Optical recording from cerebellar Purkinje cells using intracellularly injected voltage-sensitive dyes.

We evaluated several techniques for their ability to record membrane potential changes with voltage-sensitive dyes introduced into CNS neurons in the brain slice preparation. Using a probe designed for intracellular application, JPW1114, we found that iontophoresis or pressure pulses could not push the lipophilic dye through electrodes whose resistance was sufficiently high to produce good electrical recordings in cerebellar Purkinje neurons. However, properly selected patch electrodes could introduce the dye into the cell and still give good electrical records. Using this technique we recorded depolarizing and hyperpolarizing transients and climbing fiber responses using either a single photodiode or a fast, cooled CCD camera. While these results are promising, there are still problems due to the slow diffusion of the dye in the dendrites and a low sensitivity which requires signal averaging to acquire traces with a good signal to noise ratio.

Imaging membrane potential with voltage-sensitive dyes.

Membrane potential can be measured optically using a variety of molecular probes. These measurements can be useful in studying function at the level of an individual cell, for determining how groups of neurons generate a behavior, and for studying the correlated behavior of populations of neurons. Examples of the three kinds of measurements are presented. The signals obtained from these measurements are generally small. Methodological considerations necessary to optimize the resulting signal-to-noise ratio are discussed.

Optical measurement of action potential activity in invertebrate ganglia.

With a voltage-sensitive dye and an array of silicon photodiodes we monitored the action potential activity of a large fraction of the cells in the Aplysia abdominal ganglion during the gill-withdrawal reflex. This capability allowed a direct determination of the spread of sensory information within the 1,000 neurons of the ganglion. Surprisingly, approximately 30% of the neurons are activated by a light touch to a small area of siphon skin. It is likely that many other neurons also receive large synaptic potentials, either excitatory or inhibitory, and thus even a very mild and restricted stimulus will have widely distributed effects. It seems to us that these results will force a more pessimistic view of the present understanding of the neuronal basis of apparently simple behaviors.

Suicidal strangulation with a double-knotted noose.

The paper describes an unquestionable case of suicide by strangulation. The case is of special interest because in addition to two single knots there were two double knots in the noose. As a rule strangulation with a double-knotted noose is seen only in cases of murder. The data attained at autopsy and the findings of the detailed police investigation are discussed.

Prognostic significance of clinical parameters and Epstein-Barr virus infection in non-endemic undifferentiated carcinoma of nasopharyngeal type: a Serbian report.

Undifferentiated carcinoma of nasopharyngeal type (UCNT) is very rare tumour in Serbia, like in most of the countries of Europe, with incidence less than 0.5 per 100,000 people per year. The aim of this study was to assess the presence of Epstein-Barr virus (EBV) in the UCNT of a non-endemic population in Serbia and identify the main clinical parameters that interfere with patients' survival rate. This study included 102 patients with UCNT who were diagnosed between 1996 and 2003. Biopsies were analysed for EBV-encoded RNA (EBER) by in situ hybridization of tumour tissue microarray. Of 102 patients, 76 were men and 26 were women with ages ranging between 18 and 82 years (median 52.5, mean 53.0±14.1). Survival rates were 80, 39 and 31% for one, three and five years, respectively. Ninety-three of 102 cases were EBER positive (92%). Factors with unfavourable prognostic values were age over 50 years at the time of diagnosis, advanced clinical stage, therapy other than chemoradiotherapy and EBER negative status. In regard to the clinical data, EBER expression in UCNT was shown to be a strong independent predictor of overall and progression-free survival. To our knowledge, the current report constitutes the largest European non-endemic series of UCNT samples from a single institution with correlation between survival and clinical parameters/EBER status.

Multiple spike-initiation zones in single neurons revealed by voltage-sensitive dyes.

The primary function of the nerve cell is to process electrical signals. Over the past fifteen years there has been renewed interest in the detailed spatial analysis of signalling in individual neurons owing to experimental evidence that the regional electrical properties of neurons are complex. Thus the behaviour of many nerve cells cannot be understood on the basis of microelectrode measurements from the soma. Regional electrical properties of neurons have been studied using sharp microelectrode and patch-electrode recordings from neuronal processes, high-resolution multisite optical recordings of Ca2+ concentration changes and by using models to predict the distribution of membrane potential in the entire neuronal arborization. Additional direct evidence about electrical signalling in neuronal processes in situ can now be obtained by recording membrane potential changes using voltage-sensitive dyes. Here I demonstrate the existence of multiple action potential trigger zones in separate regions of the neuronal arborization of an identified molluscan neuron.

Optical signals from neurons with internally applied voltage-sensitive dyes.

