Biomedical subjects

U Kuhnt

Publications and source records attributed to U Kuhnt.

At least 37 records · Page 2Linked to original sources

PubMed1993

Evoked changes of membrane potential in guinea pig sensory neocortical slices: an analysis with voltage-sensitive dyes and a fast optical recording method.

Coronal slices from guinea pig visual neocortex were stained with voltage-sensitive fluorescent dyes RH414 or RH795. Activity was evoked by electrical stimulation of either white matter or layer I. Emitted-light intensity changes representing summated changes of membrane potential were recorded by a 10 x 10 photodiode array with a temporal resolution of 0.4 ms and a spatial resolution of 60 microns or 94 microns. Following either stimulation of layer I or of white matter, maximal activity was located close to the respective stimulation electrode, in upper layer III/II, and between layer IV and V. With stimulation of the white matter, additional peak activity was recorded from upper layer VI. Non-synaptic activity was separated from mixed (synaptic and non-synaptic) activity by comparing responses obtained in standard perfusion medium with those obtained in perfusion medium from which the calcium was omitted, such that synaptic transmission was blocked. With stimulation of the white matter, most of the evoked activity in lower cortical layers was of non-synaptic origin. This non-synaptic activity consisted of early and fast potentials, which were predominant in layer VI and probably represented presynaptic fibre activity, and of slower components that were presumably of antidromic origin. Significant postsynaptic activity was only found in upper layer III/II. In contrast, with stimulation of layer I, most of the evoked activity was of postsynaptic origin. Early and fast non-synaptic potentials consisting of presynaptic fibre activity were confined to layer I. Slower non-synaptic activity, that might reflect direct dendritic activation, was minimal and was confined to upper cortical layers. Thus, following either stimulation of layer I or of white matter, the major postsynaptic components were found in upper layer III/II. It is suggested that the postsynaptic response following stimulation of white matter resulted from di- or polysynaptic activation by afferent fibres. The postsynaptic response to stimulation of layer I was presumably a monosynaptic activation of apical dendrites from pyramidal cells by layer I horizontal fibres. Activity following stimulation of white matter spread faster than activity following stimulation of layer I. This might reflect the difference in active conduction along afferent and efferent fibres on the one hand and in passive conductance along the dendritic tree on the other hand.

Animals↗

PubMed1993

Algorithm for semi-automatic sorting of objects to specified tissue domains. An aid for co-ordinating morphometric data with identified tissue components.

A personal computer-based technique was developed that reduces the extent of human efforts in obtaining data, like numerical density of profiles on area, describing the distribution of specific features of histological sections, e.g., density of synaptic profiles or of histochemical reaction products in specified tissue compartments. The procedure consists of (i) marking the objects to be counted, (ii) recording the borderlines of (reference) tissue domains of interest, and (iii) allocating the marked objects to the corresponding domains automatically. This automatic sorting of objects into defined tissue domains is achieved by an algorithm operating on two sets of coordinates: (i) coordinates of points that constitute the boundary of tissue domains (perimeter points) and (ii) coordinates of points marking the particular objects to be sorted. The principle of the sorting calculation is to construct 'segments' of the loop by lines parallel to the ordinate which pass through neighbouring perimeter points. The 'items' to be sorted are classified by the sign of marker flags allocated to each point depending on which side of the perimeter segment they are located. Segmentation and classification procedures are sequentially repeated along the entire perimeter of each domain specified by the operator which may result during the procedure in multiple changes of the sign of the flags. The internal or external location of each item finally is represented by the last sign of its flag. Objects, allocated to domains can be counted and processed further for numerical density determination.

Algorithms↗

PubMed1993

The astrocytic localization of protein kinase C in the neocortex, hippocampus and in vitro hippocampal slices of the guinea pig.

Immunohistochemical studies prove the most of the neurons of the central nervous system express one or more protein kinase C isoenzymes. However, there are only a few descriptions of the glial localization. In the present study, we detected protein kinase C immunohistochemically in guinea pig sensory neocortex, hippocampus and hippocampal slices following in vitro maintenance. Monoclonal antibodies against types II and III of protein kinase C were used. Consecutive sections were stained with polyclonal anti-glial-fibrillary-acidic protein serum. Scattered astrocytes contained protein kinase C in layers I and VI of the cerebral cortex, in the subcortical white matter, the subiculum, alveus and molecular layer of Ammon's horn, in the hilus and molecular layer of the dentate fascia. Ultrastructurally, immunostaining of glial processes in the neuropil and around blood vessels was observed. Strong staining of the endoplasmic reticulum and nuclear envelope was noted in glial cell bodies. The ultrastructural localization of protein kinase C suggests its participation in receptor-mediated processes, which may influence the shape and function of astrocytes.

