuclear centre ligands, such as cyanide or peroxide, and is assigned to vectorial electron transfer from CuA to heme a. The two slow phases kinetically match reoxidation of heme a, require added H2O2 or methyl peroxide for full development, and are completely inhibited by cyanide; evidently, they are associated with the reduction of Compound F to the Ox state by heme a. The charge transfer steps associated with the F to Ox conversion are likely to comprise (i) electrogenic uptake of a 'chemical' proton from the N phase required for protonation of the reduced oxygen atom and (ii) electrogenic H+ pumping across the membrane linked to the F to Ox transition. Assuming heme a 'electrical location' in the middle of the dielectric barrier, the ratio of the rapid to slow electrogenic phase amplitudes indicates that the F to Ox transition is linked to transmembrane translocation of 1.5 charges (protons) in addition to an electrogenic uptake of one 'chemical' proton required to form Fe(3+)-OH- from Fe4+ = O2-. The shortfall in the number of pumped protons and the biphasic kinetics of the millisecond part of the electric response matching biphasic reoxidation of heme a may indicate the presence of 2 forms of Compound F, reduction of only one of which being linked to full proton pumping.
A confocal spot detection optical setup was used to record fluorescence signals in response to calcium pulses, elicited by flash photolysis of DM-nitrophen, with the calcium indicators CaOrange-5N and Fluo-3. Our results yield the following conclusions: [Ca2+] changes are almost perfect spikes at pCa 9 and broader transients followed by a step at pCa 7. The [Ca2+] spikes were used to measure the dissociation rate constant of the Ca2+ dyes. Experiments at pCa 7 were used to verify the kinetic rate constants of the dyes and to obtain those of DM-nitrophen. The association rate constant of this compound was found to be more than one order of magnitude faster than that suggested previously. CaOrange-5N was able to track changes in [Ca2+] more accurately than Fluo-3. This latter dye introduced severe distortions which preclude a quantitative deconvolution of the fluorescence transients into changes in the free [Ca2+].
The kinetics of nucleotide release have been measured at the level of isolated synthetic myosin filaments. This was achieved by displacing a rhodamine nucleotide analog from a filament by flash photolysis of caged-ATP with selective observation using total internal reflectance fluorescence microscopy. The procedure gave improved time resolution over previously used flow methods. Kinetics were measured both in the presence of the ADP analog and during steady-state hydrolysis of the ATP analog. These studies open the way for kinetic measurements during actin filament sliding on myosin tracks.
Microcystins were extracted from 7 l (equivalent to 313 g dry weight) of cyanobacterial scum collected from Rutland Water in Leicestershire, UK in 1989. The resulting aqueous extract was rapidly concentrated on a C18 flash chromatography cartridge and microcystins were eluted using a step gradient. Fractions were collected manually and monitored by UV spectrophotometer and analytical HPLC. Fractions containing microcystins of similar polarity were pooled to give three fractions. Simple isocratic methods for separating each fraction were developed on an analytical column and scaled up to a 15 x 7.5 cm I.D. column. Closed-loop recycling was used to maximise yield and purity of two hydrophobic microcystins.
The hypothesis that a light-flash made contingent on stuttering would reduce stuttering to the extent that it called subjects' attention to their stuttering responses was investigated with two adult female stutterers. Attention to stuttering was measured by asking subjects to depress a switch each time they detected a stuttering in their speech. For one subject, the contingent light produced a marked reduction in stuttering but had no effect on switch-pressing. For the other subject, the light had little effect on stuttering but resulted in an increase in switch-pressing. For both subjects, reductions in stuttering occurred when they were asked to note their stutterings by pressing the switch. The results suggested that the effect of the light on stuttering was not due to its role in calling attention to stuttering.