We carried out experiments to monitor optically the generation and spread of action potentials and subthreshold potentials in the processes of individual neurons in ganglia of the snail, Helix aspersa. The neurons were selectively stained by intracellular pressure injection of voltage-sensitive dyes. Optical signals were detected by a system for fast, multiple-site optical monitoring, utilizing a silicon photodiode array. After testing 30 voltage-sensitive dyes using absorption, we concluded that this mode was probably not sensitive enough to allow monitoring neuronal signals from distal processes. Satisfactory signals were obtained in fluorescence measurements using a newly synthesized styryl dye, JPW1114, specifically designed for intracellular application. There was an improvement in sensitivity (as defined by the signal-to-noise ratio) by a factor of about 50 over previously reported absorption and fluorescence signals from neuronal processes stained by either intra- or extracellular application of dyes. Recordings with good signal-to-noise ratio and adequate spatial and temporal resolution were obtained simultaneously from the cell body and long axonal branches. From this data, the site of action potential initiation was determined. Also, the propagation velocity of the action potential was calculated for different axonal segments; the results suggest that different regions have different velocities ranging from 0.53 m/sec to 0.07 m/sec. The present sensitivity was adequate to allow the recording of a 10 mV hyperpolarizing electrotonic response along axonal branches and to observe directly the decline of this passive response with distance from the site of stimulation. Relatively modest improvements in sensitivity will allow systematic analyses of the spread and summation of synaptic potentials in individual neurons.

Fast optical measurement of membrane potential changes at multiple sites on an individual nerve cell.

In the past 15 years, there has been renewed interest in the detailed spatial analyses of signalling in individual neurons. The behaviour of many nerve cells is difficult to understand on the basis of microelectrode measurements from the soma. Regional electrical properties of neurons have been studied using sharp microelectrode and patch-electrode recordings from neuronal processes, high-resolution multisite optical recordings of Ca2+ concentration changes and by using models to predict the distribution of membrane potential in the entire neuronal arborization. Additional, direct evidence about electrical signalling in neuronal processes of individual cells in situ can now be obtained by recording of membrane potential changes using voltage-sensitive dyes. A number of recent studies have shown that active regional electrical properties of individual neurons are extraordinarily complex, dynamic and, in the general case, impossible to predict by present models. This places a great significance on measuring capabilities in experiments studying the detailed functional organization of individual neurons. The main difficulty in obtaining a more accurate description was that experimental techniques for studying regional electrical properties of neurons were not available. With this motivation, we worked on the development of multisite voltage-sensitive dye recording as a potentially powerful approach. The results described here demonstrate that the sensitivity of voltage-sensitive dye recording from branches of individual neurons was brought to a level at which it can be used routinely in physiologically relevant experiments. The crucial figure-of-merit in this approach, the signal-to-noise ratio from neuronal processes in intact ganglia, has been improved by a factor of roughly 150 over previously available signals. The improvement in the sensitivity allowed, for the first time, direct investigation of several important aspects of the functional organization of an individual neuron: (1) the direction and the velocity of action potential propagation in different neuronal processes in the neuropile was determined; and (2) the interaction of two independent action potentials (spike collision) was monitored directly in a neurite in the neuropile; (3) it was demonstrated that several action potentials are initiated in the same neuron at different sites (multiple spike trigger zones) by a single stimulus; (4) the exact location and the size of one of the remote spike trigger zones was determined; (5) the spread of passive subthreshold signals was followed in the neurites in the neuropile. This kind of information was not previously available. Preliminary experiments on vertebrate neurons indicate partial success in the effort to use intracellularly applied voltage-sensitive dyes to record from neurons in a mammalian brain slice preparation. The results suggest that, with further improvements, it may be possible to follow optically synaptic integration and spike conduction in the dendrites of vertebrate nerve cells. The main impact of these results is a demonstration of a new way of analysing how individual neurons are functionally organized. Limitations and prospects for the further refinement of the technique are discussed mostly in terms of the signal-to-noise ratio; both improvements in the apparatus and design of more sensitive dyes are addressed.

Temperature acclimation: effects on membrane physiology of an identified snail neuron.

The neuronal basis for thermal acclimation was examined by comparing the short- and long-term effects of temperature change on the physiological properties of an identified neuron in the isolated ganglion of Hexis aspersa. Using intracellular electrophysiological techniques, we found that the frequency of spontaneous action potentials and excitability of neurons from warm-acclimated animals was depressed by abruptly cooling from 20 to 5 degrees C. After a 2-wk period of acclimation to 5 degrees C, the levels of spontaneous activity and excitability were comparable to those of warm-acclimated neurons at 20 degrees C. Conversely, abrupt warming of neurons from cold-acclimated animals greatly increased the frequency of spontaneous activity, but after acclimation to 20 degrees C the frequency decreased. Although the duration of the action potential and the cell's electrogenic Na-K pump were temperature sensitive, thermal acclimation had no obvious effects on these parameters. Membrane permeability to Na and PNa/PK decreased with cooling, whereas PRb/PK and PCs/PK increased. Warming had the opposite effect on the relative alkali cation permeability (PX/PK). With acclimation PX/PK underwent compensatory changes.