Animals↗

PubMed1992

Reliability of quantal parameter estimates and their changes during long-term potentiation in guinea pig hippocampal slices.

Previously we found increases in quantal content (m) and smaller increases in quantal size (v) during long-term potentiation (LTP) in CA1 of hippocampal slices. However, the validity of the deconvolution technique was questioned recently because v estimates correlated with the noise standard deviation (Sn). In computer simulations we show a double-step dependence of v on Sn/v: correct v estimates (within +/- 20%) for Sn/v < or = 0.5 and overestimates (correlated with Sn) for Sn/v > 0.5. A novel 'noise addition' procedure is proposed for accepting reliable solutions on the basis of the double-step relationship between v and Sn. Quantal analysis of LTP for more reliable solutions confirmed previous conclusions.

Animals↗

PubMed1992

Quantal parameters of "minimal" excitatory postsynaptic potentials in guinea pig hippocampal slices: binomial approach.

Binomial distributions of amplitudes of excitatory postsynaptic potentials (EPSPs) mixed with Gaussian noise were simulated. The objective of Monte Carlo simulations was, firstly, to study influences of sampling size (N) and noise standard deviation (Sn) on estimates of mean quantal content (m), quantal size (v) and binomial parameters (n and p) by four methods of quantal analysis (histogram, variance, failures and combined method) based on the binomial model and, secondly, to modify these methods on the basis of comparison of estimated with simulated parameters. Reliable estimates (within +/- 10% of the simulated values) were obtained for large sample sizes (N = 500-1000) with Sn less than or equal to v by the histogram (deconvolution) method and with Sn less than or equal to 2v by the other three methods. Similar results were obtained by averages from about 10 simulations if smaller samples were used (N = 50-200). In electrophysiological experiments on slices, "minimal" EPSPs were recorded from CA1 pyramidal cells after low-intensity stimuli to stratum radiatum or stratum oriens. Amplitudes of minimal EPSPs fluctuated in a manner predicted by the quantum hypothesis. Amplitude distributions of EPSPs in the non-facilitated state were adequately described either by binomial statistics with an average p equal to about 0.4 (a range of 0.3-0.7) and an average n of about 3 (range 2-6) or by Poisson statistics with m of about 1. The quantal analysis suggests that typical values of m and v for a single activated fibre in stratum radiatum might be about 0.5-1 and 300-400 microV, respectively, with low p (0.1-0.3) and n (2-4). However, the estimates of binomial parameters should be considered as coarse approximations in view of the simulation results and a possible nonuniformity of parameter p. The comparison of results of various methods based on the binomial model, in both simulation and physiological experiments, indicates the reliability of estimates of basic quantal parameters (m and v) under realistic conditions of physiological experiments. The methods are considered to be sufficiently sensitive to make use of them for studies on mechanisms of long-term synaptic plasticity.

Animals↗

PubMed1992

Statistical analysis of long-term potentiation of large excitatory postsynaptic potentials recorded in guinea pig hippocampal slices: binomial model.

Excitatory postsynaptic potentials (EPSPs) were recorded in guinea pig hippocampal slices (area CA1) from 15 neurons after stimulation of stratum radiatum (str. rad.) and stratum oriens. EPSP amplitudes increased in 8 neurones (10 post-tetanic regions) recorded 15 to 45 min after tetanic stimulation of str. rad. The increase was considered to represent long-term potentiation (LTP). Quantal analysis was performed by two methods assuming binomial statistics: the histogram method using deconvolution of noise and the variance method. According to both methods, LTP was associated with an increase in mean quantal content (m) which correlated with LTP magnitude. A statistically significant increase in quantal size (v) was found only by the histogram method and the increase was not correlated with LTP magnitude. A separate analysis of EPSPs with small LTP magnitude demonstrated that with the histogram method only v was increased but not m. A smaller increase in m for the pooled data of both methods did not correlate with LTP magnitude for this EPSP subset. The increase in m for the whole EPSP set corresponds to previous results on the quantal analysis of LTP in in vivo preparations and favours a presynaptic location of major mechanisms underlying LTP maintenance. The increase in v indicates the existence of another mechanism responsible for the maintenance of a small part of LTP. This mechanism might involve either pre- or postsynaptic changes or both.

Algorithms↗

PubMed1992

Quantal analysis of long-term potentiation of "minimal" excitatory postsynaptic potentials in guinea pig hippocampal slices: binomial approach.

"Minimal" excitatory postsynaptic potentials (EPSPs) were recorded from 13 neurones in area CA1 of guinea pig hippocampal slices after double-pulse stimulation of stratum radiatum (str. rad.) and stratum oriens (str. or.). Amplitudes of EPSPs significantly increased in 8 neurones 5 to 55 min after 9 tetanizations in str. rad.. The increase was considered to represent long-term potentiation (LTP). Altogether 26 EPSPs (42 post-tetanic regions) were statistically analysed by four methods of the quantum hypothesis assuming the binomial model of transmitter release: the deconvolution (histogram), the variance, the failures, and the combined (variance-failures) methods. The mean quantal content (m) significantly increased after LTP induction according to all methods used. Quantal size (v) also tended to increase but according to some methods, the increase was not statistically significant and it did not correlate with LTP magnitude. However, for an EPSP subset with a LTP magnitude of less than 1.55, the increase in v correlated with LTP magnitude, whereas the increase in m did not. The relative contribution of the increase in v to LTP magnitude was larger for cases with small LTP than for the whole EPSP set. In general, the increase in m corresponds to previous studies and favours the presynaptic location of major mechanisms of LTP maintenance, i.e. an increase in the average number of transmitter quanta released by each presynaptic volley. The post-tetanic increase in v might reflect some additional mechanisms which presumably include an increase in the amount of transmitter in one quantum.

Animals↗

PubMed1992

Analysis of fluctuations of "minimal" excitatory postsynaptic potentials during long-term potentiation in guinea pig hippocampal slices.

In previous studies, quantal analysis assuming a simple binomial model has shown that long-term potentiation (LTP) is accompanied by an increase in both mean quantal content (m) and quantal size (v), whereby the increase in m predominates. In the present study, "compound" binomial distributions with variable probabilities were convolved with Gaussian distributions in computer experiments to simulate amplitude histograms of intracellular excitatory postsynaptic potentials (EPSPs). A deconvolution procedure assuming equal "quantal" separation (v) between discrete components, but without assuming binomial statistics, was applied to the simulated distributions to determine v. It was found that with a small ratio of standard deviation of noise to v (Sn/v less than 0.4), a reliable estimate of v can be obtained even for small samples (N = 100). When Sn/v was larger (0.4-0.6), approximate v estimates (within +/- 10-20% of the simulated v) could be obtained by averaging estimates from about 10 small samples (N = 100). "Minimal" EPSPs were recorded in area CA1 of guinea pig hippocampal slices. 37 EPSP amplitude samples of 9 neurones were measured before and up to 55 min after 10 tetanizations of stratum radiatum. In accordance with the previous data, the increase in v accounted for only about 10% of the average post-tetanic increase in EPSP amplitude and was not correlated with the latter. However, for an EPSP subset with small LTP magnitude, the increase in v accounted for an essential part of the LTP magnitude while the increase in m did not correlate with it. The results are in agreement with previous data obtained in the context of the simple binomial model and are interpreted as indicating primarily a presynaptic mechanism of LTP maintenance. The results suggest two types of synaptic mechanism of LTP maintenance related to the increases in m and v, respectively. The latter mechanism is saturated at about 10 to 30% increase in post-tetanic amplitude above the pre-tetanic EPSP amplitude.

Animals↗

PubMed1992

Presynaptic calcium transients evoked by paired-pulse stimulation in the hippocampal slice.

The fluorescent dye Calcium Green and optical recording techniques were used to record intracellular Ca2+ transients resulting from paired-pulse stimulation in stratum moleculare of area CA1 in guinea-pig hippocampal slices. Presumed presynaptic calcium transients were recorded while glutamatergic synaptic transmission was blocked by kynurenic acid. Peak responses to paired-pulses (10-160 ms interval) were higher than responses to single pulses of same stimulation strength (42-23% increase). The isolated response to the second pulse, however, was of smaller magnitude in comparison to the first one; the difference in magnitude depended on the interstimulus interval. Thus, the residual presynaptic free calcium concentration may be responsible for paired-pulse facilitation of synaptic transmission in hippocampus. At the same time, a use-dependent inactivation of presynaptic calcium channels may occur.