Myocardial perfusion imaging using 99mTc-MIBI in patients with dilated cardiomyopathy--methodology and its clinical use.

The aim of the study was to demonstrate the methodology of investigation of myocardial perfusion and viability using 99mTc-MIBI in patients with dilated cardiomyopathy and to estimate its clinical use. Myocardial perfusion and viability were investigated by using the planar and SPECT scintigraphy and by estimating the wall motion of the myocardium. The foci of decreased myocardial viability were found in 7 patients. One patient had perfusion defect only at stress. Analyzing the patients data, by using the gated studies, we found various degrees of wall motion myocardial abnormalities.

Fast optical recordings of membrane potential changes from dendrites of pyramidal neurons.

Understanding the biophysical properties of single neurons and how they process information is fundamental to understanding how the brain works. A technique that would allow recording of temporal and spatial dynamics of electrical activity in neuronal processes with adequate resolution would facilitate further research. Here, we report on the application of optical recording of membrane potential transients at many sites on neuronal processes of vertebrate neurons in brain slices using intracellular voltage-sensitive dyes. We obtained evidence that 1) loading the neurons with voltage-sensitive dye using patch electrodes is possible without contamination of the extracellular environment; 2) brain slices do not show any autofluorescence at the excitation/emission wavelengths used; 3) pharmacological effects of the dye were completely reversible; 4) the level of photodynamic damage already allows meaningful measurements and could be reduced further; 5) the sensitivity of the dye was comparable to that reported for invertebrate neurons; 6) the dye spread approximately 500 micron into distal processes within 2 h incubation period. This distance should increase with longer incubation; 7) the optically recorded action potential signals from basolateral dendrites (that are difficult or impossible to approach by patch electrodes) and apical dendrites show that both direct soma stimulation and synaptic stimulation triggered action potentials that originated near the soma. The spikes backpropagated into both basolateral dendrites and apical processes; the propagation was somewhat faster in the apical dendrites.

[Ischemic heart disease in young women. Its importance and possibilities of prevention]. / Ishemijska bolest srca u mladih zena. Vaznost i mogucnost prevencije.

Ischemic heart disease in young women is a rare disease, but nowadays it is more frequently detected. The investigation involved 83 women up to the age of 19 with ischemic heart disease. The most frequent risk factors were as follows: hypertension, hyperlipoproteinaemia, smoking, stress and obesity. The association of three factors was most often observed. Electrocardiogram at rest pointed to the changes as follows: anteroseptal region, the whole anterior wall, postero-inferior region and subendocardial localization. Out of 11 exercise tests 36 (81.81%) of them were positive. Coronary arteriography was performed in 72 (86.71%), with positive changes in 61 (81.72%) patients. Occlusive changes were most frequently found on anterior descendent artery. Since a great number of risk factors in women may be easily corrected by an adequate diet and healthier way of life, their detection and implementation of measures of primary and secondary prevention have been attached a great importance.

[Hypertension as a risk factor in acute infarct in young people]. / Hipertenzija kao rizik u akutnom infarktu mladih.

INTRODUCTION: Hypertension is one of the most frequent risk factors for acute myocardial infarction. The aim of this paper was to establish both the incidence of hypertension as a risk factor for myocardial infarction in young people and its characteristics. METHODS: The investigation involved patients younger than 40 years of age with acute myocardial infarction treated at the Institute of Cardiovascular Diseases in Sremska Kamenica from 1989 to 1994. RESULTS: The investigation involved 80 patients with acute myocardial infarction younger than 40 years--40 male and 40 female. Hypertension as a risk factor was found in 22 (55%) women and in 14 (35%) men and it was a moderate hypertension of systolic-diastolic type or unstable hypertension. In 10 (27.77%) investigated patients hypertension was detected during hospitalization (before that, it had not been controlled) while in 22 (61%) it was not seriously taken into account either by the patients, or their physicians and therefore it was not treated. As a single risk factor hypertension was found in 4 (10%) women, while it was not found in men. DISCUSSION: Hypertension is found in 10-15% of population, and in 10% young men and women. The existence of hypertension as the only risk factor for the occurrence of acute myocardial infarction was sufficient in these women, although it was considered that women in their generative period are protected from ishaemic heart diseases by their hormonal status. All that points to hypertension as a secret and mysterious killer. CONCLUSION: Hypertension as a risk factor was found in 22 (55%) women, and in 14 (35%) men. In 10% of women hypertension was the only risk factor, while it was not found as such in men. It was quite sufficient in these women for the occurrence of acute myocardial infarction, although it was considered that women in their generative period are protected from ishaemia in heart diseases by their hormonal status. Detection and adequate treatment of hypertension are of great importance as well as elimination of other risk factors.