Animals↗

PubMed1991

Induction of raf-1 protein immunoreactivity in guinea pig hippocampal slices during the in vitro maintenance.

The cellular raf-1 protooncogene encodes a cytoplasmic serine/threonine protein kinase; raf-1 is expressed not only in tumour cells, but also in normal brain tissue. However, the substrate of raf-1 kinase is unknown and its function is still hypothetical. In cell cultures, mitogen stimulation may alter its properties, including its kinase activity and subcellular distribution. The aims of the present study were: (1) light- and electron microscopic immunohistochemical localization of raf-1 kinase in hippocampal slices; (2) the follow-up of the possible changes in localization and detectability during the in vitro maintenance and low-frequency stimulation. Our results suggested the induction of neuronal raf-1 kinase during the in vitro maintenance. The possibilities of increased transcription and enzyme activation are discussed.

Animals↗

PubMed1991

Algorithm for point-to-point correlation of geometrically nearly similar microscopic objects.

An algorithm is presented that compares two quasi similar images by correlating selected points on them--assuming their coordinates are available. The procedure involves translational, magnificational and rotational operations to find corresponding point pairs on the pictures. The algorithm automatically compensates for slight dissimilarities between images and constructs a reference point database for correlation during the evaluation process. Establishment of the reference point networks on the images prior to the examination is avoided.

Algorithms↗

PubMed1991

Time dependent loss of tissue GABA content and immunoreactivity in hippocampal slices.

Immunohistochemical detection of GABA was used to evaluate changes of the GABA innervation in hippocampal slices maintained in vitro. In parallel experiments the amount of GABA, glutamate and aspartate was measured with high performance liquid chromatography. The results showed that while glutamate and aspartate levels remained fairly constant, GABAergic neurons suffered remarkable alterations. During 8 hours' incubation the GABA content of the tissue and the number of GABA containing neuronal cell bodies decreased by 79.7% and 84.6%, respectively. The qualitative features of the immunoreactivity of the neuropil did not change. In conclusion, while in hippocampal slices tissue glutamate and aspartate levels are only slightly affected by the in vitro maintenance, more than half of the tissue GABA content is lost during prolonged in vitro incubation. As a consequence of the GABA loss, the ratio of endogenous inhibitory and excitatory amino acid transmitters has been altered, which could influence the viability of adult hippocampal tissue in vitro conditions.

Animals↗

PubMed1990

Transient increase of raf protein kinase-like immunoreactivity in the rat dentate gyrus during long-term potentiation.

Altered levels of cellular raf proteins (products of the raf protooncogenes) have been shown in the neurons of the dentate fascia of rats in response to high-frequency stimulation, with light microscopic immunohistochemistry by using polyclonal antibodies. No raf-1-like staining was seen in unstimulated tissue, while the pan-raf antibodies revealed immunoreactivity in the cytoplasm of neurons in the Ammon's horn and dentate fascia of rats and guinea pigs. The induction of long-term potentiation in the dentate fascia of freely-moving rats triggered the appearance of raf-1-like staining and increased the number of granule cells with pan-raf-like immunoreactivity. Since these proteins are serine/threonine-specific protein kinases, their appearance in long-term potentiation may indicate the activation of important cell membrane - nucleus transduction pathways.

Animals↗

PubMed1990

Optical recording of epileptiform voltage changes in the neocortical slice.

Voltage sensitive probes were used to monitor the development, distribution, and spread of epileptiform potentials with a photodiode array in neocortical slices of guinea pigs. Epileptiform activity was induced by bath application of bicuculline-methiodide or 3,4-diaminopyridine and electrical stimulation of white matter or cortical layer I. Stimulation evoked a primary or early potential which was followed by a delayed epileptiform potential with a larger spatial extent. Shape, duration and amplitude of the delayed epileptiform potential varied strongly among slices and across the recording area and could reach largest amplitudes at a distance from the stimulation point. At a specific recording site, however, with repeated stimulation, potentials were generated in a stereotyped way. Intracellularly recorded delayed epileptiform potentials corresponded very closely at least to the early part of the optical response. Epileptiform activity appeared in layer III as soon as the primary potential reached sufficient amplitude there. Apart from this relationship, the distribution and spread of maximal amplitudes of delayed epileptiform potentials were segregated from those of early potentials. Early potentials reached maximal amplitudes close to the stimulation site. In contrast, the largest amplitudes of delayed epileptiform potentials were always found in layer III. A second maximum occasionally occurred in layer V. The horizontal amplitude distribution of epileptiform potentials was asymmetric, i.e. amplitudes increased to one side and decreased to the other. Early potential maxima spread from deeper to upper layers when initiated by white matter stimulation and from upper to deeper layers when initiated by layer I stimulation. In contrast, delayed epileptiform potentials always spread from layer III to lower layers and to the sides. Velocity of spread of early potentials and delayed epileptiform potentials differed systematically along the vertical and horizontal axis. The distribution of maximal amplitudes, shape, and pattern, of spread of epileptiform potentials was the same whether white matter or layer I was stimulated. The independence of delayed epileptiform potential characteristics from the point of stimulation and from early potential characteristics suggests that epileptiform activity is determined by intrinsic properties of the cortex and not by afferent activation.

4-Aminopyridine↗

PubMed1990

[A quantum analysis of the long-term posttetanic changes in the minimal postsynaptic potentials in surviving hippocampal slices].

Excitatory postsynaptic potentials (EPSPs) were recorded in guinea pig hippocampal slices (area CA1) from 13 neurons after single or double-pulse stimulation of stratum radiatum (the Schaffer collaterals) and stratum oriens. Amplitudes of 23 EPSPs (9 neurons, 12 pathways) grew 5 to 55 min after 10 tetanic stimulations of the Schaffer collaterals. This increase has been considered as a long-term potentiation (LTP). A statistical analysis was carried out by four methods of the quantal hypothesis based on the binomial distribution. It revealed an increase in the mean quantal content (m) during LTP. An increase in the quantal size was also observed in the majority of the cases but it showed only a weak correlation with the LTP magnitude and for some methods it was statistically significant only for the periods later than 15 min after tetanic stimulation. A well expressed increase in m demonstrated by various methods corresponds to data of the previous in vivo studies and favours the presynaptic location of mechanisms of the LTP maintenance.

Animals↗

PubMed1989

Elevation of intracellular calcium content in area CA1 of hippocampus is not directly correlated with the development of long-term potentiation.

Electron microscopic localization of calcium-containing mitochondria in stratum radiatum of CAl of hippocampal slices was performed after (1) low-frequency stimulation, (2) high-frequency stimulation, and (3) blocking N-methyl-D-aspartate (NMDA) receptors during high-frequency stimulation. Dendritic mitochondria containing Ca deposits were found in a narrow band of stratum radiatum 280-350 microns distant from stratum pyramidale. Axonal mitochondria containing Ca deposits were evenly distributed in stratum radiatum. The total number of calcium containing-mitochondria was highest in long-term potentiated slices, and less in slices treated with APV; the lowest values were obtained with low-frequency stimulation.

Animals↗

PubMed1989

Low intensity tetanic stimulation induces long-term potentiation in hippocampal neurons.

A minimal intensity of the stimulation necessary for the induction of long-term potentiation of synaptic transmission (LTP) was investigated by intracellular recording in guinea pig in vitro hippocampal slices. High frequency stimulation of afferent fibres at intensities evoking in CA 1 neurons control excitatory postsynaptic potentials (EPSPs) of amplitudes 1-5 mV, resulted usually in a long-lasting increase in response amplitude. LTP was not observed at lower stimulus strength. The coactivation of a certain, though small number of synaptic contacts is thus necessary for the production of LTP.

Action Potentials↗

PubMed1988

Increased retention of calcium in the dendrites of long-term potentiated CA1 neurons of the hippocampal slice. A combined electrophysiological and electron histochemical study.

In view of the importance of calcium in the induction of long-term potentiation (LTP), experiments were carried out to localize calcium at the electron microscopic level in the CA1 region of guinea pig hippocampal slices, following high-frequency stimulation of the Schaffer collaterals. Apart from the ultrastructural localization, a semi-quantitative method was used to count the calcium-containing deposits in electron micrographs. Significantly more calcium-containing deposits were seen in the dendrites of the stratum radiatum in slices with LTP than in those without it. A moderate increase of the extradendritic deposits was observed, too. The calcium content of the deposits was determined by means of EGTA incubation and X-ray analysis. The presented results, together with the relevant literature data, underline the importance of calcium-activated processes in postsynaptic structures probably involved in the generation of LTP.

Animals↗